Journal: bioRxiv

Article Title: Senescence rewires microenvironment sensing to facilitate anti-tumor immunity

doi: 10.1101/2022.06.04.494739

Figure Lengend Snippet: A, GSEA (Hallmark) of RNA-Seq data from proliferating (PRO, p53 Off) vs. senescent (SEN, p53 On for 8 days) NSP liver tumor cells in vitro. B, Transcriptomic analysis of differential expressed genes (DEGs) encoding secretory factors (SASP factors) in NPS cells in the presence or absence of JQ-1 treatment. C, Cytokine array of conditioned medium collected from proliferating and senescent (p53 On Day 6 to Day 8) cells in vitro. Samples are from 2 independent biological replicates. D, GO analysis of DEGs encoding plasma membrane proteins upregulated in senescent cells. Related to E, Subcellular localization of detected DEGs (TPM > 1; p < 0.05; fold change > 2) from in vitro NSP cells treated with trametinib/palbociclib (T+P) or vehicle. F, Calculation of p value of Fisher exact test using data from the supplementary Fig. S4E. A more stringent criteria for statistic significance is used, p < 0.01. Related to . G, Graphic illustration of the protocol of plasma membrane-enriched mass spectrometry (MS). H, Correlation plot of MS samples. I, Left panel, XY plot of total proteins profiled by MS against corresponding transcriptomic expression profiled by RNA-seq. Right panel, summary of MS profiling and RNA-seq comparison J and K, validation of two MS hits using flow cytometry. PRO, proliferating. SEN, senescent. N.D., not detected. Data is presented as mean ± s.e.m.

Article Snippet: For drug-induced senescence experiments, p53-suppressed (on-dox) NSP cells were treated with trametinib (25 nM, S2673 Selleck Chem) + Palbociclib (500 nM, S1116, Selleck Chem), Nutlin (10 μM, S1061, Selleck Chem) or Cisplatin (1 μM), changed every 2-3 days, during 7 days.

Techniques: RNA Sequencing, In Vitro, Clinical Proteomics, Membrane, Mass Spectrometry, Expressing, Comparison, Biomarker Discovery, Flow Cytometry

Journal: bioRxiv

Article Title: Senescence rewires microenvironment sensing to facilitate anti-tumor immunity

doi: 10.1101/2022.06.04.494739

Figure Lengend Snippet: A, Pathway analysis of cluster 1 (C1, senescence-specific) shown in against MsigDB Hallmark genesets. B, IFNGR1 and IFNGR2 level validated in 3 independent p53-restorable liver cancer cell lines. NSP is predominantly used in this study. C, SA-β-gal staining of NSP cells treated with different senescence triggers. D, Overlapping DEGs from RNA-seq in NSP cells treated with different senescence triggers to identify common signatures upregulated (UP) and downregulated (DN) in proliferating (PRO) vs. senescent (SEN) cells, which is composed of 111 and 470 genes respectively. p53, p53 restoration; T+P, trametinib plus palbociclib. E, GSEA of the combined RNA-seq results from all human cell lines trigger to senesce in SENESCopedia showing enrichment of genes upregulated (ALL_UP) or downregulated (ALL_DN) in our common senescence signature from (D). F, GSEA of same RNA-seq results in (E) showing an enrichment in MSigDB Hallmark IFN-γ response pathway in senescent cells. G, p53 mutation status of human cell lines used in SENESCopedia. H, Viability assay of proliferating and p53-restored senescent NSP cells treated with indicated dose of IFN-γ for 48 hours. I, Cytokine array of conditioned medium collected from proliferating and senescent (p53 On Day 6 to Day 8) cells in vitro. Same data collected from . J, Cytometric bead array (CBA) assay for IFN-γ level from in vivo tumor tissue lysate samples. K, MHC-I (H2-Kb) level of proliferating and senescent NSP cells in vitro . L, RT-qPCR of selected antigen presentation pathway genes in proliferating and senescent NSP cells in vitro . Data is presented as mean ± s.e.m. Two-tailed student t-test is used.

Article Snippet: For drug-induced senescence experiments, p53-suppressed (on-dox) NSP cells were treated with trametinib (25 nM, S2673 Selleck Chem) + Palbociclib (500 nM, S1116, Selleck Chem), Nutlin (10 μM, S1061, Selleck Chem) or Cisplatin (1 μM), changed every 2-3 days, during 7 days.

