ht1080 cells Search Results


ht 1080  (CLS Cell Lines Service GmbH)
93
CLS Cell Lines Service GmbH ht 1080
Quantitative EM analysis of ER–mitochondria contacts <t>in</t> <t>HT-1080</t> and COS-7 cells. (A) Representative EM images of HT-1080 and COS-7 cells. Insets show rough ER–mitochondria contacts (RER-mito) in HT-1080 cells (green arrowheads) and smooth ER–mitochondria contacts (SER-mito) in HT-1080 and COS-7 cells (red arrowheads). (B) Quantification of contact width, contact length, contact length relative to mitochondria perimeter, and number of contacts per mitochondria profile are shown for SER-mito and RER-mito contacts in HT1080 and COS-7 cells. (C) The relative ratio of SER-mito and RER-mito contacts in HT-1080 and COS-7 cells based on the number of contacts per mitochondria or length of contacts. (D) The number of ribosomes per RER-mito contact is plotted versus the length of the contact in nm for HT-1080 and COS-7 cells. RER-mito contacts with five or less ribosomes are shown in red; those with more than five ribosomes are specific to HT-1080 cells and are shown in blue and defined as riboMERCs. n = 27 images from two independent biological replicates; ±SEM; ***P < 0.001; ****P < 0.0001; B: one-way ANOVA; C: Chi2 test. Bar = 500 nm; inset: 200 nm.
Ht 1080, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pmc10638097-176-0-4?v=CLS+Cell+Lines+Service+GmbH
Average 93 stars, based on 1 article reviews
ht 1080 - by Bioz Stars, 2026-07
93/100 stars
  Buy from Supplier
ht1080 cas9 aavs1  (Genecopoeia)
94
Genecopoeia ht1080 cas9 aavs1
(A) Correlation between log2-fold change (L2FC) in RAD51 foci number and the Rule Set 2 on-target score. All guides targeting RAD51 regulators including RAD51 paralogs (RAD51D, RAD51C, XRCC3), BRCA1, and BRCA2 in the library are shown. Splice and nonsense variants with high Rule Set 2 score shows more significant L2FC. Pearson correlation (r) equals −0.30. (B) Quantification of RAD51 foci in irradiated S/G2-phase cells with guides targeting RAD51 regulators that have low Rule Set 2 score. Cells are grouped by sgRNA category. No. or moderate significant separation from the cells with control guides is observed. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. (Outliers are not shown in the CDFs.) (C) as in B) for guides with high on-target score, showing significant reduction in RAD51 foci in cells with nonsense and splice guides, compared to the control guides. (D) as in A) for L2FC in BRCA1 foci for BRCA1-targeting guides, showing a strong negative correlation with the Rule Set 2 score. Pearson correlation (r) equals −0.75. (E) same as B) for BRCA1 foci and guides targeting BRCA1 that have low Rule Set 2 score, showing no significant separation from the control guides. (F) same as E) for guides with high Rule Set 2 score, showing significant reduction in BRCA1 foci in cells with missense, nonsense, and splice guides. (G) Volcano plot showing no guides targeting <t>AAVS1</t> and non-targeting control (NTC) guides shows statistically significant changes in RAD51 foci under irradiation. Significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.55, all AAVS1-targeting non-targeting control (NTC) guides are shown. Benjamini-Hochberg corrected two-sided KS test. (H) Same as G) showing guides that result in significant changes in RAD51 foci. (I) Gene enrichment analysis of guides targeting RAD51D among guides causing significant changes in RAD51 foci in the irradiated condition. Fisher exact test. Enriched genes are defined as p.adj <0.05. (J) same as G) for BRCA1 foci. (K) same as H) for guides that result in significant changes in BRCA1 foci (L) same as I) for BRCA1 foci.
Ht1080 Cas9 Aavs1, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/bio_rxiv__2023__12__26__572587-222-17-19?v=Genecopoeia
Average 94 stars, based on 1 article reviews
ht1080 cas9 aavs1 - by Bioz Stars, 2026-07
94/100 stars
  Buy from Supplier
ht1080 cells  (Revvity)
91
Revvity ht1080 cells
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Ht1080 Cells, supplied by Revvity, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pmc10199230-224-0-10?v=Revvity
Average 91 stars, based on 1 article reviews
ht1080 cells - by Bioz Stars, 2026-07
91/100 stars
  Buy from Supplier
ht 1080 whole cell lysate  (Santa Cruz Biotechnology)
92
Santa Cruz Biotechnology ht 1080 whole cell lysate
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Ht 1080 Whole Cell Lysate, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pmc06405706-99-27-31?v=Santa+Cruz+Biotechnology
Average 92 stars, based on 1 article reviews
ht 1080 whole cell lysate - by Bioz Stars, 2026-07
92/100 stars
  Buy from Supplier
cell culture hela.s-fucci  (BioResource International Inc)
90
BioResource International Inc cell culture hela.s-fucci
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Cell Culture Hela.S Fucci, supplied by BioResource International Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pmc03322266-41-0-36?v=BioResource+International+Inc
Average 90 stars, based on 1 article reviews
cell culture hela.s-fucci - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier
ht-1080 cells expressing optogef-rhoa and caax-cibn-gfp  (Johns Hopkins HealthCare)
90
Johns Hopkins HealthCare ht-1080 cells expressing optogef-rhoa and caax-cibn-gfp
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Ht 1080 Cells Expressing Optogef Rhoa And Caax Cibn Gfp, supplied by Johns Hopkins HealthCare, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pm39207902-745-16-44?v=Johns+Hopkins+HealthCare
Average 90 stars, based on 1 article reviews
ht-1080 cells expressing optogef-rhoa and caax-cibn-gfp - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier
ht1080 cells  (JCRB Cell Bank)
90
JCRB Cell Bank ht1080 cells
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Ht1080 Cells, supplied by JCRB Cell Bank, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pm38334180-51-31-38?v=JCRB+Cell+Bank
Average 90 stars, based on 1 article reviews
ht1080 cells - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier
human ht1080 epithelial-like fibrosarcoma cell line  (DS Pharma Biomedical)
90
DS Pharma Biomedical human ht1080 epithelial-like fibrosarcoma cell line
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Human Ht1080 Epithelial Like Fibrosarcoma Cell Line, supplied by DS Pharma Biomedical, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/10__4236_slash_pp__2011__24034-32-1-10?v=DS+Pharma+Biomedical
Average 90 stars, based on 1 article reviews
human ht1080 epithelial-like fibrosarcoma cell line - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier
ht-1080 cells  (China Center for Type Culture Collection)
90
China Center for Type Culture Collection ht-1080 cells
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Ht 1080 Cells, supplied by China Center for Type Culture Collection, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/10__1016_slash_s1995___7645_ascii40_11_ascii41_60131___4-21-0-8?v=China+Center+for+Type+Culture+Collection
Average 90 stars, based on 1 article reviews
ht-1080 cells - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier
ht1080 cells  (European Collection of Authenticated Cell Cultures)
90
European Collection of Authenticated Cell Cultures ht1080 cells
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Ht1080 Cells, supplied by European Collection of Authenticated Cell Cultures, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pm30463010-242-131-135?v=European+Collection+of+Authenticated+Cell+Cultures
Average 90 stars, based on 1 article reviews
ht1080 cells - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier
fibrosarcoma cells ht-1080  (National Centre for Cell Science)
90
National Centre for Cell Science fibrosarcoma cells ht-1080
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Fibrosarcoma Cells Ht 1080, supplied by National Centre for Cell Science, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pm28513646-78-1-9?v=National+Centre+for+Cell+Science
Average 90 stars, based on 1 article reviews
fibrosarcoma cells ht-1080 - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier
ht1080 cells  (ibidi GmbH)
90
ibidi GmbH ht1080 cells
RSS suppress oxidative stress–induced parthanatos. A , <t>HT1080</t> cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.
Ht1080 Cells, supplied by ibidi GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ht1080+cells/pmc07564779__cancers___12___02404___s001-1-62-80?v=ibidi+GmbH
Average 90 stars, based on 1 article reviews
ht1080 cells - by Bioz Stars, 2026-07
90/100 stars
  Buy from Supplier

