Estimationofprostaglandins,PGEzandPGD

Statistical Analysis

The DFD between an atom pair is the normalized frequency distribution of the interatomic distances sampled from equal time snapshots taken from the MD simulations. DFDs of corresponding atoms taken from the different simulations were used to qualitatively compare the effect of mutational perturbations on the HbS fiber. Considering that the fiber simulations were carried out only for a relatively short time scale of 1.2ns, the calculated DFDs might suffer from errors due to limited sampling. Hence a quantitative comparison of the different DFDs were not attempted, however given that the global parameters monitored during the simulation had already become reasonably stable after 0.2ns (Chapter!, Table 5), it is expected that the gross features of the DFDs would remain unaltered even in much longer simulations.

Distance Frequency Distribution (DFD)

Strains were grown overnight in LB containing 0.4% maltose and 10 mM MgSO4,subcultured and grown to early stationary phase in the same medium. 100 μl of the culture was mixed with 2.5 ml of soft agar and overlaid on LB agar plates supplemented with 0.4% maltose and 10 mM MgSO4. Serial dilutions of λcI857 lysate were prepared (in LB) and 10 μl were spotted from each dilution (and the undiluted) on the soft agar lawn and allowed to dry. The plates were incubated at the appropriate temperature overnight, and the plating efficiency determined

Determination of λ-plating efficiency

ammonium molybdate, respectively, to the assay buffer.For specific inhibition of vacuolar membrane H+-ATPaseactivity, vacuolar membrane fractions were incubatedwith 1-2.5 μM bafilomycin for 5 minprior to the activity assay.ATPase activity was initiatedby adding ATP to the assay buffer to afinal concentration of 5 mM and incubating the reactionat 30 ̊C for 30-60 min.Reaction was stopped by adding an equal volumeof a stop-developing solution (1% (w/v)SDS, 0.6 M H2SO4, 1.2%(w/v)ammonium molybdate and 1.6%(w/v)ascorbic acid). Amount of inorganic phosphate (Pi) liberated was measured at A750nmafter 10 minincubation at room temperature. Standard curve prepared with 0-50 micromoles of KH2PO4 was used for the determination of total Pi. The ATPase activity of the vacuolarmembrane H+-ATPase was expressed in micromoles of Pireleased per milligram protein per min

Vacuolar membrane H+-ATPase activitywas measured inbothcrude membrane fraction and purifiedvacuolar membrane fraction asdescribed previously(Woolfordet al.,1990).Activity inthe crude membrane fractions was carried out with 2.5-10 μgprotein in 50 μl assay buffer (5 mM MgCl2, 25 mM MES/Tris-HCl(pH 6.9)and 25 mM KCl). For activity inthe purified vacuolar membrane fraction, a totalof300 μl reactionmix was setup with of 2.5-10 μgprotein samples.Residual activities from other ATPases such as mitochondrial ATPases, plasma membrane H+-ATPase and phosphataseswere inhibited by adding 2 mM NaN3, 200 μM NaVO4and 0.2 mM

Vacuolar H+-ATPase activity measurement

Vacuole membraneswere isolatedwith slight modifications of Cabrera’s method(Cabrera et.al.,2008). Log-phase, YPD medium-grown cells wereinoculated in 1 lt YPDmedium to an initialOD600of 0.1. Cells were incubated at 30 ̊C with shaking at 200 rpm till the cell density reached to OD600of 0.8-1.0.Cells were harvested by centrifugation at 5,000 g and washed once with 30 ml 2% ice-cold glucose solution. Cells were incubated in 15 ml solution containingglycine-NaOH(50 mM; pH10)andDTT(2 mM) at 30 ̊C for 10 min. After incubation, cells were normalized to adensity of1000OD600and resuspendedin 15 ml spheroplasting buffer containing 10-15mg of zymolyase20T.Cells were incubated at 30 ̊C for 45-60 minor till the spheroplasting was completed.Spheroplasts werecollected by centrifugation at 4,500 rpmfor 5 minat 4 ̊C, washed gently with15 ml 1.2 M sorbitol solutionandresuspendedin 3.5 ml 15%ficoll solution made in PS buffercontaining 1X protease inhibitor cocktail. This suspension was homogenized on ice with 20-25 strokes in a loose-fitting Dounce homogenizer. Homogenate was transferred to an ice-cold,ultra-clear Beckman ultracentrifuge tube, overlaid witha gradient of3 ml 8%ficoll solution, 2.5 ml 4%ficoll solutionand 2.5 ml PS buffer lacking ficoll and centrifuged at 1,10,000g(30,000 rpm)for 90 minat 4 ̊Cin a pre-cooled Beckman ultracentrifuge with SW41-Ti swinging bucket rotor.Centrifugation was carried out with slow acceleration and deceleration settings.White creamy vacuole membrane layer wascollected from the interfaceof 0and4% ficoll gradientwithout mixing the layers.Total protein concentration in thevacuole fraction was estimated using BCAprotein assay kit as described earlier

