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 ICFTR/C”. The normalized with regard to cell surface evaluated by measuring the cell capacity (C, pF) values of ICFTR 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 ICFTR/C”. See legend for Table “BasaI ICFTR/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 ICFTR/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 ICFTR 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 ICFTR/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 ICFTR/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：

在本实验中，使用了稳定转染了 F508del-CFTR 质粒的 HeLa 细胞。
a) 第一泳道代表未经处理的 F508del-CFTR HeLa 细胞。第二泳道代表经 50ng/ml TNFa 处理 10 分钟的 F508del-CFTR HeLa 细胞。第三泳道代表经 50ng/ml TNFa 处理 3 小时的 F508del-CFTR HeLa 细胞。第四泳道代表经 50ng/ml TNFa 处理 24 小时的 F508del-CFTR HeLa 细胞。第五泳道与本次实验无关。最后一泳道代表未转染标记蛋白的 HeLa 细胞。所使用的抗-CFTR 为 MM-13-4（小鼠抗体）。
b) 膜已脱色，并使用 α-微管蛋白进行检测。这体现在第二张蛋白质印迹图中。脱色程序：在印迹上首次检测到 CFTR 蛋白后，硝酸纤维素膜在含有 2% SDS、625mM TRIS pH 6.7 的脱色缓冲液中孵育 30 分钟，然后膜用 PBS 洗涤 3 次，每次 10 分钟。接下来，膜按照蛋白质印迹实验方案进行再次封闭，随后使用新的第一抗体并按照蛋白质印迹实验方案进行检测。

原始数据集 2：

首张工作表：图 1B 的原始数据

在本实验中，使用了稳定转染了 F508del-CFTR 质粒的 HeLa 细胞。
· 第一张表格（橙色）代表未经处理的 F508del-CFTR HeLa 细胞。

A泳道代表实验编号，对于橙色表格：已进行 8 次实验。C带和 B 带的强度已使用 ImageJ 软件（见方法部分）进行量化。测量的强度显示在 C 列和 D 列中。E 列代表比率：C带的强度/(B带的强度+C带的强度)。

G5 方框代表 C/C+B 的平均值。

G6 方框代表平均值的标准差。

· 第二张表格（黄色）展示了 F508del-CFTR HeLa 细胞经 50ng/ml TNFa 处理 10 分钟获得的个体值。

· 第三张表格（蓝色）展示了 F508del-CFTR HeLa 细胞经 50ng/ml TNFa 处理 3 小时获得的个体值。

· 第四张表格（粉红色）展示了 F508del-CFTR HeLa 细胞经 50ng/ml TNFa 处理 24 小时获得的个体值。

第二张工作表：图 1D 的原始数据

· 在本实验中，使用了稳定转染了 F508del-CFTR 质粒的 HeLa 细胞。

· 第一张表格（黄色）代表未经处理的 F508del-CFTR HeLa 细胞。

A 列代表实验编号：已进行 4 次实验。使用 ImageJ 软件 v1.47 对 C 带和B带的强度进行了量化。结果显示在 B 列和 C 列中。D 列代表比率：C带的强度/(B带的强度+C带的强度)。

F12 方框代表 C/C+B 的平均值。

F13 方框代表平均值的 SD。

第三张工作表：图 8B 的原始数据

· 在本实验中，使用了稳定转染了 F508del-CFTR 质粒的 HeLa 细胞。

· C 列代表实验条件。

蓝色表格代表未经处理的 F508del-CFTR HeLa 细胞。

粉红色表格代表经 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 列代表选定区域内的点数计数，E 列代表选定区域内的细胞计数。

蓝色方框：

F7-F9：是点数计数除以细胞数。

橙色方框是平均值的平均值。

绿色方框是平均值的 SD。

第四张工作表：补充图 S1 的原始数据

在本实验中，使用了稳定转染了 WT-CFTR 和 F508del-CFTR 质粒的 HeLa 细胞。测试了不同的条件：50ng/ml TNFa 在不同时间点的处理：30’-3h。

