Vol. 215, No. 1 | https://doi.org/10.1084/jem.20161881 | December 20, 2017
The authors wish to clarify that duplication of the “BRAFV600E + MEKi” plot in Fig. 3 B and the “MEKi” plot in Fig. 3 G was intentional. These panels represent the same experimental dataset, which was deliberately reused to facilitate comparison across different treatment conditions. Specifically, in panel B, the plot shows BRAFV600E bone marrow–derived dendritic cells treated with a MEK inhibitor. In panel G, the identical plot was intentionally included as the reference MEK inhibitor condition (right panel) to allow direct comparison with the ABT-treated condition (middle panel) using the same experimental dataset. This was indicated in the figure legend; however, the authors recognize that the wording could have been clearer. The authors apologize for any confusion and appreciate the opportunity to clarify this point.
Panel A shows flow cytometry dot plots of Annexin V versus Propidium Iodide for Control and BRAFV600E cells, alongside bar graphs showing the percentage of CD11b positive CD11c positive cells positive for Propidium Iodide and Annexin V at Baseline Day 7 and Starving Day 7. Panel B shows flow cytometry dot plots of Caspase 3/7 versus Propidium Iodide for Control, BRAFV600E, and BRAFV600E plus MEKi cells, with a bar graph showing the percentage of Caspase 3/7 positive cells. Panel C shows a Western blot of BCLXL in Control and BRAFV600E cells at Day 6 and Day 7 under starving and non-starving conditions. Panel D shows a histogram of BCL XL fluorescence intensity. Panel E shows a bar graph of BCLXL mean fluorescence intensity, with the x-axis labeled Control and BRAFV600E and the y-axis labeled BCLXL mean fluorescence intensity. Panel F shows bar graphs of the percentage of CD11b positive CD11c positive singlets positive for Annexin V and Propidium Iodide under Dimethyl sulfoxide, 100 nanomolar ABT, 1 micromolar ABT, and 1 nanomolar MEKi treatments in BRAFV600E starving and BRAFV600E non-starving cells. Panel G shows flow cytometry dot plots of Caspase 3/7 versus Propidium Iodide for Dimethyl sulfoxide, ABT-263, and MEKi treatments, with a bar graph showing the percentage of Caspase 3/7 positive cells in Control and BRAFV600E groups. Panel H shows a Western blot of BCL2L1 and COX2 in wild type, MAP2K1 mutant, and BRAFV600E mutant patient samples treated with Dimethyl sulfoxide, BRAF inhibitor, or MEKi. Panel I shows stacked bar graphs of the percentage of cells classified as Dead, Late Apoptotic, and Early Apoptotic for LCH wild type and LCH BRAFV600E samples treated with MEKi. Panel J shows stacked bar graphs of the percentage of cells classified as Dead, Late Apoptotic, and Early Apoptotic for LCH wild type and LCH BRAFV600E samples treated with ABT263.
BCL-XL up-regulation via RAF/MEK/ERK signaling contributes to suppressed apoptosis in DCs. (A) Apoptosis measured in control and BRAFV600E BMDCs by Annexin V/propidium iodide (PI) staining. On D6 of culture, GM-CSF cytokine was removed from the BMDC culture, and BMDC viability was measured on D7 by flow cytometry. Dot plots show representative frequency of apoptotic Annexin+PI+ CD11c+CD11b+ BMDCs from two experiments. Bar graphs show the mean of triplicate samples representative of two experiments ± SEM (n = 3–5; control vs. BRAFV600E starved PI positivity: **, P = 0.0055; unpaired t test; baseline vs. starved control Annexin V positivity: *, P = 0.0419; unpaired t test). (B) Caspase 3/7 activation measured in control and BRAFV600E BMDCs starved of the GM-CSF growth factor overnight (***, P = 0.0008; unpaired t test), ±1 nM GSK1120212 (*, P = 0.0161; unpaired t test). Representative samples are shown in FACS plots. Bar graphs show the mean of three biological replicates representative of two experiments ± SEM. (C) BCL-XL expression was measured by western blot in BRAFV600ECD11c BMDCs starved (st) or not starved (nst) of the GM-CSF growth factor in overnight culture and harvested on D6 or D7 of culture. Representative data from two independent experiments are shown. (D and E) BCL-XL protein levels in control and BRAFV600E BMDCs as measured by flow cytometry. Representative data from at least two experiments with three biological replicates are shown. The bar graph in E represents quantification of triplicate conditions within one experiment ± SEM (**, P = 0.0089; unpaired t test). (F) Percentage of apoptotic BMDCs among control or BRAFV600E BMDCs cultured overnight with 100 nM BCL2-family inhibitor ABT-263, 1 µM ABT-263 (Annexin V: *, P = 0.0211; unpaired t test; PI: **, P = 0.0032; unpaired t test), or 1 nM GSK1120212 MEKi (Annexin V: *, P = 0.0268; unpaired t test; PI: **, P = 0.0030; unpaired t test). BMDCs were starved or nonstarved of the GM-CSF growth factor during overnight drug treatment and analyzed for apoptosis using Annexin V/PI staining by flow cytometry. Bar graphs show the mean of three biological replicates ± SEM, representative of two independent experiments. (G) Caspase 3/7 activation measuring BRAFV600E BMDCs treated with a control vehicle (***, P = 0.0004; unpaired t test) or with 1 nM GSK1120212 (*, P = 0.0118; unpaired t test), as shown in B, or in the presence of 1 µM ABT-263 (*, P = 0.0330; unpaired t test) overnight. Bar graphs show the mean results of triplicate conditions from two independent experiments ± SEM. (H) Western blot showing BCL2L1 protein levels in human LCH lesions cultured without serum overnight, then treated with BRAF or MEKi’s for 2 h. (C and H) Molecular mass is indicated in kilodaltons. (I and J) Viability of human LCH lesions cultured overnight without serum, then treated for 2 h with 1 nM GSK1120212 MEKi (I) or 1 µM ABT-263 BCL2-family inhibitor (J). Three patient samples are shown in each treatment group. Data represent means shown ± SEM. D6, day 6; D7, day 7.