Image Search Results


Quantitative EM analysis of ER–mitochondria contacts in HT-1080 and COS-7 cells. (A) Representative EM images of HT-1080 and COS-7 cells. Insets show rough ER–mitochondria contacts (RER-mito) in HT-1080 cells (green arrowheads) and smooth ER–mitochondria contacts (SER-mito) in HT-1080 and COS-7 cells (red arrowheads). (B) Quantification of contact width, contact length, contact length relative to mitochondria perimeter, and number of contacts per mitochondria profile are shown for SER-mito and RER-mito contacts in HT1080 and COS-7 cells. (C) The relative ratio of SER-mito and RER-mito contacts in HT-1080 and COS-7 cells based on the number of contacts per mitochondria or length of contacts. (D) The number of ribosomes per RER-mito contact is plotted versus the length of the contact in nm for HT-1080 and COS-7 cells. RER-mito contacts with five or less ribosomes are shown in red; those with more than five ribosomes are specific to HT-1080 cells and are shown in blue and defined as riboMERCs. n = 27 images from two independent biological replicates; ±SEM; ***P < 0.001; ****P < 0.0001; B: one-way ANOVA; C: Chi2 test. Bar = 500 nm; inset: 200 nm.

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Quantitative EM analysis of ER–mitochondria contacts in HT-1080 and COS-7 cells. (A) Representative EM images of HT-1080 and COS-7 cells. Insets show rough ER–mitochondria contacts (RER-mito) in HT-1080 cells (green arrowheads) and smooth ER–mitochondria contacts (SER-mito) in HT-1080 and COS-7 cells (red arrowheads). (B) Quantification of contact width, contact length, contact length relative to mitochondria perimeter, and number of contacts per mitochondria profile are shown for SER-mito and RER-mito contacts in HT1080 and COS-7 cells. (C) The relative ratio of SER-mito and RER-mito contacts in HT-1080 and COS-7 cells based on the number of contacts per mitochondria or length of contacts. (D) The number of ribosomes per RER-mito contact is plotted versus the length of the contact in nm for HT-1080 and COS-7 cells. RER-mito contacts with five or less ribosomes are shown in red; those with more than five ribosomes are specific to HT-1080 cells and are shown in blue and defined as riboMERCs. n = 27 images from two independent biological replicates; ±SEM; ***P < 0.001; ****P < 0.0001; B: one-way ANOVA; C: Chi2 test. Bar = 500 nm; inset: 200 nm.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

MCS-DETECT analysis of sub-precision contacts. (A) 3D STED images of HT-1080 and COS-7 showing overlap between mitochondria (magenta) and ER (green). Insets show STED sections at 0.5 μm Z spacing. Bars = 10 μm. (B) Two objects (red and green discs) are shown at corresponding sub-precision distances. Intensity profiles (top row), second derivatives (Laplacian), and Spearman correlations of the negative part of the Laplacian (bottom row) are shown. Note how the Spearman response overlaps and changes consistently with the sub-precision distance. (C) The detection algorithm (orange) with additional stages that each address a specific confounding factor introduced by the acquisition (bleed through) or sample (vesicle removal).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: MCS-DETECT analysis of sub-precision contacts. (A) 3D STED images of HT-1080 and COS-7 showing overlap between mitochondria (magenta) and ER (green). Insets show STED sections at 0.5 μm Z spacing. Bars = 10 μm. (B) Two objects (red and green discs) are shown at corresponding sub-precision distances. Intensity profiles (top row), second derivatives (Laplacian), and Spearman correlations of the negative part of the Laplacian (bottom row) are shown. Note how the Spearman response overlaps and changes consistently with the sub-precision distance. (C) The detection algorithm (orange) with additional stages that each address a specific confounding factor introduced by the acquisition (bleed through) or sample (vesicle removal).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

Parameter study of the proposed method. (A) We test two representative HT-1080 and COS-7 cells, with known distinct contact types. We vary the analysis window, a 3D cube of 5k × 5k × k over the mitochondria and contact channel. (a) The surface coverage ratio stays stable for a range of k. (b) The user can set a significance and statistical power threshold. This increases the minimum significance at which voxels can be detected, as well as the minimum correlation considered to be observable. As expected, when this threshold increases, expected differences between two representative cells decrease, as does the overall number of correlation voxels. At the limit of 100% confidence, no information would be left. To avoid false responses by bleed-through of signal in nearby Z-planes, as well as high background intensity in low SNR conditions, we apply an adaptive threshold in z-space. (c) We observe that at low values, the inclusion of false responses masks any differences (z = 1.5). At high values (3.5) the ER channel was visually degraded, we see that after z = 3 the difference between the two cells is maximal and converged. (B) The full reference algorithm pseudocode listing of each stage of the proposed method, enabling reproduction in any implementation. The actual Julia implementation used adds non-algorithm stages to deal with parallelization, optimization, error handling, and recording intermediate stages, which are out of scope for the purposes of this listing. The source code is available under AGPL v3 license at https://github.com/bencardoen/SubPrecisionContactDetection.jl .