Techniques: Staining, RNA Sequencing, Mutagenesis, Viability Assay, In Vitro, In Vivo, Quantitative RT-PCR, Immunopeptidomics, Two Tailed Test

Journal: bioRxiv

Article Title: Senescence rewires microenvironment sensing to facilitate anti-tumor immunity

doi: 10.1101/2022.06.04.494739

Figure Lengend Snippet: A and B, IFNGR1 level on proliferating and senescent cells profiled by mass spectrometry and validated by flow cytometry (B). AU, arbitrary unit. Data is presented as mean ± s.e.m. n = 6 for both proliferating and senescent group. C, Transcriptomic analysis of selected genes regulating IFN-γ signaling from RNA-seq data of 3 independent p53-restorable cell lines (NSP, NSM2, NSP5) restoring p53 along with NSP cells treated with two other senescence triggers. T+P, trametinib plus palbociclib. D, mRNA expression of selected genes involved in IFN-γ signaling in human cell lines triggered to senesce. Treatment: Ali, alisertib; Eto, etoposide; number indicates the length of treatment (days). Data is obtained from the public dataset SENESCOPEDIA . E, Immunoblot analysis of NSP cells under different senescent triggers, in presence or absence of IFN-γ (1 ng/ml). p-Stat1, phospho-Stat1 (Tyr701)

Article Snippet: For drug-induced senescence experiments, p53-suppressed (on-dox) NSP cells were treated with trametinib (25 nM, S2673 Selleck Chem) + Palbociclib (500 nM, S1116, Selleck Chem), Nutlin (10 μM, S1061, Selleck Chem) or Cisplatin (1 μM), changed every 2-3 days, during 7 days.

Techniques: Mass Spectrometry, Flow Cytometry, RNA Sequencing, Expressing, Western Blot

Journal: bioRxiv

Article Title: Senescence rewires microenvironment sensing to facilitate anti-tumor immunity

doi: 10.1101/2022.06.04.494739

Figure Lengend Snippet: A, Human liver cancer cell lines and isogenic p53 KO clones were treated with indicated drugs to induce senescence. Cells were treated with human recombinant IFN-γ (1 ng/ml) and HLA-A/B/C was measured after 24h of treatment. “HLA-index” was determined by calculating the HLA level difference between drug-treated vs. untreated cells, in the presence or absence of IFN-γ treatment. Here shown is an example of HLA-index calculation. B, p53 and RAS pathway mutation status of the human cell lines used in this experiment. C, The summary of 5 human cell lines (including two isogenic p53 KO clones) treated with nutlin or trametinib + palbociclib (T+P). S, senescent; P, proliferating. Data is presented as mean ± s.e.m.

Article Snippet: For drug-induced senescence experiments, p53-suppressed (on-dox) NSP cells were treated with trametinib (25 nM, S2673 Selleck Chem) + Palbociclib (500 nM, S1116, Selleck Chem), Nutlin (10 μM, S1061, Selleck Chem) or Cisplatin (1 μM), changed every 2-3 days, during 7 days.

Techniques: Clone Assay, Recombinant, Mutagenesis

Journal: bioRxiv

Article Title: Senescence rewires microenvironment sensing to facilitate anti-tumor immunity

doi: 10.1101/2022.06.04.494739

Figure Lengend Snippet: A, Representative microscopic images of IGS reporter-expressing proliferating and senescent NSP tumor cells triggered by p53 restoration or trametinib + palbociclib (T+P) in combination with IFN-γ (1 ng/ml) treatment. B, Quantification of ZsGreen1 intensity from (A). C, Quantification of ZsGreen1 intensity of NSP tumor cells in the OT-I T cells and SIINFEKL-expressing tumor cells co-culture experiment (E/T ratio 5:1) after 20 h of co-culture. Signal measured by flow cytometry. T+P, trametinib plus palbociclib. MFI, median fluorescence intensity. Data is presented as mean ± s.e.m.

Article Snippet: For drug-induced senescence experiments, p53-suppressed (on-dox) NSP cells were treated with trametinib (25 nM, S2673 Selleck Chem) + Palbociclib (500 nM, S1116, Selleck Chem), Nutlin (10 μM, S1061, Selleck Chem) or Cisplatin (1 μM), changed every 2-3 days, during 7 days.

