Journal: The EMBO Journal

Article Title: Reduced ER–mitochondria connectivity promotes neuroblastoma multidrug resistance

doi: 10.15252/embj.2021108272

Figure Lengend Snippet: Tumor models were derived at DX and REL following treatment. Mitochondria‐rich fractions were exposed to tBid or BimBH3 peptide and cytochrome c release measured as a surrogate for apoptotic commitment. Maximal cytochrome c release in response to tBid or BimBH3 peptide for each replicate of a DX/REL tumor pair (1–9 biological replicates per DX/REL pair; n = 44 total). Maximal cytochrome c release for all DX/REL pairs with ≥ 3 biological replicates; box‐whisker plots summarize data (box 25–75%; belt = median; dot = mean; and whiskers = minimum and maximum values). Bak oligomerization in response to escalating tBid concentration for DX/REL pairs. Relative mitochondrial protein loading per lane is assessed by densitometry, showing ration of loading control in REL lane with patient‐matched DX lane at same tBid exposure (no REL lane is underloaded compared to DX lane). Data information: Statistical analyses in C were performed using an unpaired two‐sided Student’s t ‐test, with significance P < 0.05 (trend P < 0.10). Cyto c, cytochrome c; mito, mitochondria.

Article Snippet: Mitochondria (50 μl of 1 μg/μl protein) were treated with caspase‐8‐cleaved tBid from 5 to 500 nM (882‐B8‐050, R&D Systems, Minneapolis, MN), Bim‐BH3 from 10 to 10,000 nM (Ac‐DMRPEIWIAQELRRIGDEFNAYYARR‐amide; New England Peptide, Gardner, MA), or 300 μM BidAltBH3 (Ac‐EDIIRNIARHAAQVGASMDR‐amide; New England Peptide, Inc.).

Techniques: Derivative Assay, Whisker Assay, Concentration Assay, Control

Journal: The EMBO Journal

Article Title: Reduced ER–mitochondria connectivity promotes neuroblastoma multidrug resistance

doi: 10.15252/embj.2021108272

Figure Lengend Snippet: Summary of paired diagnostic (DX) and relapse (REL) neuroblastoma models.

Article Snippet: Mitochondria (50 μl of 1 μg/μl protein) were treated with caspase‐8‐cleaved tBid from 5 to 500 nM (882‐B8‐050, R&D Systems, Minneapolis, MN), Bim‐BH3 from 10 to 10,000 nM (Ac‐DMRPEIWIAQELRRIGDEFNAYYARR‐amide; New England Peptide, Gardner, MA), or 300 μM BidAltBH3 (Ac‐EDIIRNIARHAAQVGASMDR‐amide; New England Peptide, Inc.).

Techniques: Diagnostic Assay, Micro-arrays for Mass Spectrometry

Journal: The EMBO Journal

Article Title: Reduced ER–mitochondria connectivity promotes neuroblastoma multidrug resistance

doi: 10.15252/embj.2021108272

Figure Lengend Snippet: A Bax oligomerization in response to escalating tBid concentration by immunoblot for DX/REL pairs. Blue boxes highlight key oligomerization differences. B, C DX/REL pair whole‐cell lysates and heavy‐membrane (HM) fractions immunoblotted for Bak and Bax with relevant loading controls. D Immunoblot of CHLA15 HM fraction, with and without tryptic digestion to deplete MAMs, compared with CHLA20 HM fraction, for Bak, Bax, and Vdac1 (loading control). E Similar to D, but with immunomagnetic separation (MACS) to deplete MAMs, immunoblotted for Bak, Bax, Facl4 (MAM marker), and Vdac1 (loading control). F Bax oligomerization in response to escalating tBid concentration by immunoblot for CHLA15 mitochondria pre‐incubated for 30 min with or without the neutral sphingomyelinase inhibitor, GW4869. Blue boxes highlight key oligomerization differences. Data information: For B and C, relative fold expression (ratio of target gene band to control band normalized to the DX expression for each patient‐matched cell line pair) is shown. For D and E, relative fold expression for each condition is shown, normalized to the first lane for each.

Article Snippet: Mitochondria (50 μl of 1 μg/μl protein) were treated with caspase‐8‐cleaved tBid from 5 to 500 nM (882‐B8‐050, R&D Systems, Minneapolis, MN), Bim‐BH3 from 10 to 10,000 nM (Ac‐DMRPEIWIAQELRRIGDEFNAYYARR‐amide; New England Peptide, Gardner, MA), or 300 μM BidAltBH3 (Ac‐EDIIRNIARHAAQVGASMDR‐amide; New England Peptide, Inc.).

Techniques: Concentration Assay, Western Blot, Membrane, Control, Immunomagnetic Separation, Marker, Incubation, Expressing

Journal: The EMBO Journal

Article Title: Reduced ER–mitochondria connectivity promotes neuroblastoma multidrug resistance

doi: 10.15252/embj.2021108272

Figure Lengend Snippet: A In vitro viability curves after 72 h exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated cytochrome c release (CHLA15/CHLA20 and SKNBE1/SKNBE2C), and one without (CHLA122/CHLA136). B DNA damage induced by 2 Gy ionizing radiation as measured by γ‐H2AX foci at 1 h ( n = 53–77 cell nuclei/condition scored; mean shown); statistical analyses performed using two‐tailed Mann–Whitney U test. C–E In vitro viability for CHLA15 (DX) and CHLA20 (REL) following ionizing radiation at 7 days, 48 h exposure to the Bcl2/Bclx inhibitor, ABT‐737, or 120 h exposure to the Alk inhibitor, crizotinib. F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643‐ALKR and SY5Y‐ALKR). G In vitro viability of NB1643 and NB1643‐ALKR cells following 72 h exposure to ABT‐737. Data information: For A, C–E, and G, data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates, dotted line represents 50% viability. For F, data points are mean of duplicate wells (SD < 0.05 at all points) in a representative experiment from at least two biological replicates.