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Parameter study of the proposed method. (A) We test two representative HT-1080 and COS-7 cells, with known distinct contact types. We vary the analysis window, a 3D cube of 5k × 5k × k over the mitochondria and contact channel. (a) The surface coverage ratio stays stable for a range of k. (b) The user can set a significance and statistical power threshold. This increases the minimum significance at which voxels can be detected, as well as the minimum correlation considered to be observable. As expected, when this threshold increases, expected differences between two representative cells decrease, as does the overall number of correlation voxels. At the limit of 100% confidence, no information would be left. To avoid false responses by bleed-through of signal in nearby Z-planes, as well as high background intensity in low SNR conditions, we apply an adaptive threshold in z-space. (c) We observe that at low values, the inclusion of false responses masks any differences (z = 1.5). At high values (3.5) the ER channel was visually degraded, we see that after z = 3 the difference between the two cells is maximal and converged. (B) The full reference algorithm pseudocode listing of each stage of the proposed method, enabling reproduction in any implementation. The actual Julia implementation used adds non-algorithm stages to deal with parallelization, optimization, error handling, and recording intermediate stages, which are out of scope for the purposes of this listing. The source code is available under AGPL v3 license at https://github.com/bencardoen/SubPrecisionContactDetection.jl .

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

Subprecision contact detection identifies distinct contact profiles in HT-1080 and COS-7 cells. (A) Volume-rendered MCS-DETECT views of cells expressing ERmoxGFP (green) and labeled for TOM20 (magenta) with contact sites overlaid (white) are shown for COS-7, HT-1080, and OMM–ER linker transfected COS-7 cell ROIs from the whole view image are shown volume rendered in adjacent panels. COS-7 mitochondria display numerous small contact zones while mitochondria in HT-1080 and OMM–ER linker transfected COS-7 cells present more extended contact zones (bar = 10 µm whole cell; 1 µm insets). (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in COS-7, HT-1080, and OMM–ER linker transfected COS-7 cells (averaged over cell, two-sided non-parametric Mann Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; ***P < 0.001). (C) 2D KDE plots of mean contact size over mean anisotropy and mean Spearman response, with a linear regression overlayed, are shown for COS-7 (red) versus HT-1080 (blue) cells or COS-7 (red) versus OMM–ER linker transfected COS-7 (blue) cells.

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Subprecision contact detection identifies distinct contact profiles in HT-1080 and COS-7 cells. (A) Volume-rendered MCS-DETECT views of cells expressing ERmoxGFP (green) and labeled for TOM20 (magenta) with contact sites overlaid (white) are shown for COS-7, HT-1080, and OMM–ER linker transfected COS-7 cell ROIs from the whole view image are shown volume rendered in adjacent panels. COS-7 mitochondria display numerous small contact zones while mitochondria in HT-1080 and OMM–ER linker transfected COS-7 cells present more extended contact zones (bar = 10 µm whole cell; 1 µm insets). (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in COS-7, HT-1080, and OMM–ER linker transfected COS-7 cells (averaged over cell, two-sided non-parametric Mann Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; ***P < 0.001). (C) 2D KDE plots of mean contact size over mean anisotropy and mean Spearman response, with a linear regression overlayed, are shown for COS-7 (red) versus HT-1080 (blue) cells or COS-7 (red) versus OMM–ER linker transfected COS-7 (blue) cells.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Expressing, Labeling, Transfection, MANN-WHITNEY

Identifying and filtering mitochondria. (A) For each contact, size and mean intensity of the adjacent mitochondria is plotted for two replicates, to indicate consistency across replicates. Note that this is by default larger than a segmentation method would compute. The size and mean intensity of mitochondria in HT-1080 and COS-7 cells present a clearly separable group of small, low-intensity mitochondria structures. To report results on what are clearly and unambiguously mitochondria, corresponding to the mitochondria observed by EM, mitochondrial structures smaller than thresholds 9 (ln size) and 0.2 (mean intensity) were eliminated. (B) 3D STED images show labeling of ER (green), mitochondria (magenta), and contact sites (white) of representative COS-7 and HT-1080 cells before and after mitochondrial filtering. Bar = 10 μM (insets: 1 μM).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Identifying and filtering mitochondria. (A) For each contact, size and mean intensity of the adjacent mitochondria is plotted for two replicates, to indicate consistency across replicates. Note that this is by default larger than a segmentation method would compute. The size and mean intensity of mitochondria in HT-1080 and COS-7 cells present a clearly separable group of small, low-intensity mitochondria structures. To report results on what are clearly and unambiguously mitochondria, corresponding to the mitochondria observed by EM, mitochondrial structures smaller than thresholds 9 (ln size) and 0.2 (mean intensity) were eliminated. (B) 3D STED images show labeling of ER (green), mitochondria (magenta), and contact sites (white) of representative COS-7 and HT-1080 cells before and after mitochondrial filtering. Bar = 10 μM (insets: 1 μM).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Labeling

Gp78 regulation of riboMERCs. (A) Volume-rendered MCS-DETECT views of cells expressing ERmoxGFP and labeled for TOM20 (magenta) with contact sites overlaid (white) are shown for HT-1080 and Gp78 KO HT-1080 cells and for untransfected COS-7 cells and COS-7 cells overexpressing WT Gp78 or Gp78 RM. Bar = 10 μm whole cell; 1 μm insets. (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in HT-1080 and Gp78 KO HT-1080 cells and for untransfected COS-7 cells and COS-7 cells overexpressing Gp78 WT or Gp78 RM. (C) 2D KDE plots of mean contact size over mean anisotropy and mean Spearman response, with a linear regression overlayed, are shown for HT-1080 (blue) versus Gp78 KO HT-1080 (green) cells or COS-7 (red) versus COS-7 overexpressing either Gp78 WT (green) or Gp78 RM (blue). Averaged over cell, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001, two-sided non-parametric Mann–Whitney test. (D) COS-7 cells were transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Integrated density of MitoView 633 per cell was quantified. n = 3 independent biological replicates; >35 cells/condition per experiment; *P < 0.05; ****P < 0.0001; Tukey post hoc test.

