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

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. Raw dataset 2: First sheet: Raw data for Figure 1B HeLa cells stably transfected with the plasmid F508del-CFTR were used in this experiment. · The first table (in orange) represents F508del-CFTR HeLa cells non-treated. 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). The square G5 represents the mean of C/C+B. The square G6 represents the SD of the mean. · The second table (yellow) presents the individual values obtained in F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 10’. · The third table (bleu) presents the individual values obtained F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 3h. · The forth table (pink) represents the individual values obtained F508del-CFTR HeLa cells treated with TNFa at 50ng/ml for 24h. Second sheet: Raw data for Figure 1D · HeLa cells stably transfected with the plasmid F508del-CFTR were used in this experiment. · The first table (yellow) represents F508del-CFTR HeLa cells non-treated. · 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). · The square F12 represents the mean of C/C+B. · The square F13 represents the SD of the mean. Third sheet: Raw data for Figure 8B · HeLa cells stably transfected with the plasmid F508del-CFTR were used in this experiment. · The column C represents the conditions of the experiments · The blue table represents F508del-CFTR HeLa cells non-treated. · The pink table represents F508del-CFTR HeLa cells treated with 50ng/ml of TNFa during 30 min. · The yellow table represents F508del-CFTR HeLa cells treated with 50ng/ml of TNFa during 3h. · Column D represents the number of dots counted in the area chosen and the column E the number of cells counted in the area. · Blue square: · F7-F9: is the number of dots counted divided by the number of cells. · Orange square is the average of the means. · Green square is the SD of the means. Fourth sheet: Raw data for Supplementary figure S1 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. Blue represents controls: non-treated cells either: WT or F508del-CFTR HeLa cells. Pink represents cells treated with 50ng/ml of TNFa for 30’. Green represents cells treated with 50ng/ml of TNFa for 3h. Column C: C8-10, C16-19, C24-28 represents the number of cells counted in the area for WT-heLa cells Column I: I8-11, I16-20, I26-30 represents the number of cells counted in the area for F508del-CFTR. Column D: D8-10, D16-19, D24-28 represents the number of cells counted in the area for WT-HeLa cells. Column J: J8-11, J16-20, J26-30 represents the number of cells counted in the area for F508del-CFTR HeLa cells. 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. Column K: K8-11, K16-20, K26-30 represents the number of cells counted in the area for F508del-CFTR HeLa cells. Totals of dots per area are divided by total of dots per area for each condition. Means and SD are calculated and sum up in the last table. SD are in purple and means in orange. Raw dataset 3: Table “BasaI I_CFTR/C”. The normalized with regard to cell surface evaluated by measuring the cell capacity (C, pF) values of I_CFTR 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 Table “10 min 50 ng/ml TNFa I_CFTR/C”. See legend for Table “BasaI I_CFTR/C”. Here the cells were exposed to 50 ng/ml TNFa for 10 min. 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). Raw dataset 5: Individual values of normalized I_CFTR/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. Raw dataset 6: Table “I _CFTR /C (pA/pF) with IL1b + cAMP/IBMX”. Shown are the values of I_CFTR 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 _CFTR /C (pA/pF) cAMP/IBMX control”. See legend for table “I _CFTR /C (pA/pF) with IL1b + cAMP/IBMX”. The cells were exposed only to CPTcAMP/IBMX without IL1b and served as controls. Table “I _CFTR /C (pA/pF) at -60 mV”. Depicted are the individual normalized current values at -60 mV measured in individual cells shown in table “I _CFTR /C (pA/pF) with IL1b + cAMP/IBMX” and “I _CFTR /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. 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. Raw dataset 8: Individual values of normalized I_CFTR/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. 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. Raw dataset 10: Individual values of normalized I_CFTR/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.

