Tweet Post Share Save Get PDF Buy Copies Print Idea in Brief The Situation The fast-changing nature of business today means that employees’ continual learning is vital for organizational success. The Response Chief learning officers are assuming a more expansive role, aiming not only to train employees but also to transform their organizations’ capabilities and make learning an integral part of the company’s strategic agenda. The Specifics Extensive interviews at 19 large companies revealed that “transformer CLOs”—those who are embracing this expanded role—are driving changes in their enterprises’ learning goals, learning methods, and learning departments. In today’s dynamic business environment, workplace learning has become a key lever for success. And with that shift, the traditional role of the chief learning officer is changing. No longer are CLOs responsible just for training—making skills-based and compliance-oriented courses available to employees and perhaps running leadership-development programs. Instead, they’re embracing a more powerful role in which they reshape capabilities and organizational culture. We call this new type of leader the transformer CLO. Transformer CLOs are strong senior managers whose mission is to help their companies and their employees thrive, even as technologies, business practices, and whole industries undergo rapid change. The transformer CLO role is not reserved for the lucky few whose CEOs see learning and development as essential; any CLO can take steps to fundamentally change the nature of learning in an organization. We recently conducted extensive interviews with 21 senior learning officers at 19 large companies to find out how they conceive of their roles and organizations. This research, which builds on our prior work on digital leadership and culture, revealed that transformer CLOs are driving three principal types of change in their enterprises. They’re transforming their organizations’ learning goals, shifting the focus from the development of skills to the development of mindsets and capabilities that will help workers perform well now and adapt smoothly in the future. They’re transforming their organizations’ learning methods, making them more experiential and immediate, and atomizing content for delivery when and where it’s needed. And they’re transforming their organizations’ learning departments, making them leaner, more agile, and more strategic. Transforming Learning Goals The need for organizations to become more adaptable means changing the goals of corporate learning. Instead of narrowly focusing on job- or compliance-related training for all but their high-potential leaders, organizations should cultivate every employee’s ability to explore, learn, and grow. The objective is not only to train people but also to position the company for success. To achieve this, CLOs should strive to do the following: Reshape leadership development. Creating a true learning organization starts at the top, with preparing executives to lead in new ways. One company that has done this well recently is Standard Chartered, a multinational financial-services company. Three years ago, under a new CEO, Standard Chartered launched a strategy that fundamentally changed the way it does business—and required its leaders to build new strengths. “We’d been doing executive development for years,” said Ewan Clark, the company’s global head of leadership effectiveness and organizational development. “But a lot of it had been about either pure self-actualization or aspects of coaching. This time we’ve put the organizational agenda right in the center of executive development, and we’ve said that leadership is about developing the skills, capabilities, and value behaviors to lead this agenda.” As part of that effort, the company began teaching leaders to augment their experience and intuition with investigation, experimentation, and data-driven analysis when making decisions about their parts of the organization. Their instructions, according to Clark, were straightforward: “Articulate a hypothesis. Go out and experiment. And if it doesn’t work, then why not? What did you learn? Add to it. Capture your learning. Share it with other people.” This new approach required changes in the leaders’ mindsets, not just their skills and procedures. Organizations should cultivate every employee’s ability to learn and grow. It’s not enough, though, to improve leadership capabilities at the very top of the organization. To effect widespread change, organizations need strong leadership to cascade down. Cargill, a privately held food and agriculture business, achieved this by democratizing learning. As Julie Dervin, the company’s global head of corporate learning and development, told us, “We really only had the capacity to reach about 10% to 15% of the total relevant population in a given year when delivering a particular learning program. Unintentionally, we were creating a learning culture where only a select few got access to high-quality training.” Dervin and her team resolved to fix that problem. “We’ve been fundamentally changing how we design, deliver, and shape those learning experiences to be able to reach exponentially more learners with high-impact learning,” she said. Concentrate on capabilities, not competence. In their change programs, transformer CLOs focus less on teaching currently needed skills and more on developing mindsets and behaviors that can enable employees to perform well in tasks that may not yet be defined. This shift may also mean moving away from comprehensive skills inventories and competency maps, which can lead people to check boxes rather than build capabilities. “We don’t really know enough about what the world will look like in the next couple of years to be able to predict exactly what skills we will need,” said Amelie Villeneuve, the head of the corporate university at UBS, the multinational financial-services firm. “If you focus on building individual microskills, you may be missing the bigger picture.” Emphasize digital thinking. The transformer CLOs we interviewed have sought to develop digital awareness and aptitude in their employees. Singapore-based DBS Bank, for example, created a learning curriculum that