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 regulation of riboMERCs. (A) Volume-rendered MCS-DETECT views of cells expressing ERmoxGFP and labeled for TOM20 (magenta) with contact sites overlaid (white) are shown for HT-1080 and Gp78 KO HT-1080 cells and for untransfected COS-7 cells and COS-7 cells overexpressing WT Gp78 or Gp78 RM. Bar = 10 μm whole cell; 1 μm insets. (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in HT-1080 and Gp78 KO HT-1080 cells and for untransfected COS-7 cells and COS-7 cells overexpressing Gp78 WT or Gp78 RM. (C) 2D KDE plots of mean contact size over mean anisotropy and mean Spearman response, with a linear regression overlayed, are shown for HT-1080 (blue) versus Gp78 KO HT-1080 (green) cells or COS-7 (red) versus COS-7 overexpressing either Gp78 WT (green) or Gp78 RM (blue). Averaged over cell, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001, two-sided non-parametric Mann–Whitney test. (D) COS-7 cells were transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Integrated density of MitoView 633 per cell was quantified. n = 3 independent biological replicates; >35 cells/condition per experiment; *P < 0.05; ****P < 0.0001; Tukey post hoc test.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Expressing, Labeling, MANN-WHITNEY, Transfection, Control

Gp78 overexpression western blot and representative Mito V iew images. (A) Western blots of COS-7 cells transfected with pcDNA3, Flag-Gp78 WT, or Flag-Gp78 RM were probed with antibodies to Flag tag to reveal Gp78 and to β-actin. (B) Representative images of COS-7 cells transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Corresponding GFP or RFP images (FP) are shown and cell boundaries outlined . (C) Representative images of HeLa cells transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Corresponding GFP or RFP images (FP) are shown and cell boundaries outlined . (D) Representative images of HT-1080 cells transfected with either siControl or siRRBP1 and labeled with MitoView633 are shown and cell boundaries outlined . Bars (B, C, D) = 20 μm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 overexpression western blot and representative Mito V iew images. (A) Western blots of COS-7 cells transfected with pcDNA3, Flag-Gp78 WT, or Flag-Gp78 RM were probed with antibodies to Flag tag to reveal Gp78 and to β-actin. (B) Representative images of COS-7 cells transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Corresponding GFP or RFP images (FP) are shown and cell boundaries outlined . (C) Representative images of HeLa cells transfected with EGFP (as a control), Gp78 WT IRES-GFP, Gp78 RM IRES-GFP, or the OMM–ER linker (RFP) and labeled with MitoView 633. Corresponding GFP or RFP images (FP) are shown and cell boundaries outlined . (D) Representative images of HT-1080 cells transfected with either siControl or siRRBP1 and labeled with MitoView633 are shown and cell boundaries outlined . Bars (B, C, D) = 20 μm. Source data are available for this figure: .

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Over Expression, Western Blot, Transfection, FLAG-tag, Control, Labeling

Gp78 expression in COS-7 and HeLa cells, siRRBP1 western blot, and shape features for Q95 Gp78 RM overexpressing cells. (A) COS-7 and HeLa cells were transfected in parallel with Flag-Gp78 WT, Flag-Gp78 RM, or the RFP-tagged OMM–ER linker and fixed after 24 h. The Flag-Gp78 transfected cells were labeled for anti-Flag and the OMM–ER linker transfected cells left unlabeled. Cells were imaged and anti-Flag or RFP labeling density was quantified. n = 3; >36 cells per sample; ****P < 0.0001; Student t test. (B) Western blots of RRBP1 and β-actin show a reduction of RRBP1 in HT-1080 and Gp78 KO HT-1080 cells following transfection of siRRBP1 relative to siCTL. Graph shows densitometric quantification of band intensity. n = 3; *P < 0.05; **P < 0.01. (C) Shape features, height, sphericity, and planarity for the Q95V contacts of a representative cell closest to the mean Q95V for control HeLa cells or HeLa cells overexpressing Gp78 WT or Gp78 RM were analyzed. The comparison shows that the large contacts induced by Gp78 RM in HeLa cells have a shape signature comparable to control cells and not to the riboMERCs induced by Gp78 overexpression. Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3; ***P < 0.001. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 expression in COS-7 and HeLa cells, siRRBP1 western blot, and shape features for Q95 Gp78 RM overexpressing cells. (A) COS-7 and HeLa cells were transfected in parallel with Flag-Gp78 WT, Flag-Gp78 RM, or the RFP-tagged OMM–ER linker and fixed after 24 h. The Flag-Gp78 transfected cells were labeled for anti-Flag and the OMM–ER linker transfected cells left unlabeled. Cells were imaged and anti-Flag or RFP labeling density was quantified. n = 3; >36 cells per sample; ****P < 0.0001; Student t test. (B) Western blots of RRBP1 and β-actin show a reduction of RRBP1 in HT-1080 and Gp78 KO HT-1080 cells following transfection of siRRBP1 relative to siCTL. Graph shows densitometric quantification of band intensity. n = 3; *P < 0.05; **P < 0.01. (C) Shape features, height, sphericity, and planarity for the Q95V contacts of a representative cell closest to the mean Q95V for control HeLa cells or HeLa cells overexpressing Gp78 WT or Gp78 RM were analyzed. The comparison shows that the large contacts induced by Gp78 RM in HeLa cells have a shape signature comparable to control cells and not to the riboMERCs induced by Gp78 overexpression. Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3; ***P < 0.001. Source data are available for this figure: .

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Expressing, Western Blot, Transfection, Labeling, Control, Comparison, Over Expression, MANN-WHITNEY

RRBP1 knockdown reduces riboMERCS independent of Gp78. (A) Representative EM images of HT-1080 and HT-1080 Gp78 KO cells treated with either siControl or siRRBP1. Images highlight the presence of riboMERCS in both HT-1080 WT and Gp78 KO cells, which are almost completely lost upon RRBP1 knockdown. (B) Quantification of the number of riboMERCs per mitochondria and the ratio of riboMERC length to mitochondrial perimeter for the conditions in A. (C) Quantification of the MERC width for both riboMERCs and smooth MERCs for the conditions in A. n = 31 images from two independent biological replicates; **P < 0.01; ***P < 0.001; ****P < 0.0001; unpaired t test. Bar = 200 nm.