Purified vacuole membrane isolation

Crude fractionation of total membraneswas carried outviadifferential centrifugation asdescribed previously (Moranoand Klionsky,1994)with slight modifications. Cells grown tolog-phase in YPDmedium werecollected, washed,normalizedto 10 OD600and resuspendedin 1 ml spheroplast buffer containing 1-2mg of zymolyase20T (MP Biomedicals).Following incubation at 30 ̊Cfor 30-45 min,spherolplastswerecollected by centrifugation at 800 g for 3 minat 4 ̊C and resuspendedin 1 mlice-cold Tris-EDTA (pH 7.5). Spheroplastswere lysed with 100 μl 0.5mm glass beads on a vortex mixer with 10 secpulsegiven thricewith intermittent ice-breaks.Cellsuspension was centrifuged at 800 g for 5 minat 4 ̊C to pellet unbrokenspheroplastsdown andthesupernatant was centrifuged at 15,000 g for 5 minat 4 ̊C to obtainthemembrane fraction pellet.Pellet was washed once with ice-cold Tris-EDTA (pH 7.5), resuspendedin 50 μl of the samebuffer and stored at -20 ̊Ctill further use. Protein concentration of pellet fraction was estimated using BCAprotein assay kit with BSA as thestandard

Crude vacuolar membrane extraction

Vacuolar H+-ATPase activity measurement

Total RNA was isolated from the treated cells usingTRIzol method, essentially described by Donald and his co-worker (Donald et al., 2010).TRIzol is a single-phase solution of guanidinium isothiocyanate and phenolthat can concomitantly denature proteins and other biological material. Addition of chloroform to this leads to phase separation: proteins remains in organic phase whereas, DNA and RNA resolves to interphase and aqueous phase, respectively. Before starting the experiment, area was sanitized with RNAZap toremove any contamination of DNases. After treatment, culture media was gently removed from the dish without disturbing the cell monolayer. TRIzolreagentwas added directly on to the dish andcellswere allowed tosuspend in it by repeated pipetting. The cellular homogenate was then transferred into amicrofuge tubes. For each ml of TRIzolused, 200μl of chloroform was added andvortexedfor about 30 seconds, followed bycentrifugation at maximum speed of 13,000 rpm for 10 minutes. The upper aqueous phase wastransferred into a fresh micro-centrifuge tube and 500μl of ice-cold iso-propanol was addedto precipitate RNA.The RNA was pelleted by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant was decanted and the pelletwas allowed towash with 1ml of ice-cold 70% ethanol followed by centrifugation at maximum speed for 10 minutes. Finally, the supernatant was removed and the pellet was allowed toair-dryfor about 5-10 minutes and solubilized in 50μl RNase free deionized (DEPC-treated Milli-Q) water and quantified by spectrophotometry for further use

Isolationof total RNAfrom cultured cells

Immunoprecipitation of chromatin was performed by incubating the lysate with 3 μg of anti-GST antibody overnight at 4°C followed by50 μL of 1:1 suspension of Protein A beads for 4 h. Beads were washedtwice each in wash buffer I, wash buffer II, and TE buffer(Section 2.1.6.5). Each wash was performed for 15 min at 4oC by rotating on a Lab-net end over mixer. Chromatin was eluted in 100 μL of elution buffer(Section 2.1.6.5) by rigorous mixing on a Thermo mixer (Eppendorf) for 30 min at 65oC. 90 μLof this eluted sample was incubated overnight at 65oCto reverse the cross linking. 10 μLlysate taken as input was diluted to 90 μLwith elution buffer and incubated overnight at 65oC to reverse the crosslinking.DNA was extractedfrom the input and immunoprecipitated samplesintoafinal volumeof40μLusing aPCR purification kit (Qiagen)