Techniques: Expressing, Co-Culture Assay, Flow Cytometry, Fluorescence

Journal: Science Advances

Article Title: Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models

doi: 10.1126/sciadv.abj5405

Figure Lengend Snippet: ( A ) Bubble plot showing the dysregulated signaling pathways with P values in −log 10 . ( B ) Bubble plot showing the dysregulated signaling pathways with P values in −log 10 . ( C ) RNA splicing network node from essential fitness gene network, in which RBM39 is highlighted in yellow. ( D ) DEMETER2 score (mean ± SD) of an RNAi screen across 25 cancer lineages. Neuroblastoma versus others, P = 0.0023. ( E ) Western blot to validate the RBM39 knockdown after lentiviral transduction of shRNA control [green fluorescent protein (GFP)] and two different RBM39 shRNAs (#188 and #190) for 72 hours. ( F ) Colony formation for neuroblastoma cells transduced with lentiviral shRNA control and two different RBM39 shRNAs followed by puromycin selection for 7 days. Cells stained with crystal violet for visualization. ( G ) Tumor volume comparison between shRNA control and RBM39 knockdown by two different shRNAs in four tumor models. P values calculated using unpaired Student’s t test. ( H ) RNA splicing analyses for different events altered by RBM39 knockdown in BE2C and SK-N-AS cells. The y axis indicates numbers for each splicing event induced by RBM39 knockdown in comparison with shRNA control. A3SS, alternative 3′ splicing; A5SS, alternative 5′ splicing; MXE, mutually exclusive exon; RI, intron retention; SE, skipped exon. ( I ) Sashimi plot using IGV program shows the splicing changes of EED gene in BE2C and SK-N-AS cells after RBM39 knockdown, which indicates the missplicing among exons 7 to 10. The numbers indicate the read counts of exon-intron junctions of RNA-seq.

Article Snippet: The following antibodies were used: ß-actin (Sigma-Aldrich, A5441, mouse antibody, 1:5000 dilution), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, 5174s, rabbit antibody, 1:5000 dilution), RBM39 (Atlas Antibodies, HPA-001591, rabbit antibody, 1:2000 dilution), c-Myc (Cell Signaling Technology, 5605s, rabbit antibody, 1:2000 dilution), MYCN (Santa Cruz Biotechnology, 53993, mouse antibody, 1:200 dilution), FLAG (Sigma-Aldrich, F1804, mouse antibody, 1:2000 dilution), ATM (D2E2) (Cell Signaling Technology, 2873, rabbit antibody, 1:1000 dilution), EZH2 (D2C9) (Cell Signaling Technology, 5246, rabbit antibody, 1:1000 dilution), CDK4 (D9G3E) (Cell Signaling Technology, 12790, rabbit antibody, 1:1000 dilution), RET (C31B4) (Cell Signaling Technology, 3223, rabbit antibody, 1:1000 dilution), and EED (Millipore 09-774, rabbit antibody, 1:1000 dilution).

Techniques: Protein-Protein interactions, Western Blot, Knockdown, Transduction, shRNA, Control, Selection, Staining, Comparison, RNA Sequencing

Journal: Science Advances

Article Title: Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models

doi: 10.1126/sciadv.abj5405

Figure Lengend Snippet: ( A ) Differential mean toxicity plot for neuroblastoma cell lines ( n = 5) treated with single doses of 4686 compounds by PRISM. ( B ) AUC screen by PRISM showing indisulam selectivity against neuroblastoma cell lines ( n = 4). ( C ) AUC of indisulam against neuroblastoma cell lines ( n = 14) and other cancer lineages by CTRP . ( D ) Neuroblastoma cells were treated with indicated concentrations of indisulam for 72 hours, followed by Western blot analysis of RBM39. ( E ) Analysis of splicing events in BE2C cells altered by 250 nM indisulam treatment for 72 hours. ( F ) Sashimi plot shows EED missplicing in exons 7 to 10 in BE2C cells by indisulam. The numbers indicate the read counts of exon-intron junctions of RNA-seq. ( G ) Genome-wide pairwise correlation analysis of altered splicing events induced by indisulam and RBM39 knockdown. ( H ) Dose-response curve shows the differential effect of indisulam on neuroblastoma cells and normal human cell BJ and HS6. ( I ) Colony formation assay for cell lines treated with indisulam for 7 days. ( J ) Mean tumor volume for BE2C (vehicle = 5; indisulam = 6), SK-N-AS (vehicle = 7; indisulam = 8), HCT116 (vehicle = 5; indisulam = 5), and Rh30 (vehicle = 5; indisulam = 5) xenografts treated with indisulam (25 mg/kg), 5 days on and 2 days off for two cycles. Cyan arrow indicates when therapy discontinued. ( K to M ) Kaplan-Meier survival curve for BE2C (K), Rh30 (L), and HCT116 (M) xenografts.