aims to build seven priority skills for digital-business success. “While not everyone needs to be an expert at each of these,” said David Gledhill, who served as the company’s chief information officer until August 2019, “we want them to know enough so that they understand the transformation we’re driving and contribute great ideas.” Vital Skills for a Digital World To equip its employees for success in today’s digital business environment, DBS Bank focuses on imparting skills ... One priority, for instance, is to get people more comfortable using data in decision-making. Data-driven thinking is key for almost everyone in an organization, but in different ways. Frontline sales and service reps need to be aware of information about customer preferences and behaviors. Executives must learn to trust and value data even when it contradicts their past experiences and gut feelings. Leaders often don’t know what to do with all the data that digital innovations are making available to them, said Nancy Robert, who, as the executive vice president of the American Nurses Association, led the design and delivery of training for millions of the organization’s members. As Robert put it, nurses don’t necessarily have the “digital-data competency” to answer the questions that confront them. “How am I going to interpret that data and integrate it into the rest of the care?” she said. “That takes a very different cognitive skill.” Cultivate curiosity and a growth mindset. CLOs can amplify their teams’ energies and capabilities by fostering a “pull” model of learning, in which employees set their own agendas for gaining knowledge and skills. Doing that, however, requires an environment that sparks employees’ curiosity and ignites in them the desire to learn and grow. Villeneuve has worked on this at UBS and previously at Google, where, she said, she learned how it is possible to “accelerate wisdom more effectively by providing a series of contexts where people can play and learn at the same time.” Leaders at DBS Bank launched a number of programs to find out what would inspire curiosity among their employees. One notable success is GANDALF Scholars, in which employees can apply to receive grants of $1,000 toward training on any work-related topic, as long as they agree to teach what they learn to at least 10 other people. When you engage employees in teaching, as DBS is doing, you expand and deepen learning. Rahul Varma, the senior managing director for talent at Accenture, calls this a “leaders teaching leaders” philosophy. “You learn the most,” he said, “when you actually have to teach somebody what you learn.” This approach turns the natural curiosity and energy of any single employee into learning opportunities for many others. It certainly seems to be working at DBS: As of early 2019, 120 grant recipients had gone on to train more than 13,500 people—4,000 in person and the rest through digital channels. According to Gledhill, many GANDALF Scholars report that the teaching component of the program is their favorite part. “What they enjoyed most,” he said, “was the empowerment.” Transformer CLOs are personalizing, digitizing, and atomizing learning. UBS, DBS, Accenture, and other companies that have embraced a growth mindset subscribe to two beliefs: that everyone’s abilities can and must be developed if the organization is to thrive in a fast-moving environment, and that innate talent is just the starting point. But for a growth mindset to become part of the company’s culture, all employees must internalize those beliefs. That won’t happen unless learning is pervasive, available to everybody who might benefit from it. And that requires rethinking the way learning is delivered. Transforming Learning Methods Until recently, providing learning to all employees was too expensive, and there weren’t enough trainers. Employees almost always had to be physically present at training sessions, which often meant traveling and missing time at work. That naturally limited the number of participants, making learning an exclusive rather than a democratic opportunity. Now things have changed. Peer teaching greatly expands the number of trainers and expert content developers. And digital instruction expands the reach of learning opportunities to more employees without the company’s having to worry about enrollment numbers, scheduling conflicts, or travel costs. Employees can access learning when and where they need it, often from colleagues who live the topic every day. Transformer CLOs are taking advantage of all these developments. Perhaps most visibly, they are moving away from traditional classroom training in which people are exposed to the same content for the same amount of time regardless of their particular needs and levels of understanding. Instead, these CLOs are personalizing, digitizing, and atomizing learning. They are shifting their attention from specific courses to the whole learning experience. To accommodate the different preferences employees have for how they consume and absorb information, a growing number of companies now make training available through a variety of media—text, audio, video, and more. Transformer CLOs go even further. They’re introducing innovations such as programs that set aside learning time on people’s calendars, and mobile apps that pose leadership questions to managers during their day. They’re offering games and simulations and encouraging the company’s own subject-matter experts to produce YouTube-type instructional videos. They’re even exploring the use of artificial intelligence to develop recommendation engines that, guided by individual and peer behavior, will suggest tailored learning activities to employees. In short, transformer CLOs do everything possible to create engaging and effective experiences that meet employees wherever they happen to be, geographically, temporally, or intellectually. Optimize the inventory of learning resources. CLOs need to be selective about what learning materials to stock and how to supply them. At GE Digital, Heather Whiteman, the company’s former head of learning, used analytics with her team to study hundreds of courses taken by thousands of employees—and then systematically rooted out those found