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: RRBP1 knockdown reduces riboMERCS independent of Gp78. (A) Representative EM images of HT-1080 and HT-1080 Gp78 KO cells treated with either siControl or siRRBP1. Images highlight the presence of riboMERCS in both HT-1080 WT and Gp78 KO cells, which are almost completely lost upon RRBP1 knockdown. (B) Quantification of the number of riboMERCs per mitochondria and the ratio of riboMERC length to mitochondrial perimeter for the conditions in A. (C) Quantification of the MERC width for both riboMERCs and smooth MERCs for the conditions in A. n = 31 images from two independent biological replicates; **P < 0.01; ***P < 0.001; ****P < 0.0001; unpaired t test. Bar = 200 nm.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Knockdown

MCS-DETECT captures MERC changes induced by RRBP1 knockdown. (A) Volume-rendered MCS-DETECT views of HT-1080 WT and Gp78 KO cells treated with either siControl or siRRBP1. Mitochondria are labeled with TOMM20 (red) and MERCS are visualized in white. Bar = 10 µm whole cell; 1 µm insets. (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in HT-1080 WT and Gp78 KO cells treated with either siControl or siRRBP1. Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001. (C) HT-1080 cells transfected with either siControl or siRRBP1 were labeled with MitoView633. Integrated density of MitoView633 per cell was quantified. n = 3 independent biological replicates; >50 cells/condition per experiment; ***P < 0.0001; ****P < 0.0001; Tukey post hoc test.

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: MCS-DETECT captures MERC changes induced by RRBP1 knockdown. (A) Volume-rendered MCS-DETECT views of HT-1080 WT and Gp78 KO cells treated with either siControl or siRRBP1. Mitochondria are labeled with TOMM20 (red) and MERCS are visualized in white. Bar = 10 µm whole cell; 1 µm insets. (B) Mitochondria surface coverage ratio and the number of contacts per sampled mitochondria window are shown for contact zones in HT-1080 WT and Gp78 KO cells treated with either siControl or siRRBP1. Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001. (C) HT-1080 cells transfected with either siControl or siRRBP1 were labeled with MitoView633. Integrated density of MitoView633 per cell was quantified. n = 3 independent biological replicates; >50 cells/condition per experiment; ***P < 0.0001; ****P < 0.0001; Tukey post hoc test.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Knockdown, Labeling, MANN-WHITNEY, Transfection

Large MERCs induced by Gp78 and the OMM–ER linker present distinct shape signatures. (A) The 95th quantile of MERC volume per cell (Q95V; largest 5% of MERCs per cell) and number of MERCs per cell larger than the average 500-voxel size of HT-1080 Q95V MERCs are shown for HT-1080 and COS-7 cells, COS-7, and COS-7 cells overexpressing either Gp78 WT, Gp78 RM, or the OMM–ER linker, HeLa and HeLa cells overexpressing either Gp78 WT, Gp78 RM, or the OMM–ER linker, HT-1080, and Gp78 KO HT-1080 cells, HT-1080 cells transfected with siCTL and siRRBP1, and Gp78 KO HT-1080 cells transfected with siCTL and siRRBP1. (B) Representative cells whose Q95V is closest to the mean Q95V for HT-1080 cells, for COS-7 or HeLa cells overexpressing Gp78 WT, and for COS-7 cells overexpressing the OMM–ER linker were selected for analysis. For the Q95V contacts of each cell, we compute shape features: height, sphericity, and planarity. The comparison shows that the COS-7 OMM–ER linker–induced contacts have a markedly different shape signature compared to those present in HT-1080 and Gp78 overexpressing COS-7 or HeLa cells (i.e., riboMERCS). Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001.

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Large MERCs induced by Gp78 and the OMM–ER linker present distinct shape signatures. (A) The 95th quantile of MERC volume per cell (Q95V; largest 5% of MERCs per cell) and number of MERCs per cell larger than the average 500-voxel size of HT-1080 Q95V MERCs are shown for HT-1080 and COS-7 cells, COS-7, and COS-7 cells overexpressing either Gp78 WT, Gp78 RM, or the OMM–ER linker, HeLa and HeLa cells overexpressing either Gp78 WT, Gp78 RM, or the OMM–ER linker, HT-1080, and Gp78 KO HT-1080 cells, HT-1080 cells transfected with siCTL and siRRBP1, and Gp78 KO HT-1080 cells transfected with siCTL and siRRBP1. (B) Representative cells whose Q95V is closest to the mean Q95V for HT-1080 cells, for COS-7 or HeLa cells overexpressing Gp78 WT, and for COS-7 cells overexpressing the OMM–ER linker were selected for analysis. For the Q95V contacts of each cell, we compute shape features: height, sphericity, and planarity. The comparison shows that the COS-7 OMM–ER linker–induced contacts have a markedly different shape signature compared to those present in HT-1080 and Gp78 overexpressing COS-7 or HeLa cells (i.e., riboMERCS). Averaged over cell, two-sided non-parametric Mann–Whitney test, n = 3 independent biological replicates, ≥30 cells/condition per experiment; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Comparison, MANN-WHITNEY

Gp78 induces convoluted, tubular riboMERCs. Representative whole-cell views of Q95V MERCs (color-coded for increasing size from 500 to 5,613 voxels) from HT-1080 cells, COS-7, or HeLa cells overexpressing Gp78 WT and COS-7 cells overexpressing the OMM–ER linker as well as representative individual Q95V MERCs alone or adjacent to transparent (pink) or solid mitochondria (magenta) to highlight intercalation of riboMERCs with mitochondria. Rotating videos of these MERCs are included as Supplemental , , , , , , , , , , , and . Bar = 1 μm.

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: Gp78 induces convoluted, tubular riboMERCs. Representative whole-cell views of Q95V MERCs (color-coded for increasing size from 500 to 5,613 voxels) from HT-1080 cells, COS-7, or HeLa cells overexpressing Gp78 WT and COS-7 cells overexpressing the OMM–ER linker as well as representative individual Q95V MERCs alone or adjacent to transparent (pink) or solid mitochondria (magenta) to highlight intercalation of riboMERCs with mitochondria. Rotating videos of these MERCs are included as Supplemental , , , , , , , , , , , and . Bar = 1 μm.