Article Snippet: The following antibodies were used: ß-actin (Sigma-Aldrich, A5441, mouse antibody, 1:5000 dilution), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, 5174s, rabbit antibody, 1:5000 dilution), RBM39 (Atlas Antibodies, HPA-001591, rabbit antibody, 1:2000 dilution), c-Myc (Cell Signaling Technology, 5605s, rabbit antibody, 1:2000 dilution), MYCN (Santa Cruz Biotechnology, 53993, mouse antibody, 1:200 dilution), FLAG (Sigma-Aldrich, F1804, mouse antibody, 1:2000 dilution), ATM (D2E2) (Cell Signaling Technology, 2873, rabbit antibody, 1:1000 dilution), EZH2 (D2C9) (Cell Signaling Technology, 5246, rabbit antibody, 1:1000 dilution), CDK4 (D9G3E) (Cell Signaling Technology, 12790, rabbit antibody, 1:1000 dilution), RET (C31B4) (Cell Signaling Technology, 3223, rabbit antibody, 1:1000 dilution), and EED (Millipore 09-774, rabbit antibody, 1:1000 dilution).

Techniques: Western Blot, RNA Sequencing, Genome Wide, Knockdown, Colony Assay

Journal: Science Advances

Article Title: Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models

doi: 10.1126/sciadv.abj5405

Figure Lengend Snippet: ( A ) Generation of G268V RBM39 knock-in cell lines. Cells modified with CRISPR-Cas9 and single-stranded oligo (ssODN) carrying the G268V mutation were selected under 2 μM indisulam treatment. Surviving cells were stained with crystal violet. ( B ) Cells with RBM39 G268V mutants generated by CRISPR-Cas9 knock-in were treated with 2 μM indisulam for 18 hours, followed by Western blot analysis. ( C ) Dose-response curve of cell viability assay for cells engineered to express the RBM39 G268V mutant. ( D and E ) Colony formation assays for SK-N-AS (D) and BE2C (E) and their RBM39 mutant G268V derivatives that were treated with different concentrations of indisulam. ( F ) Mean tumor volume for SK-N-AS xenografts (vehicle = 3; indisulam = 5) and SK-N-AS with RBM39 G268V knock-in xenografts (vehicle = 5; indisulam = 4) that were treated with indisulam (5 mg/kg), 5 days on and 2 days off for two cycles via tail vein injection. Cyan arrow indicates when therapy was discontinued. ( G ) Mean tumor volume for BE2C xenografts (vehicle = 4; indisulam = 5) and BE2C with RBM39 G268V knock-in xenografts (vehicle = 5; indisulam = 5) that were treated with indisulam (5 mg/kg), 5 days on and 2 days off for two cycles via tail vein injection. Cyan arrow indicates when therapy was discontinued. ( H ) Western blot assessment of RBM39 in SK-N-AS parental and G268V xenografts (vehicle = 3; indisulam = 3) harvested 24 hours after 3-day dosing treatment.

Article Snippet: The following antibodies were used: ß-actin (Sigma-Aldrich, A5441, mouse antibody, 1:5000 dilution), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, 5174s, rabbit antibody, 1:5000 dilution), RBM39 (Atlas Antibodies, HPA-001591, rabbit antibody, 1:2000 dilution), c-Myc (Cell Signaling Technology, 5605s, rabbit antibody, 1:2000 dilution), MYCN (Santa Cruz Biotechnology, 53993, mouse antibody, 1:200 dilution), FLAG (Sigma-Aldrich, F1804, mouse antibody, 1:2000 dilution), ATM (D2E2) (Cell Signaling Technology, 2873, rabbit antibody, 1:1000 dilution), EZH2 (D2C9) (Cell Signaling Technology, 5246, rabbit antibody, 1:1000 dilution), CDK4 (D9G3E) (Cell Signaling Technology, 12790, rabbit antibody, 1:1000 dilution), RET (C31B4) (Cell Signaling Technology, 3223, rabbit antibody, 1:1000 dilution), and EED (Millipore 09-774, rabbit antibody, 1:1000 dilution).