lacking, not just in terms of usage and ratings but in their effects on employee growth. “If a course didn’t move the dial for capabilities that lead to performance,” she told us, “we would drop it in favor of one that did.” Similarly, Villeneuve and her team at UBS used analytics to optimize the learning inventory. The bank had a wealth of training materials online, but analysis showed that many employees who searched for those materials gave up before finding what they needed. Armed with that knowledge, Villeneuve and her team focused on developing a core of fewer but better resources. Then, applying principles of behavioral science, they designed a user interface that put no more than six items on a page, with no more than three clicks needed to get to any item. The results have been remarkable: Ten times more employees now engage with the materials on the company’s core learning shelf. Balance face-to-face and digital learning. CLOs should experiment to get the right mix of face-to-face and digital learning. Cargill, which until recently allocated 80% of its budget to in-person training and only 20% to digital training, is in the process of flipping that ratio around. Dervin and her team have redesigned the company’s leadership-development programs to put some of the coursework online. Senior leaders initially had reservations about the effectiveness of digital instruction and worried about losing opportunities to network and build relationships. But those misgivings were short-lived. The first three cohorts who tried the online learning ended up enjoying the experience so much that they engaged in more training than was required. “What we’re seeing,” Dervin said, “is that this goes hand in glove with the pace and the rhythms of their day-to-day, and they’re loving the flexibility it provides.” Deutsche Telekom, for its part, has developed a matrix to help determine whether a given offering might be better handled with face-to-face instruction, a purely digital approach, or a blend of the two. The matrix helps leaders weigh multiple factors: the type of content, the target audience, and development and delivery considerations. Digital or Face-to-Face Training? Deutsche Telekom considers a number of factors when deciding how best to present specific learning programs. FORMAT CONTENT TARGET AUDIENCE DEVELOPMENT AND DELIVERY CONSIDERATIONS Purely digital formats Best suited for: Hard skills Mandatory training Simple topics Durable, reusable material Larger groups Geographically dispersed or mobile employees, such as those in sales and field service More time required to produce nonstandard material Higher up-front cost to produce nonstandard material Lower cost to deliver per user No need for trainers or videoconferencing facilities at the location Face-to-face or blended formats Best suited for: Soft skills Ad hoc training Complex topics Material that changes frequently Smaller groups Geographically concentrated employees Employees being onboarded Less time required to produce nonstandard material Lower up-front cost for course preparation Potential higher cost to deliver, but possibility of using existing staff as trainers Need for training rooms or videoconferencing at the location   Source: Adapted from company documents © HBR.org Rethink face-to-face learning. As engaging and effective as digital learning experiences can be, face-to-face learning is still important—although it may take new forms. Accenture employs some very sophisticated digital learning platforms and tools and has a vast library of online content, but Varma’s experience is that digital learning goes only so far. “What we’ve found,” he said, “is that there is no substitute for getting people together in cohorts that are cross-cultural and cross-functional.” To achieve that without requiring employees to be in the same physical space, Accenture has created more than 90 “connected classrooms” around the world. These enable the company to offer all employees some types of training—classes in design thinking, for example—that are taught by in-house experts in several different locations. “One facilitator could be in Bangalore, another could be in Manila, and another in Dalian, China,” Varma told us. People are still learning from people, but thanks to videoconferencing and other interactive technologies, along with more-collaborative approaches to learning, traditional geographic constraints no longer apply. Teams all over the world now coach one another and solve problems together. “That is how we do learning, every single day,” Varma said. Some companies have pursued another approach for their face-to-face learning: They’ve created hands-on simulations in which participants must solve real-life problems. At UBS, employees take part in “three-dimensional case studies” in order to develop key capabilities, such as the ability to influence stakeholders or rethink a company product. The interactive case studies test not only their knowledge and intellectual skills but also how they engage with others and react as the situation unfolds. As Villeneuve told us, “They have to do it all together, and they get feedback on everything at the same time.” Face-to-face learning is still important—although it may take new forms. Similarly, operational professionals at DBS spend three days in a simulation exercise that involves transforming a hypothetical old-school bank into a full-fledged digital bank. They work with trainers and colleagues from other parts of the business to tackle staffing and resourcing issues and handle crisis situations unique to the digital world. An element of competition heightens the intensity and engagement. Go beyond instruction. Transformer CLOs believe that instruction alone is not sufficient for meaningful learning. Accenture’s Varma anchors his approach in what he calls the three I’s: instruction, introspection, and immersion. Instruction comes first, of course. But then trainees need to engage in reflection—the introspection part of Varma’s three I’s. This might involve giving employees time to privately mull over what they’ve learned, having them talk it over with a fellow trainee on a walk, or providing a formal opportunity during class to discuss it with a whole cohort.