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques:

360° views of MERCs larger than the 500 voxels in complete HT-1080 cell, transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . Video was rendered in Imaris 10.0 software using the contacts channel outputted from MCS-DETECT (30 frames/s). MERCs are color-coded for increasing size from 500 (blue) to 5,613 voxels (red).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of MERCs larger than the 500 voxels in complete HT-1080 cell, transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . Video was rendered in Imaris 10.0 software using the contacts channel outputted from MCS-DETECT (30 frames/s). MERCs are color-coded for increasing size from 500 (blue) to 5,613 voxels (red).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

360° views of an individual 95th quantile MERC in an HT-1080 cell transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . MERCs display a high degree of complexity, with multiple branch points and extending over several Z slices. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in an HT-1080 cell transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . MERCs display a high degree of complexity, with multiple branch points and extending over several Z slices. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

360° views of an individual 95th quantile MERC in an HT-1080 cell transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . MERCs display a high degree of complexity, with multiple branch points and extending over several Z slices. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in an HT-1080 cell transfected with ERmoxGFP, labeled for anti-TOM-20, and imaged using 3D STED . MERCs display a high degree of complexity, with multiple branch points and extending over several Z slices. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

360° views of an individual 95th quantile MERC in a COS-7 cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a COS-7 cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

360° views of an individual 95th quantile MERC in a COS-7 cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a COS-7 cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (50 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (red), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

360° views of an individual 95th quantile MERC in a HeLa cells overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (10 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (magenta), and transparent ER (green).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a HeLa cells overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (10 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (magenta), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

360° views of an individual 95th quantile MERC in a HeLa cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (10 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (magenta), and transparent ER (green).

Journal: The Journal of Cell Biology

Article Title: Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria–ER contacts

doi: 10.1083/jcb.202206109

Figure Lengend Snippet: 360° views of an individual 95th quantile MERC in a HeLa cell overexpressing WT Gp78-FLAG , transfected with ERmoxGFP, labeled for anti-TOM-20 and anti-FLAG, and imaged using 3D STED. MERCs display a similar phenotype to MERCs observed in HT-1080 cells. Video was rendered in Imaris 10.0 software using the contacts, mitochondria, and ER channels outputted from MCS-DETECT (10 frames/s). All channels depict a single contact. Additional channels are rendered after each rotation in the following order: MERC channel (white), transparent mitochondria (pink), opaque mitochondria (magenta), and transparent ER (green).

Article Snippet: HT-1080 (Cat# 300216/p517_HT-1080, RRID:CVCL_0317; CLS) and HeLa cell lines were acquired from ATCC and authenticated by short tandem repeat profiling at the TCAG Genetic Analysis Facility (Hospital for Sick Kids, Toronto, ON, Canada https://www.tcag.ca/facilities/geneticAnalysis.html ).

Techniques: Transfection, Labeling, Software

(A) Correlation between log2-fold change (L2FC) in RAD51 foci number and the Rule Set 2 on-target score. All guides targeting RAD51 regulators including RAD51 paralogs (RAD51D, RAD51C, XRCC3), BRCA1, and BRCA2 in the library are shown. Splice and nonsense variants with high Rule Set 2 score shows more significant L2FC. Pearson correlation (r) equals −0.30. (B) Quantification of RAD51 foci in irradiated S/G2-phase cells with guides targeting RAD51 regulators that have low Rule Set 2 score. Cells are grouped by sgRNA category. No. or moderate significant separation from the cells with control guides is observed. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. (Outliers are not shown in the CDFs.) (C) as in B) for guides with high on-target score, showing significant reduction in RAD51 foci in cells with nonsense and splice guides, compared to the control guides. (D) as in A) for L2FC in BRCA1 foci for BRCA1-targeting guides, showing a strong negative correlation with the Rule Set 2 score. Pearson correlation (r) equals −0.75. (E) same as B) for BRCA1 foci and guides targeting BRCA1 that have low Rule Set 2 score, showing no significant separation from the control guides. (F) same as E) for guides with high Rule Set 2 score, showing significant reduction in BRCA1 foci in cells with missense, nonsense, and splice guides. (G) Volcano plot showing no guides targeting AAVS1 and non-targeting control (NTC) guides shows statistically significant changes in RAD51 foci under irradiation. Significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.55, all AAVS1-targeting non-targeting control (NTC) guides are shown. Benjamini-Hochberg corrected two-sided KS test. (H) Same as G) showing guides that result in significant changes in RAD51 foci. (I) Gene enrichment analysis of guides targeting RAD51D among guides causing significant changes in RAD51 foci in the irradiated condition. Fisher exact test. Enriched genes are defined as p.adj <0.05. (J) same as G) for BRCA1 foci. (K) same as H) for guides that result in significant changes in BRCA1 foci (L) same as I) for BRCA1 foci.

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Correlation between log2-fold change (L2FC) in RAD51 foci number and the Rule Set 2 on-target score. All guides targeting RAD51 regulators including RAD51 paralogs (RAD51D, RAD51C, XRCC3), BRCA1, and BRCA2 in the library are shown. Splice and nonsense variants with high Rule Set 2 score shows more significant L2FC. Pearson correlation (r) equals −0.30. (B) Quantification of RAD51 foci in irradiated S/G2-phase cells with guides targeting RAD51 regulators that have low Rule Set 2 score. Cells are grouped by sgRNA category. No. or moderate significant separation from the cells with control guides is observed. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. (Outliers are not shown in the CDFs.) (C) as in B) for guides with high on-target score, showing significant reduction in RAD51 foci in cells with nonsense and splice guides, compared to the control guides. (D) as in A) for L2FC in BRCA1 foci for BRCA1-targeting guides, showing a strong negative correlation with the Rule Set 2 score. Pearson correlation (r) equals −0.75. (E) same as B) for BRCA1 foci and guides targeting BRCA1 that have low Rule Set 2 score, showing no significant separation from the control guides. (F) same as E) for guides with high Rule Set 2 score, showing significant reduction in BRCA1 foci in cells with missense, nonsense, and splice guides. (G) Volcano plot showing no guides targeting AAVS1 and non-targeting control (NTC) guides shows statistically significant changes in RAD51 foci under irradiation. Significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.55, all AAVS1-targeting non-targeting control (NTC) guides are shown. Benjamini-Hochberg corrected two-sided KS test. (H) Same as G) showing guides that result in significant changes in RAD51 foci. (I) Gene enrichment analysis of guides targeting RAD51D among guides causing significant changes in RAD51 foci in the irradiated condition. Fisher exact test. Enriched genes are defined as p.adj <0.05. (J) same as G) for BRCA1 foci. (K) same as H) for guides that result in significant changes in BRCA1 foci (L) same as I) for BRCA1 foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Irradiation, Control

