Journal: Journal of Cell Science

Article Title: Charcot–Marie–Tooth type 2A variants of mitofusin 2 sensitize cells to apoptotic cell death

doi: 10.1242/jcs.263691

Figure Lengend Snippet: CMT2A variants of MFN2 cause apoptotic cell death. (A) Western blot analysis (upper panel) and quantification (lower panel) of HeLa WT cells or 2KO cells stably expressing FLAG-tagged K357N variant of MFN2, untreated or treated with Act D (1 µM, 6 h), with or without ZVAD (20 µM, 6 h), as indicated, immunoblotted with anti-PARP1 and anti-MFN2 antibodies. Staining of total protein with Ponceau S (PoS) was used as loading control. Bars represent the mean±s.d. intensity of cleaved PARP1, normalized to the PoS staining and shown relative to untreated K357N cells ( n =5 biological repeats). Individual values of each experiment are plotted as open and filled circles, squares, triangles, diamonds and hexagons. One-way ANOVA with Tukey's post-hoc test was applied. P values from left to right: ** P =0.0091; * P =0.0205; **** P <0.0001; ** P =0.0060. (B) Annexin V 568 fluorescence, measured by the Incucyte live imaging system over 48 h and plotted relative to timepoint zero, of HeLa WT or 2KO cells stably expressing FLAG-tagged K357N variant of MFN2, either untreated or treated with ABT+S (BH3 mimetic ABT-737 and the MCL1 inhibitor S63845 , 0.1 µM, 48 h) for apoptosis induction, and in presence or absence of ZVAD (20 µM, 48 h) for inhibition of caspases, as indicated. Values represent the mean±s.d. intensity per object count of three technical replicates each from three biological replicates. (C) Annexin V 568 fluorescence, analysed as in B, of HeLa 2KO cells transiently transfected with WT MFN2, MFN2 R364Q, MFN2 R707W or MFN2 K357N, either untreated or treated with ABT+S (0.1 µM, 24 h). Values represent the mean intensity per object count of a single technical replicate each from three biological replicates.

Article Snippet: The following chemicals were used for cell culture treatments: actinomyin D (Sigma-Aldrich, Schnelldorf, Germany), ABT-737 (MedChemExpress, Sollentuna, Sweden), MCL1 inhibitor S63458 (MedChemExpress Sollentuna, Sweden), ZVAD-FMK (Hölzel, Köln, Germany), ISR inhibitor (ISRIB) (Merck, Hamburg, Germany), thapsigargin (NEB, Frankfurt am Main, Germany), staurosporine (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: Western Blot, Stable Transfection, Expressing, Variant Assay, Staining, Control, Fluorescence, Imaging, Inhibition, Transfection

Journal: Journal of Cell Science

Article Title: Charcot–Marie–Tooth type 2A variants of mitofusin 2 sensitize cells to apoptotic cell death