But decentralized learning goes farther than that: in a decentralized, Collaborative Learning environment, each team member participates in the learning process. They can identify their learning needs, request courses, give feedback on existing courses, and create courses themselves. We call this a bottom-up approach

Leaders from Accenture and DBS Bank told Harvard Business Review that encouraging employees to teach newly-acquired skills to their colleagues expanded and deepened learning for all. The training of a single employee results in learning opportunities for dozens of others. Collaborative approaches to training ripple through an organization, where ideas and methodologies cross-pollinate from one part of the business to another

Many companies view L&D as a service provider for employees instead of a strategic partner for growth

Homology directed repair (HDR) assayEach variant was introduced into a HA-FLAG-tagged full-length PALB2 complementary DNA (cDNA) expression in the pOZC plasmid by site-directed mutagenesis using pfu turbo. Variants were verified by Sanger sequencing. Cotransfection of PALB2 expression constructs and the I-SceI expression plasmid into B400/DR-GFP reporter cells was performed at a 5:1 molar ratio using Xtremegene 9 transfection reagent (Roche). At least two independent clones containing each variant were analyzed in duplicate. PALB2 expression and transfection efficiency was verified by western blotting. Green fluorescence protein (GFP) expressing cells were quantified by fluorescence-activated cell sorting. Fold increases in GFP-positive cells, which are equivalent to HDR fold change, were normalized and rescaled relative to a 1:5 ratio derived from the p.Y551X pathogenic variant control and the wild-type PALB2 control.

Viability assayPALB2 variants were introduced into B400 cells using mCherry-pOZC expression vector and flow cytometry for Cherry-red was performed to select for cells expressing PALB2. Sorted cells were plated in 96-well plates and exposed to increasing amounts of Olaparib or cisplatin and incubated for a period of 5 days. Presto Blue (Invitrogen) was added and incubated for 1–2 hours before measuring fluorescence intensity on a Cytation 3 microplate reader (BioTek).