Techniques: Knock-In, Modification, CRISPR, Mutagenesis, Staining, Generated, Western Blot, Viability Assay, Injection

Journal: Science Advances

Article Title: Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models

doi: 10.1126/sciadv.abj5405

Figure Lengend Snippet: ( A ) Western blot assessment of RBM39 in tumor tissues, heart, lungs, kidney, and liver after CB17 SCID mice bearing SJNB11 PDX tumors were treated with vehicle or one of three different doses of indisulam, 5, 12.5, and 25 mg/kg, respectively ( n = 3 per dosing group), for 5 days. ( B ) Western blot assessment of RBM39 in tumor tissues, heart, lungs, kidney, and liver after CB17 SCID mice bearing SJNB19 PDX (non– MYCN -amplified) tumors were treated with vehicle or one of two different doses of indisulam, 12.5 and 25 mg/kg, respectively ( n = 3 per dosing group), for 5 days. ( C ) Mouse body weight for four neuroblastoma models that were treated with indisulam (25 mg/kg) for 5 days on and 2 days off, two cycles. ( D ) Analysis of neutrophils (NE), platelets (PLT), red blood cells (RBC), and hemoglobin (HB) from CB17 SCID mice bearing SJNB11 PDX that were treated with vehicle or one of three different doses of indisulam, 5, 12.5, and 25, respectively ( n = 4 per dosing group), for 5 days via tail injection. ( E ) Analysis of neutrophils, platelets (PLT), red blood cells (RBC), and hemoglobin (HB) from CB17 SCID mice bearing SJNB11 PDX that were treated with vehicle or one of three different doses of indisulam, 5, 12.5, and 25 mg/kg, respectively ( n = 5 per dosing group), for 12 days on and 2 days off, for two cycles via tail injection.

Article Snippet: The following antibodies were used: ß-actin (Sigma-Aldrich, A5441, mouse antibody, 1:5000 dilution), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, 5174s, rabbit antibody, 1:5000 dilution), RBM39 (Atlas Antibodies, HPA-001591, rabbit antibody, 1:2000 dilution), c-Myc (Cell Signaling Technology, 5605s, rabbit antibody, 1:2000 dilution), MYCN (Santa Cruz Biotechnology, 53993, mouse antibody, 1:200 dilution), FLAG (Sigma-Aldrich, F1804, mouse antibody, 1:2000 dilution), ATM (D2E2) (Cell Signaling Technology, 2873, rabbit antibody, 1:1000 dilution), EZH2 (D2C9) (Cell Signaling Technology, 5246, rabbit antibody, 1:1000 dilution), CDK4 (D9G3E) (Cell Signaling Technology, 12790, rabbit antibody, 1:1000 dilution), RET (C31B4) (Cell Signaling Technology, 3223, rabbit antibody, 1:1000 dilution), and EED (Millipore 09-774, rabbit antibody, 1:1000 dilution).

Techniques: Western Blot, Amplification, Injection

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Domain organization and mitochondrial localization of ChChd3. A, amino acid sequence and exon organization of ChChd3. Different exons are shown in alternate black and gray colors. The myristoylation motif at the N terminus is underlined . The consensus site for the PKA phosphorylation is shown in the box, and the previously identified PKA phosphorylation site is underlined . The C X 9 C-C X 9 C motif cysteines in the chch domain are highlighted with asterisk. B, schematic diagram of the ChChd3 protein. DUF, domain of unknown function, CHCH, coiled-coil helix-coiled-coil helix domain. C and D, ChChd3 in mitochondria is primarily localized to the IM facing toward the IMS. Mouse liver mitochondrial subfractions were separated by SDS-PAGE and analyzed by immunoblotting by using antibodies against ChChd3 and known mitochondrial marker proteins. ChChd3 is enriched in the IM fraction similar to that of the IM marker protein, NDUFS3. M, matrix ( C ). SDS-PAGE and immunoblot analysis of trypsin-treated samples of mitochondria, swollen mitochondria, and submitochondrial particles ( D ).