(A) Wasserstein distance of cells with DDR gene-targeting guides (Perturb) or control guides (Controls) to control cells for RAD51 foci in irradiated cells. Hits of RAD51 foci identified in the pooled screening are marked. (B) same as A) for BRCA1 foci. (C) Volcano plots showing variants yielding significant changes in the proportion of cells with high p21 expression in untreated (left) or irradiated (right) conditions. Significance is defined by p.adj < 0.05, Beta-Binomial test. The proportion of control cells with high p21 is marked with the vertical line (UNT: 0.376; IR: 0.576). sgRNA category and ClinVar category for significant guides are displayed by colors and shapes, respectively. (D) Correlation of RAD51 foci log2-fold change (L2FC) in guides delivered in the pooled library (pooled) and transduced individually (individual). The Pearson correlation (r) is 0.90. (E) same as D) for BRCA1 foci. The Pearson correlation (r) is 0.95. (F) MCF7-BE3 cells were lentivirally transduced with the DDR364 library, selected in puromycin-containing medium for 2 days, and cultured 2 days without puromycin. Cells were either untreated (UNT) or treated with DNA damaging agents Camptothecin (CPT), Olaparib (OLAP), Cisplatin (CISP) or Etoposide (ETOP) for 24 hours, then fixed before optical phenotyping and barcode detection. (G) Correlation between L2FC in RAD51 foci in CISP-treated cells and the Rule Set 2 on-target score, showing a negative correlation. All guides targeting RAD51 regulators including RAD51D, RAD51C, XRCC3, BRCA1, and BRCA2 are shown. The Pearson correlation (r) equals −0.29. (H) same as G) for OLAP-treated cells. The Pearson correlation (r) equals −0.30. (I) Volcano plot showing variants yielding significant changes in RAD51 foci in CISP-treated cells. Statistical significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.5, all AAVS1-targeting and non-targeting control (NTC) guides are shown. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. Same figure legend as in C). (J) same as I) for OLAP-treated cells. (K) Gene enrichment analysis in variants that result in significant changes in RAD51 foci. Fisher exact test. Enrichment is defined as p.adj <0.05. (L) Same as I) for variants yielding significant changes in large γH2AX foci in CISP-treated cells. (M) same as L) for OLAP-treated cells. (N) same as K) for large γH2AX foci.

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Wasserstein distance of cells with DDR gene-targeting guides (Perturb) or control guides (Controls) to control cells for RAD51 foci in irradiated cells. Hits of RAD51 foci identified in the pooled screening are marked. (B) same as A) for BRCA1 foci. (C) Volcano plots showing variants yielding significant changes in the proportion of cells with high p21 expression in untreated (left) or irradiated (right) conditions. Significance is defined by p.adj < 0.05, Beta-Binomial test. The proportion of control cells with high p21 is marked with the vertical line (UNT: 0.376; IR: 0.576). sgRNA category and ClinVar category for significant guides are displayed by colors and shapes, respectively. (D) Correlation of RAD51 foci log2-fold change (L2FC) in guides delivered in the pooled library (pooled) and transduced individually (individual). The Pearson correlation (r) is 0.90. (E) same as D) for BRCA1 foci. The Pearson correlation (r) is 0.95. (F) MCF7-BE3 cells were lentivirally transduced with the DDR364 library, selected in puromycin-containing medium for 2 days, and cultured 2 days without puromycin. Cells were either untreated (UNT) or treated with DNA damaging agents Camptothecin (CPT), Olaparib (OLAP), Cisplatin (CISP) or Etoposide (ETOP) for 24 hours, then fixed before optical phenotyping and barcode detection. (G) Correlation between L2FC in RAD51 foci in CISP-treated cells and the Rule Set 2 on-target score, showing a negative correlation. All guides targeting RAD51 regulators including RAD51D, RAD51C, XRCC3, BRCA1, and BRCA2 are shown. The Pearson correlation (r) equals −0.29. (H) same as G) for OLAP-treated cells. The Pearson correlation (r) equals −0.30. (I) Volcano plot showing variants yielding significant changes in RAD51 foci in CISP-treated cells. Statistical significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.5, all AAVS1-targeting and non-targeting control (NTC) guides are shown. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. Same figure legend as in C). (J) same as I) for OLAP-treated cells. (K) Gene enrichment analysis in variants that result in significant changes in RAD51 foci. Fisher exact test. Enrichment is defined as p.adj <0.05. (L) Same as I) for variants yielding significant changes in large γH2AX foci in CISP-treated cells. (M) same as L) for OLAP-treated cells. (N) same as K) for large γH2AX foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Control, Irradiation, Expressing, Transduction, Cell Culture

(A) Immunoblots on guides targeting BRCA1 showing the reduction of full-length BRCA1 protein in the splice variant BRCA1.234, compared to the missense variants BRCA1.416 and.BRCA1.476, and the AAVS1 variant AAVS1.86. Cells transduced with Firefly siRNA (siFirefly) and BRCA1 siRNA (siBRCA1) were included to show the specificity of BRCA1 detection. Cells were treated with or without irradiation and the induction of DNA damage is shown with the phospho-KAP1 (pKAP1) staining. Tubulin is used as the loading control. (B) As in A) for BRCA2 variants showing the reduction of full-length BRCA2 protein in the nonsense variant BRCA2.207, compared to the missense variant BRCA2.438 and the AAVS1 variant AAVS1.86.

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Immunoblots on guides targeting BRCA1 showing the reduction of full-length BRCA1 protein in the splice variant BRCA1.234, compared to the missense variants BRCA1.416 and.BRCA1.476, and the AAVS1 variant AAVS1.86. Cells transduced with Firefly siRNA (siFirefly) and BRCA1 siRNA (siBRCA1) were included to show the specificity of BRCA1 detection. Cells were treated with or without irradiation and the induction of DNA damage is shown with the phospho-KAP1 (pKAP1) staining. Tubulin is used as the loading control. (B) As in A) for BRCA2 variants showing the reduction of full-length BRCA2 protein in the nonsense variant BRCA2.207, compared to the missense variant BRCA2.438 and the AAVS1 variant AAVS1.86.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Western Blot, Variant Assay, Transduction, Irradiation, Staining, Control

RSS suppress oxidative stress–induced parthanatos. A , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress oxidative stress–induced parthanatos. A , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: MTS Assay, Control, Concentration Assay, Labeling, Western Blot, Software, Fluorescence, FACS

RSS suppress the ALIS formation. A , HT1080 and p62 KO HT1080 cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. B , HT1080 cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. C , HT1080 cells were treated with CTX (1 mg/ml) and/or NAC (2 mM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. D , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. Scale bar represents 10 μm. G , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. I , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were treated with the indicated reagents for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. CTX (1 mg/ml). I3MT-3 (10 μM). PAG (5 mM). Scale bar represents 10 μm. K , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, I3MT-3 0 μM PAG 0 mM cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; PAG, DL-propargylglycine; NAC, N-acetylcysteine; RSS, reactive sulfur species.