Viability assayPALB2 variants were introduced into B400 cells using mCherry-pOZC expression vector and flow cytometry for Cherry-red was performed to select for cells expressing PALB2. Sorted cells were plated in 96-well plates and exposed to increasing amounts of Olaparib or cisplatin and incubated for a period of 5 days. Presto Blue (Invitrogen) was added and incubated for 1–2 hours before measuring fluorescence intensity on a Cytation 3 microplate reader (BioTek).

ImmunofluorescenceLive cell imaging and microirradiation studies of HeLa cells transfected with peYFP-C1-PALB2 WT or variant constructs were carried out with a Leica TCS SP5 II confocal microscope. To monitor the recruitment of YFP-PALB2 to laser-induced DNA damage sites, cells were microirradiated in the nucleus for 200 ms using a 405-nm ultraviolet (UV) laser and imaged every 30 seconds for 15 minutes. Fluorescence intensity of YFP-PALB2 at DNA damage sites relative to an unirradiated nuclear area was quantified (Supplemental Materials). Cyclin A–positive HeLa cells treated with siCtrl and siRNA against PALB2 were complemented with wild-type and mutant FLAG-tagged PALB2 expression constructs, exposed to 2 Gy of γ-IR, incubated for 6 hours, and subjected to immunofluorescence for RAD51 foci. HeLa cells were fixed with 4% (w/v) paraformaldehyde for 10 minutes at room temperature, washed with tris-buffered saline (TBS), and fixed again with ice-cold methanol for 5 minutes at −20 °C. Cells were incubated for 1 hour at room temperature with the anti-RAD51 (1:7000, B-bridge International, 70-001) and anticyclin A (1:400, BD Biosciences, 611268), and incubated for 1 hour at room temperature with the Alexa Fluor 568 goat antirabbit (Invitrogen, A-11011) and Alexa Fluor 647 goat antimouse (Invitrogen, A-21235) secondary antibodies. Z-stack images were acquired on a Leica CTR 6000 microscope and the number of RAD51 foci per cyclin A–positive cells expressing the indicated YFP-PALB2 constructs was scored with Volocity software v6.0.1 (Perkin–Elmer Improvision). Results represent the mean (± SD) of three independent trials (n = 50 cells per condition). HEK293T cells transfected with PALB2 expression constructs were also subjected to immunofluorescence for PALB2 using the monoclonal anti-FLAG M2 antibody (Sigma) and the Alexa Fluor 568 goat antimouse (Life Technologies) secondary antibody.

CRISPR-LMNA HDR assayU2OS were seeded in 6-well plates at 200 000 cells per well. Knockdown of PALB2 was performed 6–8 h later with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours post-transfection, 1.5 × 106 cells were pelleted for each condition and resuspended in 100 μL complete nucleofector solution (SE Cell Line 4D-Nucleofector™ X Kit, Lonza) to which 1μg of pCR2.1-mRuby2LMNAdonor, 1 μg of pX330-LMNAgRNA2, 1 μg of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct, and 150 ρmol siRNA was added. Once transferred to a 100 ul Lonza certified cuvette, cells were transfected using the 4D-Nucleofector X-unit, program CM-104, resuspended in culture media and split into 2 60-mm dishes. One dish was harvested 24 h later for protein expression analysis as described above while cells from the other were trypsinised after 48 h for plating onto glass coverslips. Coverslips were fixed with 4% paraformaldehyde and cells analyzed for expression of mRuby2-LMNA (indicative of successful HR) by fluorescence microscopy (63×) a total of 72 h post-nucleofection. Data are represented as mean relative percentages ± SD of mRuby2-positive cells over the YFP-positive population from 3 independent experiments (total n >300 YFP-positive cells per condition).