Yeast strains carrying p416GPD GST-RPA43were grown in SC-Ura medium overnight and sub cultured at 0.2 OD600.45 mL of mid-log phase yeast cultures were subjected to cross linking with 1% formaldehyde for 15 min at room temperature(Szijgyartoet al., 2011). Cross linking was quenched by adding glycine to a final concentration of 0.1 M. Cells were washed in ice cold Tris-buffered saline and were lysed in 500 μL of ice cold lysis buffer (Section 2.1.6.5) by bead beating. Chromatin was fragmented using a Diagenode bath sonicator, 15 sec on time and 30 sec off time, for 15 min. Cell lysates were centrifuged at high speed and the supernatant was pre-cleared with 3 μg of normal rabbit IgG followed by30 μL of 1:1 suspension ofProtein A beads. Supernatant was collected and 10 μL of this lysate was taken as input

Chromatin immunoprecipitation(ChIP)

Luciferase assay was performed using luciferase assay systems (Promega #1500). Cells were transfected with shRNA. Following 24 hrs.of shRNA transfection, cells were transfected separately with FOP-Flash and TOP-Flashvectors in the absence and presence of Wnt3a plasmids. After 24 hrs, media was removed, and the cells were rinsed twice with 1X PBS. After removing the final wash, the cells were incubated with lysis buffer (1X lysis reagent: CCLR; 20μl/well for a96-wellplate, or 400 μl/60mm culture dish, or 900μl/100mm culture dish). Cells were collected in a microcentrifuge tube and were centrifuged at 14000 rpm for 10 minutes. The cell lysate (supernatant) was transferred to a new tube. 20μl of cell lysate was mixed with 100μl of Luciferase Assay Reagent(LAR), and the amount of light produced was measuredin luminometer by usinga delay time of 2 sec and a read time of 10 sec.

Luciferase reporter assay

Four week old tomato S-22 cultivar (acts as non-host for Xanthomonas oryzae pv. oryzicola) were syringe-infiltrated with a suspension of Xocstrains and water control. Plants were incubated in green house for 24 h with minimum and maxium temperature of 26 and 28°C, respectively and relative humidity of 65%. Callose deposition assay was performed as a marker for hypersensitvity response in non host plant as described previously (Hauck et al., 2003). Leaf picture was captured at this stage to observe the HR browning of leaf. For assaying callose deposition by aniline blue staining, infilterated leaves were removed from plant,dipped in lactophenol solution and incubated at 65°C in water bath until the cholorohyll is completely removed. Leaves were rehydrated by washing with 50% ethanol, and finally rinsed with water. For aniline blue staining, leaves were incubated in 0.01% aniline blue solution, prepared in 100 mM K2HPO4(pH 9.5), for 15-20 min in dark. Subsequently, leaves were washed with water and observed for callose deposition in epifluorescence microscope (Stereo, Lumar V7, Zeiss) under UV illumination

In plantahypersensitive response (HR) and callose deposition assay

Scratch wound healing assay was performed as described previously (Raoet al., 2015). Cells were seeded in 6 well plates in triplicates to attain confluence. On the next day, a scratch was made on the confluent monolayer culture using a 200 μL pipette tip. Cells were washed gently twice with PBS to remove floating cells, replaced with complete DMEM and incubated at 37ºC with 5% CO2.Images were acquired using a Zeiss phase contrast inverted microscope (ProgRes CapturePro v2.8 acquisition software, 5x 0.12 N.A. objective). Images were acquired immediately after scratch (0 h) and after a period of 4 h (MEFs) or 24 h (HeLa and HCT116) to monitor wound closure. The area of wound closure was analyzed using ImageJ software, and the data was plotted as total area covered in square μm(μm2)

Scratch wound healing assay