doi: 10.1242/jcs.263691

Figure Lengend Snippet: The ISR is not implicated in apoptosis observed in cells expressing the K357N variant. (A) Western blot analysis of HeLa WT cells or 2KO cells stably expressing FLAG-tagged K357N variant of MFN2, untreated or treated with ISRIB (5 µM, 16 h) or thapsigargin (Tg; 0.5 µM, 2 h) as indicated, immunoblotted with anti-PARP1 antibody. Staining of total protein with Ponceau S (PoS) was used as loading control. Blots shown are representative of three experiments. (B) mRNA levels of CHOP , spliced XBP1 ( XBP1s ), ATF4 , BiP and ATF6 measured by qPCR of total mRNA, extracted from HeLa WT or 2KO cells stably expressing FLAG-tagged K357N variant of MFN2, either untreated or treated with ISRIB (5 µM, 16 h). Transcript levels were quantified using the 2− ΔΔCt method and normalized to HPRT transcript levels. Individual values of each experiment are plotted as open or filled circles, squares or triangles. Bars represent the mean±s.d. of the respective mRNA transcript level relative to the untreated WT group ( n =4 biological replicates). Two-way ANOVA with Tukey's post-hoc test was applied. P values for CHOP from left to right: ns (not significant), P =0.8327; * P =0.0119; ns, P =0.9589. P values for ATF4 from left to right: * P =0.0488; * P =0.020; ns, P =0.3693. P values for XBP1s from left to right: ns, P =0.9661; * P =0.0289; ns, P =0.8301. P values for ATF6 from left to right: ns, P =0.9968; * P =0.0110; ns, P =0.6780. P values for BiP from left to right: ns, P =0.7441; * P =0.0420; ns, P =0.9608. (C) Western blot analysis (upper panel) and quantification (lower panel) of HeLa WT cells or 2KO cells stably expressing FLAG-tagged K357N variant of MFN2, untreated or treated with ISRIB (5 µM, 16 h), as indicated, immunoblotted with anti-PARP1 antibody. Staining of total protein with PoS was used as loading control. Bars represent the mean±s.d. intensity of cleaved PARP1, normalized to the PoS staining and shown relative to untreated WT ( n =3 biological replicates). Individual values of each experiment are plotted as open and filled circles, triangles and squares. One-way ANOVA with Tukey's post-hoc test was applied. P values from left to right: ns, P =0.1082; ns, P =0.3795.

Article Snippet: The following chemicals were used for cell culture treatments: actinomyin D (Sigma-Aldrich, Schnelldorf, Germany), ABT-737 (MedChemExpress, Sollentuna, Sweden), MCL1 inhibitor S63458 (MedChemExpress Sollentuna, Sweden), ZVAD-FMK (Hölzel, Köln, Germany), ISR inhibitor (ISRIB) (Merck, Hamburg, Germany), thapsigargin (NEB, Frankfurt am Main, Germany), staurosporine (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: Expressing, Variant Assay, Western Blot, Stable Transfection, Staining, Control

Journal: Clinical and Translational Medicine

Article Title: CD98 defines a metabolically flexible, proinflammatory subset of low‐density neutrophils in systemic lupus erythematosus

doi: 10.1002/ctm2.1150

Figure Lengend Snippet: CD98 + low‐density neutrophils (LDN) are relatively resistant to apoptosis, but equally susceptible to necroptosis. Cell death was evaluated using the IncuCyte S3 system. (A) Spontaneous apoptosis of normal‐density neutrophils (NDN) (white) and LDN (black) was measured over 18 h ( n = 5). (B) Quantification of apoptosis after 12 h induced by Smac‐mimetic compound A ( n = 5). (C) Spontaneous apoptosis ( n = 5), extrinsic apoptosis (stimulated with tumor necrosis factor [TNF] and a Smac‐mimetic, n = 6) or intrinsic apoptosis (stimulated with ABT‐737 and an Mcl‐1 inhibitor, n = 3) quantified after 12 h. (D) Both neutrophil subsets were treated with a necroptotic stimulus (TNF, Smac‐mimetic and IDN‐6556 [TSI]) and cell death was measured over 18 h using the IncuCyte S3 System ( n = 8). (E) The level of necroptotic cell death at 12 h was quantified ( n = 8). Data are mean ± standard error of mean (SEM). * p < .05, ** p < .01, *** p < .001, one‐way analysis of variance (ANOVA)

Article Snippet: Cells were treated with combinations of the following agonists/antagonists: 100 ng/ml recombinant human TNF (produced in‐house ), 500 nM Smac‐mimetic/compound A (Tetralogic Pharmaceuticals ), 5 μM IDN‐6556 (Idun Pharmaceuticals), 100 μg/ml cycloheximide (Sigma), 1 μM ABT‐737 (Abbott) and 0.1 μM Mcl‐1 inhibitor (also known as S63485; SYNthesis MedChem).

Techniques: