Raw data for Bitam et al., 2015 ‘An unexpected effect of TNFα on F508del-CFTR maturation and function.’

<p>Raw dataset 1:                   HeLa cells stably transfected with the plasmid F508del-CFTR were used in this experiment. a) The first lane represents F508del-CFTR HeLa cells non-treated. The second lane represents F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 10 min. The third lane represents F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 3h.The fourth lane represents F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 24h. The fifth lane is not relevant for this experiment. The last lane represents HeLa cells non transfected with markers of weight. The anti-CFTR used is MM-13-4 (mouse antibody). b) The membrane has been stripped and the a-tubulin has been used. This is represented by the second western blot. Stripping procedure: after the first detection of CFTR proteins on the blot, the nitrocellulose membrane is incubated for 30 min in a stripping buffer containing 2% SDS, 625mM TRIS pH 6.7, then the membrane is washed 3 times with PBS during 10 minutes. Next, the membrane is blocked again as described in the protocol of western blot, followed by the use of new first antibody and detected as described in the protocol of western blot.</p> <p>           </p> <p>Raw dataset 2:                        First sheet: Raw data for Figure 1B</p> <p>HeLa cells stably transfected with the plasmid F508del-CFTR were used in this experiment.</p> <p>·            The first table (in orange) represents F508del-CFTR HeLa cells non-treated.</p> <p>The lane A represents the number of the experiment, for the table orange: 8 experiments have been done. The intensity of band C and band B have been quantified with ImageJ software (see methods for version). The intensities measured are shown in the column C and D. The column E represents the ratio: intensity of the band C/ (intensity of band B+ intensity of band C).</p> <p>The square G5 represents the mean of C/C+B.</p> <p>The square G6 represents the SD of the mean.</p> <p>·            The second table (yellow) presents the individual values obtained in F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 10’.</p> <p>·            The third table (bleu) presents the individual values obtained F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 3h.</p> <p>·            The forth table (pink) represents the individual values obtained F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 24h.</p> <p> </p> <p>Second sheet: Raw data for Figure 1D</p> <p>·            HeLa cells stably transfected with the plasmid F508del-CFTR were used in this experiment.</p> <p>·            The first table (yellow) represents F508del-CFTR HeLa cells non-treated.</p> <p>·            The column A represents the number of the experiment: 4 experiments have been done. The intensity of band C and of band B have been quantified with ImageJ software v1.47. The results are in the column B and C. The column D represents the ratio: intensity of the band C/ (intensity of band B+ intensity of band C).</p> <p>·            The square F12 represents the mean of C/C+B.</p> <p>·            The square F13 represents the SD of the mean.</p> <p>Third sheet: Raw data for Figure 8B</p> <p>·            HeLa cells stably transfected with the plasmid F508del-CFTR were used in this experiment.</p> <p>·             The column C represents the conditions of the experiments</p> <p>·             The blue table represents F508del-CFTR HeLa cells non-treated.</p> <p>·            The pink table represents F508del-CFTR HeLa cells treated with 50ng/ml of TNFa during 30 min.</p> <p>·            The yellow table represents F508del-CFTR HeLa cells treated with 50ng/ml of TNFa during 3h.</p> <p>·            Column D represents the number of dots counted in the area chosen and the column E the number of cells counted in the area.</p> <p>·            Blue square:</p> <p>·            F7-F9: is the number of dots counted divided by the number of cells.</p> <p>·            Orange square is the average of the means.</p> <p>·            Green square is the SD of the means.</p> <p>Fourth sheet: Raw data for Supplementary figure S1</p> <p>HeLa cells stably transfected with the plasmid WT-CFTR and F508del-CFTR were used in this experiment. Different conditions have been tested: 50ng/ml of TNFa at different times: 30’-3h.</p> <p>Blue represents controls: non-treated cells either: WT or F508del-CFTR HeLa cells.</p> <p>Pink represents cells treated with 50ng/ml of TNFa for 30’.</p> <p>Green represents cells treated with 50ng/ml of TNFa for 3h.</p> <p>Column C: C8-10, C16-19, C24-28 represents the number of cells counted in the area for WT-heLa cells</p> <p>Column I: I8-11, I16-20, I26-30 represents the number of cells counted in the area for F508del-CFTR.