Article Snippet: Mitochondria (50 μl of 1 μg/μl protein) were treated with caspase‐8‐cleaved tBid from 5 to 500 nM (882‐B8‐050, R&D Systems, Minneapolis, MN), Bim‐BH3 from 10 to 10,000 nM (Ac‐DMRPEIWIAQELRRIGDEFNAYYARR‐amide; New England Peptide, Gardner, MA), or 300 μM BidAltBH3 (Ac‐EDIIRNIARHAAQVGASMDR‐amide; New England Peptide, Inc.).

Techniques: In Vitro, Two Tailed Test, MANN-WHITNEY, Comparison, Cell Culture

Journal: The EMBO Journal

Article Title: Reduced ER–mitochondria connectivity promotes neuroblastoma multidrug resistance

doi: 10.15252/embj.2021108272

Figure Lengend Snippet: A Heavy‐membrane (HM) fractions of CHLA15 were rendered MAM depleted by immunomagnetic separation to derive purified mitochondria (pMito), and cytochrome c release measured in response to BimBH3 peptide. B–D TEM analyses were used to quantify mitochondrial size, circularity, and roundness in CHLA15 cells transfected with a sh‐control (Ctrl), shMFN2 or shPACS2 constructs (mean ± SD shown). E Immunoblot assessment of Mfn2 and Pacs2 protein knockdown. F Proportion of mitochondria with 0–5 MAMs are shown for each. G Percentage of mitochondria perimeter with an apposed ER within defined gap widths; dotted lines denote +10% and −10% change. H In vitro viability of CHLA15‐ctrl, CHLA15‐shMFN2, CHLA15‐shPACS2, and CHLA20 cells following 72 h exposure to ABT‐737; dotted line represents 50% viability. I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15‐Ctrl, CHLA15‐shMFN2, and CHLA15‐shPACS2 cells. Data information: For B–D and F, G, n = 214–246 mitochondria per cell line. For A and I, data points are mean of duplicate wells (SD < 0.05 at all points) in a representative experiment from at least two biological replicates; for H, data points are mean and SD from triplicate wells, experiments, are representative of at least two biological replicates. For B–D, statistical analyses were performed using a two‐tailed Mann–Whitney U test, with significance P < 0.05.

Article Snippet: Mitochondria (50 μl of 1 μg/μl protein) were treated with caspase‐8‐cleaved tBid from 5 to 500 nM (882‐B8‐050, R&D Systems, Minneapolis, MN), Bim‐BH3 from 10 to 10,000 nM (Ac‐DMRPEIWIAQELRRIGDEFNAYYARR‐amide; New England Peptide, Gardner, MA), or 300 μM BidAltBH3 (Ac‐EDIIRNIARHAAQVGASMDR‐amide; New England Peptide, Inc.).

Techniques: Membrane, Immunomagnetic Separation, Purification, Transfection, Control, Construct, Western Blot, Knockdown, In Vitro, Two Tailed Test, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Reduced ER–mitochondria connectivity promotes neuroblastoma multidrug resistance

doi: 10.15252/embj.2021108272

Figure Lengend Snippet: A–C Concentration of ceramide species as measured by LC/MS from DX and REL pair whole‐cell pellets. D, E Cumulative ceramides and sphingomyelins (of C32–C36 chain length) for DX/REL pairs. F Ratio of total C32‐C36 sphingomyelins:ceramides for DX/REL pairs. G Cytochrome c release for CHLA15 and SKNBE1 mitochondria following exposure to tBid, pre‐incubated for 30 min with or without GW4869, and CHLA20 and SKNBE2C pre‐incubated without GW4869 (left panel); same experiment but with GW4869 added with tBid, after the 30 min pre‐incubation (right panel). H Summary data for CHLA15/CHLA20 and SKNBE1/SKNBE2C showing relative cytochrome c release when pre‐incubated with GW4869 to inhibit ceramide generation, compared with untreated cells. Data information: A–F show mean of three to four biological replicates plotted; three technical replicates each; D–F error bars are ± SD. For G, data points are mean of duplicate wells (SD < 0.05 at all points) in a representative experiment from at least two biological replicates. All data points from two (CHLA15) or three (SKNBE1) biological replicates are depicted in H. Statistical analyses were performed using an unpaired two‐sided Student’s t ‐test, with significance P < 0.05 (trend, P < 0.10).

Article Snippet: Mitochondria (50 μl of 1 μg/μl protein) were treated with caspase‐8‐cleaved tBid from 5 to 500 nM (882‐B8‐050, R&D Systems, Minneapolis, MN), Bim‐BH3 from 10 to 10,000 nM (Ac‐DMRPEIWIAQELRRIGDEFNAYYARR‐amide; New England Peptide, Gardner, MA), or 300 μM BidAltBH3 (Ac‐EDIIRNIARHAAQVGASMDR‐amide; New England Peptide, Inc.).

Techniques: Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Incubation