RAD51 foci assayHeLa cells were seeded on glass coverslips in 6-well plates at 225 000 cells per well. Knockdown of PALB2 was performed 18 h later with 50 nM PALB2 siRNA using Lipofectamine RNAiMAX (Invitrogen). After 5 h, cells were subjected to double thymidine block. Briefly, cells were treated with 2 mM thymidine for 18 h and release into fresh media for 9 h. Complementation using 800 ng of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct was carried out with Lipofectamine 2000 during that release time. Then, cells were treated with 2 mM thymidine for 17 h and protected from light from this point on. After 2 h of release from the second block, cells were irradiated with 2 Gy and processed for immunofluorescence 4 h post-irradiation. Unless otherwise stated, all immunofluorescence dilutions were prepared in PBS and incubations performed at room temperature with intervening washes in PBS. Cell fixation was carried out by incubation with 4% paraformaldehyde for 10 min followed by 100% ice-cold methanol for 5 min at −20°C. This was succeeded by permeabilization in 0.2% Triton X-100 for 5 min and a quenching step using 0.1% sodium borohydride for 5 min. After blocking for 1 h in a solution containing 10% goat serum and 1% BSA, cells were incubated for 1 h with primary antibodies anti-RAD51 (1 :7000, B-bridge International, #70–001) and anti-cyclin A (1:400, BD Biosciences, #611268) diluted in 1% BSA. Secondary antibodies Alexa Fluor 568 goat anti-rabbit (Invitrogen, #A-11011) and Alexa Fluor 647 goat anti-mouse (Invitrogen, #A-21235) were diluted 1:1000 in 1% BSA and applied for 1 h. Nuclei were stained for 10 min with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) prior to mounting onto slides with 90% glycerol containing 1 mg/ml paraphenylenediamine anti-fade reagent. Z-stack images were acquired on a Leica CTR 6000 microscope using a 63× oil immersion objective, then deconvolved and analyzed for RAD51 foci formation with Volocity software v6.0.1 (Perkin-Elmer Improvision). The number of RAD51 foci per cyclin A-positive cells expressing the indicated YFP-PALB2 constructs was scored using automatic spot counting by Volocity software and validated manually. Data from three independent trials (total n = 225 cells per condition) were analyzed for outliers using the ROUT method (Q = 1.0%) in GraphPad Prism v6.0 and the remaining were reported in a scatter dot plot. Intensity values, also provided by Volocity, of 500 RAD51 foci from a representative trial were normalized to the WT mean and reported in a scatter dot plot. Horizontal lines on the plots designate the mean values.

Olaparib sensitivity assayFor the sensitivity assay in HeLa, 240 000 cells were seeded into one well of a six-well plate before being transfected 6–8 h later with 50 nM control or PALB2 siRNA using Lipofectamine RNAiMAX (Invitrogen). The next morning, cells were complemented with 800 ng of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-tagged PALB2 construct using Lipofectamine 2000 (Invitrogen) for 24 h and then seeded in triplicates into a Corning 3603 black-sided clear bottom 96-well microplate at a density of 3000 cells per well. The remaining cells were kept and stored at −80°C until processed for protein extraction and immunoblotting as described above. Once attached to the plate, cells were exposed to different concentrations of olaparib (Selleckchem, #S1060) ranging from 0 (DMSO) to 2.5 μM. After 3 days of treatment, nuclei were stained with Hoechst 33342 (Invitrogen) at 10 μg/ml in media for 45 min at 37°C. Images of entire wells were acquired at 4x with a Cytation 5 Cell Imaging Multi-Mode Reader followed by quantification of Hoechst-stained nuclei with the Gen5 Data Analysis Software v3.03 (BioTek Instruments). Cell viability was expressed as percentage of survival in olaparib-treated cells relative to vehicle (DMSO)-treated cells. Results represent the mean ± SD of at least 3 independent experiments, each performed in triplicate.