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Sequencing, SDS Page, Western Blot, Marker

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Loss of ChChd3 in HeLa cells results in abnormal mitochondrial morphology. A, mitochondria in ChChd3 knockdown cells are clumpy, fragmented, and clustered around the nucleus. Representative confocal microscopic images of HeLa-control, control-shRNA, and ChChd3-shRNA HeLa cells expressing matrix-targeted RFP ( mito-RFP ). Two independent clones of ChChd3-shRNA ( ChChd3-shRNA clone 1 and ChChd3-shRNA clone 2 ) were analyzed to avoid the selection artifacts. Scale bar, 10 μm. B, quantification of mitochondrial abnormalities in ChChd3 knockdown cells. 300 cells each from the control and ChChd3 knockdown cells were analyzed under confocal microscope for mitochondrial fragmentation and clumping. Mean ± S.D. from three independent experiments is shown. C, HeLa control, control-shRNA, and ChChd3-shRNA cell lysates were assessed for the changes in the protein levels of the key regulators of mitochondrial fusion and fission. ChChd3-shRNA1 and shRNA2 represent two different clones derived from a single shRNA sequence.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: shRNA, Expressing, Clone Assay, Selection, Microscopy, Derivative Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: ChChd3 is required for mitochondrial fusion in HeLa cells. Inhibition of fission by K38A-Drp1 does not restore tubular mitochondrial network in the absence of ChChd3. Representative confocal micrographs of control-shRNA or ChChd3-shRNA HeLa cells expressing mito-RFP ( red ) and GFP or Drp1 WT GFP or dominant negative mutant of Drp1, Drp1 K38A GFP ( green ) are shown. Scale bars, 5 μm.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Inhibition, shRNA, Expressing, Dominant Negative Mutation

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: ChChd3 depletion impairs cellular bioenergetics. A and B, ChChd3 knockdown cells show a drastic decrease in the OCR and ECAR. OCR and ECAR were measured simultaneously by using the Seahorse extracellular flux analyzer in real time. Rates shown are the averages of four wells measured for 3 min after every 5 min. Also shown are the OCR and ECAR values after addition of 1 μ m oligomycin, 200 n m carbonyl cyanide p -trifluoromethoxyphenylhydrazone ( FCCP ), and 100 n m rotenone + myxothiozol ( Rot/Myx ). C, cellular ATP levels in control-shRNA and ChChd3-shRNA HeLa cells were measured by luminescence-based CellTiter-Glo® cell viability assay. ATP levels were normalized to the protein levels. ChChd3-shRNA1 and shRNA2 represent two different clones derived from a single shRNA sequence, Error bars represent standard deviation from triplicate samples. p < 0.01.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: shRNA, Viability Assay, Clone Assay, Derivative Assay, Sequencing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Loss of ChChd3 results in crista remodeling and perinuclear clumping and fragmentation of mitochondria. A, EM analysis of control-shRNA and ChChd3-shRNA HeLa cells showed that mitochondria in ChChd3-shRNA cells cluster around the nucleus. Altered cristae and an increase in autophagy/mitophagy were also prevalent. Panel a, control-shRNA-expressing HeLa cells have mitochondria distributed throughout the cytoplasm. Panel b, in contrast, ChChd3-shRNA cells show clustering of mitochondria around the nucleus. Panel c, mitochondria of control cells have predominantly lamellar crista, whereas the mitochondria in the ChChd3 knockdown cells have lower crista density and more tubular crista ( panel d ). Panel e, common in these cells is mitochondria devoid or nearly devoid of cristae. Panel f, an autophagosome in a ChChd3 knockdown cell engulfing mitochondria. Panel g, multivesicular bodies are seen high in number in ChChd3 knockdown cells. B–I, quantification of mitochondrial abnormalities in ChChd3 knockdown cells from EM analysis of control and ChChd3 knockdown cells. ChChd3 knockdown cells have significantly reduced crista surface area ( B ), higher percentage of mitochondria without crista ( C ), and altered crista ( D ) with no change in number of cristae per mitochondria ( E ). B, bar graph showing the ratio of crista membrane surface area to the OM surface area in control and ChChd3 knockdown cells. OM surface area and the crista membrane surface area from 25 mitochondria each from control and ChChd3 knockdown cells were measured. p < 0.001. C, bar graph showing the percentage of mitochondria completely devoid of cristae measured from 10 different cells as the total number of mitochondria completely devoid of cristae in a cell divided by the total number of mitochondria in the cell. A total of 414 mitochondria in control and 822 mitochondria in ChChd3 knockdown cells were counted. p = 0.0015. D , bar graph showing the percentage of mitochondria with small and rounded cristae. Measured as above in C from 100 mitochondria from control and ChChd3 knockdown cells, 10 were randomly chosen from each cell from the same 10 cells used in C. p = 0.0015. The average number of cristae per mitochondria were counted from the same 10 cells and 100 mitochondria used in C–E. F–I, mitochondria in ChChd3 knockdown cells are fragmented and clustered around the nucleus. F, statistical analysis of the closest distance between the mitochondrial outer membrane and nuclear membrane. 448 mitochondria from five control cells and 339 mitochondria from five ChChd3 knockdown cells were used for measurements. p < 0.01. G, bar graph of the average number of mitochondria per μm 2 in control and ChChd3 knockdown HeLa cells. The number of mitochondria per cell was measured as the number/cytoplasm cross-sectional area for 10 cells each of control and ChChd3 knockdown. There were a total of 414 mitochondria in the control cells and 822 mitochondria in the knockdown cells. p = 0.019. H, bar graph of quantification of mitochondrial volume over total cytoplasmic volume measured from 10 cells each of control and ChChd3 knockdown. I, bar graph of average length of mitochondria measured from the same 10 cells used in H . 165 mitochondria from each control and ChChd3 knockdown were measured. p < 0.001. Means ± S.E. used throughout.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: shRNA, Expressing