原始数据集1：本实验使用经F508del突变型囊性纤维化跨膜传导调节蛋白（CFTR）质粒稳定转染的HeLa细胞（HeLa cells）。a) 泳道1代表未处理的F508del-CFTR HeLa细胞；泳道2代表以50ng/ml肿瘤坏死因子α（TNFa）处理10分钟的F508del-CFTR HeLa细胞；泳道3代表以50ng/ml TNFa处理3小时的F508del-CFTR HeLa细胞；泳道4代表以50ng/ml TNFa处理24小时的F508del-CFTR HeLa细胞；泳道5与本实验无关；最末泳道代表未转染的分子量标记HeLa细胞。本次实验使用的抗CFTR抗体为MM-13-4（小鼠源抗体）。b) 印迹膜经洗脱处理后，以α-微管蛋白（a-tubulin）作为内参，对应第二次蛋白质印迹实验。洗脱流程如下：首次完成CFTR蛋白的印迹检测后，将硝酸纤维素膜置于含2%十二烷基硫酸钠（SDS）、625mM Tris-HCl pH 6.7的洗脱缓冲液中孵育30分钟，随后用磷酸盐缓冲液（PBS）洗涤3次，每次10分钟。后续按照蛋白质印迹实验流程再次封闭膜片，更换一抗后按照标准流程完成检测。

原始数据集2：
第一个工作表：图1B原始数据
本实验使用经F508del-CFTR质粒稳定转染的HeLa细胞。
· 橙色表格代表未处理的F508del-CFTR HeLa细胞：A列标注实验编号，该橙色表格共完成8次独立实验。使用ImageJ软件（版本详见方法部分）对条带C与条带B的灰度强度进行定量，测量结果分别列于C列与D列；E列为条带C强度/(条带B强度+条带C强度)的比值；G5单元格为C/(C+B)比值的平均值；G6单元格为该平均值的标准差（SD）。
· 黄色表格代表经50ng/ml TNFa处理10分钟的F508del-CFTR HeLa细胞的单次实验原始数值。
· 蓝色表格代表经50ng/ml TNFa处理3小时的F508del-CFTR HeLa细胞的单次实验原始数值。
· 粉色表格代表经50ng/ml TNFa处理24小时的F508del-CFTR HeLa细胞的单次实验原始数值。
第二个工作表：图1D原始数据
本实验使用经F508del-CFTR质粒稳定转染的HeLa细胞。
· 黄色表格代表未处理的F508del-CFTR HeLa细胞：A列标注实验编号，共完成4次独立实验。使用ImageJ软件v1.47对条带C与条带B的灰度强度进行定量，结果列于B列与C列；D列为条带C强度/(条带B强度+条带C强度)的比值；F12单元格为C/(C+B)比值的平均值；F13单元格为该平均值的SD。
第三个工作表：图8B原始数据
本实验使用经F508del-CFTR质粒稳定转染的HeLa细胞。
· C列为实验分组条件；蓝色表格代表未处理的F508del-CFTR HeLa细胞；粉色表格代表经50ng/ml TNFa处理30分钟的F508del-CFTR HeLa细胞；黄色表格代表经50ng/ml TNFa处理3小时的F508del-CFTR HeLa细胞。
· D列为选定视野内计数的斑点数，E列为选定视野内计数的细胞数。
· 蓝色方块：F7-F9为视野内斑点数与细胞数的比值；橙色方块为各组均值的平均值；绿色方块为各组均值的SD。
第四个工作表：补充图S1原始数据
本实验使用经野生型囊性纤维化跨膜传导调节蛋白（WT-CFTR）与F508del-CFTR质粒稳定转染的HeLa细胞，测试分组为：50ng/ml TNFa处理不同时长（30分钟~3小时）。
· 蓝色组为对照组：未处理的WT-CFTR或F508del-CFTR HeLa细胞；粉色组为经50ng/ml TNFa处理30分钟的细胞；绿色组为经50ng/ml TNFa处理3小时的细胞。
· C列（C8-10、C16-19、C24-28）为WT-HeLa细胞选定视野内的计数细胞数；I列（I8-11、I16-20、I26-30）为F508del-CFTR HeLa细胞选定视野内的计数细胞数。
· D列（D8-10、D16-19、D24-28）为WT-HeLa细胞选定视野内的计数细胞数；J列（J8-11、J16-20、J26-30）为F508del-CFTR HeLa细胞选定视野内的计数细胞数。
· E列（E8-10、E16-19、E24-28）为经50ng/ml TNFa处理3小时的细胞选定视野内的计数细胞数；K列（K8-11、K16-20、K26-30）为F508del-CFTR HeLa细胞选定视野内的计数细胞数。
将各分组的单位视野斑点总数除以对应视野的细胞总数，计算各组均值与SD，最终汇总于最后一张表格中，其中SD以紫色标注，均值以橙色标注。