To further assess the impact of the 5 selected VUS on PALB2, we examined whether they affected the accumulation of RAD51 at IR-induced DSBs by measuring the formation RAD51 foci.

analyzed several PALB2 variants in their response to the ICL-inducing agent cisplatin

sensitivity to PARPi treatment using a cellular proliferation assay

A cell-based functional assay for PALB2 variants

Automated Patch ClampingCells were patch clamped with the SyncroPatch 384PE automated patch clamping device (Nanion). To prepare cells for patch clamping, cells were washed in PBS, treated with Accutase (Millipore-Sigma) for 3 min at 37°C, then recovered in CHO-S-serum free media (GIBCO). Cells were pelleted and resuspended in divalent-free reference solution (DVF) at ∼200,000–400,000 cells/mL. DVF contained (mM) NaCl 140, KCl 4, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with NaOH. Cells were then added to a medium resistance (4–6 MΩ) 384-well recording chamber with 1 patch aperture per well (NPC-384, Nanion), which contained DVF and internal solution: CsCl 10, NaCl 10, CsF 110, EGTA 10, HEPES 10 (pH 7.2) adjusted with CsOH. Next, to enhance seal resistance, 50% of the DVF was exchanged with a calcium-containing seal enhancing solution: NaCl 80, NMDG 60, KCl 4, MgCl2 1, CaCl2 10, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with HCl. The cells were washed three times in external recording solution: NaCl 80, NMDG 60, KCl 4, MgCl2 1, CaCl2 2, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with HCl. Currents elicited in response to activation, inactivation, and recovery from inactivation protocols were then recorded (Figure S2). A late current measurement was captured every 5 s. After 1 min, 50% of the external solution was exchanged with external solution containing 200 μM tetracaine hydrochloride (Sigma; effective concentration 100 μM tetracaine). After tetracaine addition, late current measurements were obtained every 5 s for 1 additional minute. At least 10 cells expressing wild-type SCN5A were included for comparison in each SyncroPatch experiment (Figure 1), and at least 2 independent transfections and at least 10 replicate cells were studied per mutant. Recordings were performed at room temperature.We also conducted experiments to assess the effects of incubation at low temperature or mexiletine (a sodium channel blocker), interventions reported to increase cell surface expression of mistrafficked channels.27Clatot J. Ziyadeh-Isleem A. Maugenre S. Denjoy I. Liu H. Dilanian G. Hatem S.N. Deschênes I. Coulombe A. Guicheney P. Neyroud N. Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Na(v)1.5 α-subunits.Cardiovasc. Res. 2012; 96: 53-63Crossref PubMed Scopus (62) Google Scholar,  28Makiyama T. Akao M. Tsuji K. Doi T. Ohno S. Takenaka K. Kobori A. Ninomiya T. Yoshida H. Takano M. et al.High risk for bradyarrhythmic complications in patients with Brugada syndrome caused by SCN5A gene mutations.J. Am. Coll. Cardiol. 2005; 46: 2100-2106Crossref PubMed Scopus (99) Google Scholar,  29Pfahnl A.E. Viswanathan P.C. Weiss R. Shang L.L. Sanyal S. Shusterman V. Kornblit C. London B. Dudley Jr., S.C. A sodium channel pore mutation causing Brugada syndrome.Heart Rhythm. 2007; 4: 46-53Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,  30Valdivia C.R. Ackerman M.J. Tester D.J. Wada T. McCormack J. Ye B. Makielski J.C. A novel SCN5A arrhythmia mutation, M1766L, with expression defect rescued by mexiletine.Cardiovasc. Res. 2002; 55: 279-289Crossref PubMed Scopus (77) Google Scholar,  31Valdivia C.R. Tester D.J. Rok B.A. Porter C.B. Munger T.M. Jahangir A. Makielski J.C. Ackerman M.J. A trafficking defective, Brugada syndrome-causing SCN5A mutation rescued by drugs.Cardiovasc. Res. 2004; 62: 53-62Crossref PubMed Scopus (106) Google Scholar For these experiments, cells stably expressing loss-of-function variants were generated as described above. The cells were incubated for 24 h at 30°C, or at 37°C with or without 500 μM mexiletine hydrochloride (Sigma), washed with HEK media, and were patch clamped as described above.