Panel A shows flow cytometry dot plots of Annexin V versus Propidium Iodide for Control and BRAFV600E cells, alongside bar graphs showing the percentage of CD11b positive CD11c positive cells positive for Propidium Iodide and Annexin V at Baseline Day 7 and Starving Day 7. Panel B shows flow cytometry dot plots of Caspase 3/7 versus Propidium Iodide for Control, BRAFV600E, and BRAFV600E plus MEKi cells, with a bar graph showing the percentage of Caspase 3/7 positive cells. Panel C shows a Western blot of BCLXL in Control and BRAFV600E cells at Day 6 and Day 7 under starving and non-starving conditions. Panel D shows a histogram of BCL XL fluorescence intensity. Panel E shows a bar graph of BCLXL mean fluorescence intensity, with the x-axis labeled Control and BRAFV600E and the y-axis labeled BCLXL mean fluorescence intensity. Panel F shows bar graphs of the percentage of CD11b positive CD11c positive singlets positive for Annexin V and Propidium Iodide under Dimethyl sulfoxide, 100 nanomolar ABT, 1 micromolar ABT, and 1 nanomolar MEKi treatments in BRAFV600E starving and BRAFV600E non-starving cells. Panel G shows flow cytometry dot plots of Caspase 3/7 versus Propidium Iodide for Dimethyl sulfoxide, ABT-263, and MEKi treatments, with a bar graph showing the percentage of Caspase 3/7 positive cells in Control and BRAFV600E groups. Panel H shows a Western blot of BCL2L1 and COX2 in wild type, MAP2K1 mutant, and BRAFV600E mutant patient samples treated with Dimethyl sulfoxide, BRAF inhibitor, or MEKi. Panel I shows stacked bar graphs of the percentage of cells classified as Dead, Late Apoptotic, and Early Apoptotic for LCH wild type and LCH BRAFV600E samples treated with MEKi. Panel J shows stacked bar graphs of the percentage of cells classified as Dead, Late Apoptotic, and Early Apoptotic for LCH wild type and LCH BRAFV600E samples treated with ABT263.
BCL-XL up-regulation via RAF/MEK/ERK signaling contributes to suppressed apoptosis in DCs. (A) Apoptosis measured in control and BRAFV600E BMDCs by Annexin V/propidium iodide (PI) staining. On D6 of culture, GM-CSF cytokine was removed from the BMDC culture, and BMDC viability was measured on D7 by flow cytometry. Dot plots show representative frequency of apoptotic Annexin+PI+ CD11c+CD11b+ BMDCs from two experiments. Bar graphs show the mean of triplicate samples representative of two experiments ± SEM (n = 3–5; control vs. BRAFV600E starved PI positivity: **, P = 0.0055; unpaired t test; baseline vs. starved control Annexin V positivity: *, P = 0.0419; unpaired t test). (B) Caspase 3/7 activation measured in control and BRAFV600E BMDCs starved of the GM-CSF growth factor overnight (***, P = 0.0008; unpaired t test), ±1 nM GSK1120212 (*, P = 0.0161; unpaired t test). Representative samples are shown in FACS plots. Bar graphs show the mean of three biological replicates representative of two experiments ± SEM. (C) BCL-XL expression was measured by western blot in BRAFV600ECD11c BMDCs starved (st) or not starved (nst) of the GM-CSF growth factor in overnight culture and harvested on D6 or D7 of culture. Representative data from two independent experiments are shown. (D and E) BCL-XL protein levels in control and BRAFV600E BMDCs as measured by flow cytometry. Representative data from at least two experiments with three biological replicates are shown. The bar graph in E represents quantification of triplicate conditions within one experiment ± SEM (**, P = 0.0089; unpaired t test). (F) Percentage of apoptotic BMDCs among control or BRAFV600E BMDCs cultured overnight with 100 nM BCL2-family inhibitor ABT-263, 1 µM ABT-263 (Annexin V: *, P = 0.0211; unpaired t test; PI: **, P = 0.0032; unpaired t test), or 1 nM GSK1120212 MEKi (Annexin V: *, P = 0.0268; unpaired t test; PI: **, P = 0.0030; unpaired t test). BMDCs were starved or nonstarved of the GM-CSF growth factor during overnight drug treatment and analyzed for apoptosis using Annexin V/PI staining by flow cytometry. Bar graphs show the mean of three biological replicates ± SEM, representative of two independent experiments. (G) Caspase 3/7 activation measuring BRAFV600E BMDCs treated with a control vehicle (***, P = 0.0004; unpaired t test) or with 1 nM GSK1120212 (*, P = 0.0118; unpaired t test), as shown in B, or in the presence of 1 µM ABT-263 (*, P = 0.0330; unpaired t test) overnight. Bar graphs show the mean results of triplicate conditions from two independent experiments ± SEM. (H) Western blot showing BCL2L1 protein levels in human LCH lesions cultured without serum overnight, then treated with BRAF or MEKi’s for 2 h. (C and H) Molecular mass is indicated in kilodaltons. (I and J) Viability of human LCH lesions cultured overnight without serum, then treated for 2 h with 1 nM GSK1120212 MEKi (I) or 1 µM ABT-263 BCL2-family inhibitor (J). Three patient samples are shown in each treatment group. Data represent means shown ± SEM. D6, day 6; D7, day 7.
