RNA extraction and cDNA synthesis

AnalysisofAP-landNFKBactivatio

Descriptive Statistics

a very sensitive test and is often used for determining the viability of a strain in the presence or absence of a metabolite or a particular temperature. An EOP of ≤0.01 suggests lethality of the strain on the test medium. For strains carrying IPTG-dependent plasmids, EOP was determined by growing the strains overnight in medium containing IPTG and the appropriate antibiotic and plating an appropriate dilution (10─5 and 3X10─6) on +IPTG (permissive) and –IPTG (test) plates to observe growth. The ratio of the number of colonies obtained on the –IPTG plate to that on the +IPTG plate determined the efficiency of plating. The colonies from the test plate (that is, ─IPTG plate) were also subsequently subcultured on the same medium (─IPTG) to determine viability of the strain. Likewise, strains carrying Ts plasmids were cultured overnight at 30 ̊C with the appropriate antibiotic and dilutions of this culture (10─5 and 3X10─6) were plated at two temperatures 30 ̊C (permissive) and 37 ̊C or 39 ̊C (non-permissive or test). The ratio of the number of colonies obtained on the test temperature to that on the permissive temperature determined the efficiency of plating at the test temperature. Viability of the strain was subsequently confirmed after subculturing from the test plate as stated above

Efficiency of plating (EOP) is a measure of the ratio of number of colonies (obtained from a given volume of a suitable culture dilution) on a test medium to those on a control or permissive medium, and is a measure of cell viability on the former. It is

Efficiency of plating

CPY activity was measured as described previously (Jones,2002). A 2.5 mg/ml stock solution of CPY-specific substrate N-benzoyl-L-tyrosine p-nitroanilide(BTPNA, prepared in dimethyl formamide) was diluted 5 times with 0.1 M Tris-HCl (pH 7.5). 100 μl diluted substrate solution was added to a 96-well plate containing 25 μl cell suspension (5 x 107cells). After 18 h of incubation at 37 ̊C, plate contents were clarified by centrifugation and colour formation was quantified by absorbance at 405 nm. Background absorbance measured using BTPNA-free cell cultures was subtracted from BTPNA-loaded cell cultures and absorbancevalues were normalized to total number of viable cells to enumerate total cellular CPY activity

Carboxypeptidase Y(CPY) activity assay

Total RNA, isolated by TRIzol method was reverse transcribed into cDNA by One step Access RT-PCR kit (Promega, Madison, WI), as per manufacturer’sprotocol.Briefly, 1 μg of RNA, 1 μloligo-dT(500ng), 1 μl 10 mMdNTP and nuclease free water was added in a PCR tube. This reaction mixture was incubated at 65°C for 5 minutes in a thermo cyclerand then quickly transferred to ice. To the mixture, 4μl 5X firststrand buffer, 1μl 0.1M DTT,1μlRNase-OUT (40U/μl) and 1 μl (200 units/μl) of SuperScriptIII were added.The contents were mixed by gentle vortexing and incubated at 37°C for 60 minutes in a thermo cycler. Finally,the reaction was stopped by increasing the temperature to72°C for 10minuteson thermo cycler. The cDNA thus prepared, wasused as a template for PCR.PCR was then performed for either amplification of the gene of interest or relative expression of desired genes by the using gene specific primers(Table 2.5). Products were separated by agarose gel electrophoresis (2%) and visualized by ethidium bromide staining.Table2.5: List of RT-PCR primersused in the study

Reverse transcriptase (RT)-PCR

Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23

PCR reactions were set up with the immunoprecipitated DNA by using the primers thatamplify the 5’ETSand promoter regions. Real-time PCR was performed using Mesa Green 2X PCR MasterMix (Eurogentec)in a 20 μL reactionvolume using 1 μL from the input sample and 3 μL from the immunoprecipitated samplein an RT-PCR mechine (Applied Biosystems). Ct values of the immunoprecipitated samples were normalised to the adjusted Ct values of the inputsample, andthedata were plotted as ‘immunoprecipitated DNA as a percentage of input DNA (% Input)’, as described in (Haringet al., 2007; Livak and Schmittgen, 2001).Calculation of %InputThe amount of DNA taken for q-PCR analysis is inversely proportional to the Ct value% Input = 2-Ct(IP) / 2-Ct(Input)x 100, (% of gDNA immunoprecipitated from the total amount of gDNA taken)Ct(IP) = Ctvalue of immunoprecipitated DNACt(Input) = Ctvalue of Input DNAIn this experiment,10 μL of this lysate was taken as input and this has to be adjusted to the total volume of the lysate. Therefore, the formula would be,

Quantitative PCR (q-PCR) reaction

hemocytometer. 2 mL of cell suspension containing 2×104cells/mL were aliquoted in 15 mL conical tubes. To prepare top agarose (0.3%), 3 mL of prewarmed 0.5% agarose mixture was added to the 2 mL cell suspension, mixed gently by pipetting up and down to avoid air bubbles. Quickly 2 mL of this mixture (cells+0.5% agarose mixture) was transferred to the plate containing 2 mL top agarose. All the samples plates were kept at room temperature (TC laminar flow hood) for at least 60 min to solidify the top agarose. After agarose solidification, plates were incubated at 37°C and 5% CO2for 28 days. Every 2 days, spent media was replaced with 0.5 mL fresh media to prevent it from drying. After 28 days, colonies were stained with 0.005% crystal violet (in 2% ethanol) for 1 h at 37°C. Excess stain was washed with 2% ethanol until clear visible colonies were seen. Plates were imaged using AlphaImager (Alpha Innotech), and the number of colonies was determined by ImageJ software. Multiple images of stained colonies were acquired on a phase contrast inverted microscope(Zeiss)at 5x objective with 1.2 N.A using ProgRes CapturePro v2.8 acquisition software. Area of the individual colonies was determined using ImageJ software

Anchorage-independent cell growth was conducted in 6 well plates at least in triplicates. 1% of low temperature melting agarose (Difco) was prepared in sterile MQ water and mixed with 2X DMEM containing 20% FBS kept warm at 37ºC. Both the solutions were mixed in equal proportion to get 0.5% agarose in 1X DMEM and 10% FBS. This solution was kept warm throughout the experiment at 37ºCin a water bath or incubator. To prepare base agar (0.5% agarose), 2 mL of this solution (0.5% agarose+1x DMEM+10% FBS) was dispensed in 6 well plate to spread evenly without any bubbles and allowed to solidify at room temperature at least for 30 min. Cells were harvested and counted using a

Anchorage independent growth