原始数据集3：表格“Basal I_CFTR/C”。本表格以对照未处理组的CFTR电流（I_CFTR）值为基准进行归一化，归一化参数为通过测量细胞电容（C，单位pF）得到的细胞表面积。本数据集用于计算图3B所示的均值（电压范围：-100mV至+80mV），各施加电压对应的均值列于对应列的底部。其中-60mV处的数值以蓝色标注，用于图3C的统计分析。
表格“10 min 50 ng/ml TNFa I_CFTR/C”：详见表格“Basal I_CFTR/C”的说明。本表格中细胞经50ng/ml TNFa处理10分钟。

原始数据集4：本数据集为单个细胞的归一化电流值，用于计算图3C所示的平均电流。每个细胞作为自身对照：先用cAMP混合液（CPT-cAMP与IBMX，详见方法部分）灌流10分钟后记录电流，随后在灌流液中加入TNFa并维持cAMP混合液，继续灌流10分钟，最后灌流CFTR抑制剂172（CFTR inh 172，详见方法及图3C图例）。

原始数据集5：本数据集为单个细胞的归一化I_CFTR/C数值，对应0至50ng/ml TNFa处理10分钟后的剂量反应实验：于-60mV记录CFTR电流幅值，并以细胞电容进行归一化。

原始数据集6：表格“I_CFTR/C (pA/pF) with IL1b + cAMP/IBMX”：本表格展示了经IL1b灌流10分钟后的CFTR电流值，该电流值以细胞电容（C，单位pF）测得的细胞表面积为基准进行归一化，此前已使用CPT-cAMP/IBMX混合液激活基础电流。本数据集用于计算图4所示的电流-电压曲线（I/V曲线）均值（电压范围：-100mV至+80mV），各施加电压对应的均值与标准误（SEM）列于对应列的底部。其中-60mV处的数值以蓝色标注，用于图4右侧面板的直方图分析。
表格“I_CFTR/C (pA/pF) cAMP/IBMX control”：详见表格“I_CFTR/C (pA/pF) with IL1b + cAMP/IBMX”的说明。本表格中细胞仅经CPT-cAMP/IBMX混合液处理，未添加IL1b，作为对照组。
表格“I_CFTR/C (pA/pF) at -60 mV”：本表格展示了分别来自“I_CFTR/C (pA/pF) with IL1b + cAMP/IBMX”与“I_CFTR/C (pA/pF) cAMP/IBMX control”表格中单个细胞在-60mV处的归一化电流值，用于计算图4右侧面板直方图的平均电流。对照组与IL1b处理组使用不同批次的细胞。TNFa处理组的数值与图3C及图3原始数据中的数值一致。

原始数据集7：本数据集的说明与图3B原始数据一致，仅实验细胞提前用BFA进行预处理（详细说明参见方法部分与正文）。其中-60mV列以蓝色标注，对应图6C所示的直方图分析。

原始数据集8：本数据集为单个细胞的归一化I_CFTR/C数值，对应经BFA预处理或未预处理的细胞组（详细说明参见方法部分与正文）。经BFA预处理后，将细胞暴露于TNFa中10分钟：于-60mV记录CFTR电流幅值，并以细胞电容进行归一化。

原始数据集9：本数据集的说明与“图3B原始数据”一致，仅实验细胞提前用GF109203X预处理30分钟（详细说明参见方法部分与正文）。其中-60mV列以蓝色标注，对应图8C所示的直方图分析。

原始数据集10：本数据集为单个细胞的归一化I_CFTR/C数值，对应经GF109203X预处理或未预处理的细胞组（详细说明参见方法部分与正文）。经GF109203X预处理后，将细胞暴露于TNFa中10分钟：于-60mV记录CFTR电流幅值，并以细胞电容进行归一化。