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Electron tomography reveals that crista junctions in ChChd3 knockdown mitochondria are smaller than those in control mitochondria. a, 2.4-nm-thick slice through the tomographic volume of a control mitochondrion. Control mitochondria are generally longer than their ChChd3 knockdown counterpart. Two crista junctions are shown ( arrowheads ) with the one at bottom having a very large opening. Scale bar , 200 nm and applies to all panels. b, 2.4-nm thick slice through the tomographic volume of five ChChd3 knockdown mitochondria. These mitochondria are generally smaller and often found close to each other suggesting fission has occurred. The amount of cristae present can differ remarkably (compare L , similar cristae complement to control mitochondria, with M , tiny mitochondrion devoid of cristae), and the matrix density can also differ substantially (compare M with R ). Their crista junction openings are characteristically smaller ( arrowheads ). c and d, side views of a segmented and surface-rendered inner membrane from a control mitochondrial volume with numbered crista junctions. Six crista junctions are shown on one side and nine on the other side. e and f, side views of a segmented and surface-rendered inner membrane from a ChChd3 knockdown mitochondrial volume showing smaller crista junctions ( numbered ) than the control. Six crista junctions are shown on one side and four on the other side. g, crista junction openings in ChChd3 knockdown mitochondria are about half the size of control openings. The mean crista junction width at its largest opening in tomographic reconstructions is compared. The number of measurements is shown above each bar. Error bar, S.E. (**, p < 0.01). h, density of crista junctions is no different in control and ChChd3 knockdown mitochondria. The total number of crista junctions per mitochondrial volume was counted and divided by the mitochondrial surface area derived from the tomographic volume to determine the crista junction density. Error bar, S.E.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Tomography, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: ChChd3 interacts with Mitofilin, Sam50, HSP70, and OPA1. A–C, FLAG-tagged ChChd3 ( A ), mitofilin ( B ), and Sam50 ( C ) were transiently expressed in HEK293 cells and immunoprecipitated ( IP ) with FLAG resin. Eluted samples were analyzed on SDS-PAGE followed by immunoblot (IB) with the indicated antibodies. D, ChChd3 binds to mitofilin through the chch domain and to Sam50 through N-terminal myristoylation or myristoylated confirmation. E–H, ChChd3 and mitofilin preferentially interact with the shorter isoform of OPA1 in HEK293 cells ( E–G ) and in mitochondria ( H ). FLAG-tagged full-length WT ChChd3 ( E ) or FLAG-tagged mitofilin ( F ) or ChChd3 mutant proteins ( G ) were expressed in HEK293 cells and immunoprecipitated with FLAG resin. Sam50 levels are not shown in the input as the antibodies available for Sam50 failed to detect endogenous protein in total cell lysates of HEK293 cells. H, ChChd3 protein, immunoprecipitated from mouse liver mitochondria by using ChChd3 antibody, binds efficiently with mitofilin and the soluble IMS isoform of OPA1. 5% of the total input is shown throughout. I, proposed model for localization of ChChd3 in mitochondria. ChChd3 is synthesized in the cytoplasm and kept in a reduced and soluble form with the help of HSP70, before it is imported and folded in the mitochondria. In mitochondria, ChChd3 may exist at two discrete foci: 1) at CJ and (2) at contact sites ( CS ). At the CJs, ChChd3 would form a complex with mitofilin and OPA1, and at the contact sites it would associate with mitofilin and Sam50 thereby influencing the regulation of crista biogenesis and mitochondrial protein import, respectively. MF, mitofilin; IBM, inner boundary membrane.