蓝色代表对照组：未经处理的细胞，无论是 WT 还是 F508del-CFTR HeLa 细胞。

粉红色代表经 50ng/ml TNFa 处理 30 分钟的细胞。

绿色代表经 50ng/ml TNFa 处理 3 小时的细胞。

C 列：C8-10, C16-19, C24-28 代表 WT-HeLa 细胞区域内的细胞计数

I 列：I8-11, I16-20, I26-30 代表 F508del-CFTR 细胞区域内的细胞计数

D 列：D8-10, D16-19, D24-28 代表 WT-HeLa 细胞区域内的细胞计数

J 列：J8-11, J16-20, J26-30 代表 F508del-CFTR HeLa 细胞区域内的细胞计数

E 列：E8-10, E16-19, E24-28 代表经 50ng/ml TNFa 处理 3 小时的细胞区域内的细胞计数

K 列：K8-11, K16-20, K26-30 代表 F508del-CFTR HeLa 细胞区域内的细胞计数

每个区域的点数总和除以每个条件下的点数总和。

平均值和 SD 计算并汇总在最后一张表中。

SD 用紫色表示，平均值用橙色表示。

原始数据集 3：

“BasaI ICFTR/C” 表格。与细胞表面评估相关的标准化数据，通过测量控制条件下未经处理的细胞中获得的 ICFTR 电流值的细胞容量（C，pF）值，用于计算图 3B 中显示的平均值（在 -100mV 和 +80mV 之间）。每个施加电压的平均值显示在相应列的底部。在 -60mV 的值用蓝色表示，因为它们用于图 3C 中显示的统计学分析。

“10 min 50 ng/ml TNFa ICFTR/C” 表格。参见“BasaI ICFTR/C” 表格的图例。在此，细胞暴露于 50 ng/ml TNFa 10 分钟。

原始数据集 4：

用于计算图 3C 中显示的平均电流的个体细胞中测量的标准化电流值。每个细胞都作为其自身的对照。在用 cAMP 鸣管（CPTAMPc 和 IBMX，见方法）灌注 10 分钟后测量电流，然后在存在 cAMP 鸣管的情况下添加 TNFa 进行下一步 10 分钟的灌注，之后用 CFTR inh 172 灌注（见方法图 3C 的图例和说明）。

原始数据集 5：

0 到 50 ng/ml TNFα 剂量反应后的 10 分钟内，标准化 ICFTR/C 的个体值：在 -60 mV 处记录 CFTR 电流幅度，并将其归一化到细胞电容。

原始数据集 6：

“I CFTR /C (pA/pF) with IL1b + cAMP/IBMX” 表格。显示的是在用 CPTcAMP/IBMX 鸣管激活基线电流后，灌注 IL1b 10 分钟后处理的细胞中，通过测量细胞电容（C，pF）对 ICFTR 电流进行归一化后得到的 ICFTR 电流值。这些值用于计算图 4 中 I/V 曲线（在 -100 mV 和 +80 mV 之间）显示的平均值。每个施加电压的平均值和平均值的标准误差显示在相应列的底部。在 -60 mV 的值用蓝色表示，因为它们用于图 4（右侧面板）中的直方图。

“I CFTR /C (pA/pF) cAMP/IBMX control” 表格。参见“ I CFTR /C (pA/pF) with IL1b + cAMP/IBMX” 表格的图例。细胞仅暴露于 CPTcAMP/IBMX 而无 IL1b，并作为对照。

“I CFTR /C (pA/pF) at -60 mV” 表格。展示的是在“ I CFTR /C (pA/pF) with IL1b + cAMP/IBMX” 和“ I CFTR /C (pA/pF) cAMP/IBMX control” 表格中测量的单个细胞的标准化电流值，分别用于计算图 4（右侧面板）中的直方图显示的平均电流。用于对照和 IL1b 处理细胞的细胞组不同。TNFa 处理细胞的值与图 3C 和图 3 的原始数据中显示的值相同。

原始数据集 7：

除细胞预先用 BFA 处理（见方法和正文中的细节）外，其余与图 3B 原始数据的图例相同。-60mV 列用蓝色表示，因为这些值用于图 6C 中的直方图。

原始数据集 8：

标准化 ICFTR/C 的个体值，对于预先或未预先用 BFA 处理的细胞（见方法和正文中的细节）。在用 BFA 预处理之后，细胞暴露于 TNFa 10 分钟：在 -60 mV 处记录 CFTR 电流幅度，并将其归一化到细胞电容。

原始数据集 9：

除细胞预先用 GF109203X 处理 30 分钟（见方法和正文中的细节）外，其余与图 3B 原始数据的图例相同。-60mV 列用蓝色表示，因为这些值用于图 8C 中的直方图。

原始数据集 10：

标准化 ICFTR/C 的个体值，对于预先或未预先用 GF109203X 处理的细胞（见方法和正文中的细节）。在用 GF109203X 预处理之后，细胞暴露于 TNFa 10 分钟：在 -60 mV 处记录 CFTR 电流幅度，并将其归一化到细胞电容。