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress the ALIS formation. A , HT1080 and p62 KO HT1080 cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. B , HT1080 cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. C , HT1080 cells were treated with CTX (1 mg/ml) and/or NAC (2 mM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. D , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. Scale bar represents 10 μm. G , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. I , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were treated with the indicated reagents for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. CTX (1 mg/ml). I3MT-3 (10 μM). PAG (5 mM). Scale bar represents 10 μm. K , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, I3MT-3 0 μM PAG 0 mM cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; PAG, DL-propargylglycine; NAC, N-acetylcysteine; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence, Staining, Ubiquitin Proteomics

RSS disaggregate the ALIS by inducing HSP70. A , HT1080 cells were treated with the indicated reagents for 24 h and then incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. CTX (1 mg/ml). PAG (5 mM). I3MT-3 (20 μM). Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM, I3MT-3 0 μM cells). B , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h and then incubated with 10 μM DCFH-DA. Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), N.S. p > 0.05 ( versus CTX 1 mg/ml Na 2 S 4 0 μM cells). C , HT1080 cells were treated with the indicated reagents for 24 h, and then the detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. Bafilomycin A1 (5 nM). CTX (1 mg/ml). Na 2 S 4 (100 μM). D , HT1080 cells were treated with CTX (1 mg/ml) for 20 h and then treated with MG132 (10 μM) and/or Na 2 S 4 (100 μM) for 4 h. The detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the indicated concentration of Na 2 S 4 for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. G , HT1080 cells were treated with CTX (1 mg/ml) and Na 2 S 4 (100 μM) for 18 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then whole cell lysate were subjected to immunoblotting with the indicated antibodies. I , HT1080 and PARP-1 KO cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. L , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05, ∗∗ p < 0.01, ( versus siRNA Ctr, CTX 1 mg/ml cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; HSP, heat shock protein; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; ROS, reactive oxygen species; RSS, reactive sulfur species.

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS disaggregate the ALIS by inducing HSP70. A , HT1080 cells were treated with the indicated reagents for 24 h and then incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. CTX (1 mg/ml). PAG (5 mM). I3MT-3 (20 μM). Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM, I3MT-3 0 μM cells). B , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h and then incubated with 10 μM DCFH-DA. Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), N.S. p > 0.05 ( versus CTX 1 mg/ml Na 2 S 4 0 μM cells). C , HT1080 cells were treated with the indicated reagents for 24 h, and then the detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. Bafilomycin A1 (5 nM). CTX (1 mg/ml). Na 2 S 4 (100 μM). D , HT1080 cells were treated with CTX (1 mg/ml) for 20 h and then treated with MG132 (10 μM) and/or Na 2 S 4 (100 μM) for 4 h. The detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the indicated concentration of Na 2 S 4 for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. G , HT1080 cells were treated with CTX (1 mg/ml) and Na 2 S 4 (100 μM) for 18 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then whole cell lysate were subjected to immunoblotting with the indicated antibodies. I , HT1080 and PARP-1 KO cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. L , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05, ∗∗ p < 0.01, ( versus siRNA Ctr, CTX 1 mg/ml cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; HSP, heat shock protein; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; ROS, reactive oxygen species; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Incubation, Fluorescence, Control, Western Blot, Concentration Assay, Transfection, Negative Control, Immunofluorescence, Staining, Ubiquitin Proteomics

RSS activate HSF1 by promoting its dissociation from HSP90. A , HT1080 cells were treated with the Na 2 S 4 (100 μM) for indicated period, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗∗ p < 0.001, ( versus control cells). B , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. C , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. D , HT1080 and PARP-1 KO cells were treated with the Na 2 S 4 (100 μM) for 6 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus control cells), ## p < 0.01 ( versus Na 2 S 4 100 μM, KRIBB11 0 μM cells). G , HT1080 cells were transfected with FLAG-HSP90 and/or Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with indicated concentration of Na 2 S 4 for 4 h, then immunoprecipitated protein G-Sepharose beads with the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. I , HSP90 was immunoprecipitated using anti-HSP90 antibody with protein G beads. Beads were washed four times with PBS and then treated with Na 2 S 4 (100, 1000 μM) for 1 h. After reaction, beads were washed four times with PBS and subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with FLAG-HSP90 (WT/C412A/C564A/C521A) and Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with FLAG-Empty or FLAG-HSP90 (WT/C521A) plasmid for 24 h and treated with Na 2 S 4 (100 μM) for indicated periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. All data are representative of at least three independent experiments. HSF, heat shock factor; HSP, heat shock protein; PARP-1, poly (ADP-ribose) polymerase-1; RSS, reactive sulfur species.

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS activate HSF1 by promoting its dissociation from HSP90. A , HT1080 cells were treated with the Na 2 S 4 (100 μM) for indicated period, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗∗ p < 0.001, ( versus control cells). B , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. C , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. D , HT1080 and PARP-1 KO cells were treated with the Na 2 S 4 (100 μM) for 6 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus control cells), ## p < 0.01 ( versus Na 2 S 4 100 μM, KRIBB11 0 μM cells). G , HT1080 cells were transfected with FLAG-HSP90 and/or Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with indicated concentration of Na 2 S 4 for 4 h, then immunoprecipitated protein G-Sepharose beads with the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. I , HSP90 was immunoprecipitated using anti-HSP90 antibody with protein G beads. Beads were washed four times with PBS and then treated with Na 2 S 4 (100, 1000 μM) for 1 h. After reaction, beads were washed four times with PBS and subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with FLAG-HSP90 (WT/C412A/C564A/C521A) and Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with FLAG-Empty or FLAG-HSP90 (WT/C521A) plasmid for 24 h and treated with Na 2 S 4 (100 μM) for indicated periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. All data are representative of at least three independent experiments. HSF, heat shock factor; HSP, heat shock protein; PARP-1, poly (ADP-ribose) polymerase-1; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Concentration Assay