</p> <p>Column D: D8-10, D16-19, D24-28 represents the number of cells counted in the area for WT-HeLa cells.</p> <p>Column J: J8-11, J16-20, J26-30 represents the number of cells counted in the area for F508del-CFTR HeLa cells.</p> <p>Column E: E8-10, E16-19, E24-28 represents the number of cells counted in the area treated with 50ng/ml of TNFa for 3h.</p> <p>Column K: K8-11, K16-20, K26-30 represents the number of cells counted in the area for F508del-CFTR HeLa cells.</p> <p>Totals of dots per area are divided by total of dots per area for each condition.</p> <p>Means and SD are calculated and sum up in the last table.</p> <p>SD are in purple and means in orange.</p> <p>Raw dataset 3:            Table “BasaI I<sub>CFTR</sub>/C”. The normalized with regard to cell surface evaluated by measuring the cell capacity (C, pF) values of I<sub>CFTR</sub> current values obtained in control, non-treated conditions, used to calculates the mean values showed in Fig 3B (between -100mV and +80mV) the mean values for each imposed voltage are presented in the bottom of the corresponding column. The values at -60mV are in blue as they served for the statistics shown in Figure 3C</p> <p>Table “10 min 50 ng/ml TNFa I<sub>CFTR</sub>/C”. See legend for Table “BasaI I<sub>CFTR</sub>/C”. Here the cells were exposed to 50 ng/ml TNFa for 10 min.</p> <p>Raw dataset 4:            The normalized current values measured in individual cells which were used to calculate the mean currents shown in Figure 3C. Each cell served as its own control. The currents were measured after 10 min of perfusion with cAMP cocktail (CPTAMPc and IBMX, see methods), then the TNFa was added to the perfusion in the presence of cAMP cocktail for next 10 min followed by perfusion of CFTR inh 172 (see methods and legends of fig 3C for details).</p> <p>Raw dataset 5:         Individual values of normalized I<sub>CFTR</sub>/C for dose-response of 0 to 50 ng/ml TNFα after 10 min: CFTR current amplitudes were recorded at -60 mV and normalized to cell capacitance.</p> <p>Raw dataset 6:        Table “I <sub>CFTR</sub> /C (pA/pF) with IL1b + cAMP/IBMX”.  Shown are the values of I<sub>CFTR</sub> current, normalized with regard to the cell surface evaluated by measuring the cell capacity (C, pF) in cells treated with IL1b in the perfusion for 10 min after activation of the baseline current with a cocktail of CPTcAMP/IBMX. These values were used to calculate the mean values shown in the I/V curve in Figure 4 (between -100 mV and +80 mV). The mean values and the standard error of the mean for each imposed voltage are presented in the bottom of the corresponding column. The values at -60 mV are shown in blue as they served for the histogram shown in Figure 4 (right panel). Table “I <sub>CFTR</sub> /C (pA/pF) cAMP/IBMX control”. See legend for table “I <sub>CFTR</sub> /C (pA/pF) with IL1b + cAMP/IBMX”. The cells were exposed only to CPTcAMP/IBMX without IL1b and served as controls. Table “I <sub>CFTR</sub> /C (pA/pF) at -60 mV”. Depicted are the individual normalized current values at -60 mV measured in individual cells shown in table “I <sub>CFTR</sub> /C (pA/pF) with IL1b + cAMP/IBMX” and “I <sub>CFTR</sub> /C (pA/pF) cAMP/IBMX control”, respectively, which were used to calculate the mean currents shown in the histogram of Figure 4 (right panel). A different set of cells served for control and IL1b treated cells. The values for TNFa treated cells are the same as shown in Fig 3C and in the raw data for Figure 3.</p> <p>Raw dataset 7:         As for the legend for Figure 3B raw data except that the cells were pretreated with BFA (see methods and text for details). The column -60mV is in blue as these values served for histograms shown in Fig 6C.</p> <p>Raw dataset 8:         Individual values of normalized I<sub>CFTR</sub>/C for cells pre-treated or not with BFA (see methods and text for details). After pre-treatment with BFA the cells were exposed to TNFa for 10 min: CFTR current amplitudes were recorded at -60 mV and normalized to cell capacitance.</p> <p>Raw dataset 9:         See legends “Fig 3B raw data” except that the cells were pretreated with GF109203X for 30min (see methods and text for details). The column -60mV is in blue as these values served for histograms shown in Fig 8C.</p> <p> </p> <p>Raw dataset 10:       Individual values of normalized I<sub>CFTR</sub>/C for cells pre-treated or not with GF109203X (see methods and text for details). After pre-treatment with GF109203X the cells were exposed to TNFa for 10 min: CFTR current amplitudes were recorded at -60 mV and normalized to cell capacitance.</p> <p> </p>