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Mutagenesis, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Depletion of ChChd3 leads to major loss of mitofilin and Sam50 and reduction in specific mitochondrial proteins. A, equal amounts of protein from control and ChChd3 knockdown HeLa cell lysates were analyzed on the immunoblot against the antibodies indicated. Protein load was normalized against tubulin. B, ChChd3 knockdown cells show reduced levels of mitochondrial inner membrane proteins AIF, prohibitin, Cox-IV, and Cox-II and the outer membrane protein VDAC. Cytoplasmic and mitochondrial fractions from control and ChChd3 knockdown cells were separated as described under “Experimental Procedures,” and equal amounts of protein (15 μg) from each of these fractions were analyzed on SDS-PAGE followed by Western blotting. HSP90 and MFN1 were used as loading controls for cytoplasmic and mitochondrial fractions, respectively. ChChd3-shRNA1 and shRNA2 represent two different clones derived from a single shRNA sequence.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Western Blot, SDS Page, Clone Assay, Derivative Assay, shRNA, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Loss of ChChd3 results in reduced cellular proliferation. A, rate of cellular proliferation in control and ChChd3 knockdown cells measured by plating an equal number of cells and counting them periodically for 4 days. Error bars represent standard deviation from triplicate samples. B, reduced growth rate is not due to the increased apoptosis or sensitivity toward apoptosis in ChChd3 knockdown cells. Control and ChChd3 knockdown cells were treated with staurosporine to induce apoptosis, and the cells were lysed after the indicated time periods and analyzed for the changes in poly(ADP-ribose) polymerase ( PARP ) cleavage. C, ChChd3 knockdown cells show reduced p70 S6 kinase ( S6k ) and phospho-p70 S6 kinase protein levels. Equal amounts of protein from the control and ChChd3 knockdown cells were analyzed for p70 S6 kinase and phospho-p70 S6 kinase. D, ChChd3 knockdown cells show elevated autophagy. Equal amounts of protein from the total cell lysates of control and ChChd3 knockdown cells were separated on a SDS-PAGE and immunoblotted against LC3 antibody. Tubulin is used as the loading control.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Standard Deviation, SDS Page

Journal:

Article Title: gp-91 mediates histone deacetylase inhibition-induced cardioprotection

doi: 10.1016/j.bbamcr.2010.04.007

Figure Lengend Snippet: Western blot analysis of gp-91 in H9c2 cardiomyoblasts transfected with or without gp-91 siRNA. (A): H9c2 cardiomyoblast were transfected with gp-91 (500 nmol/L) or scrambled siRNA (500 nmol/L) for 24 h to prepare protein lysates and following immunoblotting assay. (B) The LDH release of H9c2 myocytes in culture medium at the end of reperfusion (n=4/group). (C): Cell viability was measured using the MTT cell viability assay (n=4/group). H9c2 cardiomyoblast cells were exposed to simulated ischemia (KCN and 2-deoxy-D-glucose) and description of simulated ischemia is provided under METHODS. HDAC inhibitor, trichostatin A (50 nmol/L) was maintained in culture medium. Gp-91 and negative control siRNA transfections are described as under METHODS. SI: simulated ischemia. LDH: lactate dehydrogenase. Results are expressed as means ± SE and the values expressed as percentages of control values. *P < 0.05.

Article Snippet: Gp-91 polyclonal rabbit antibody, β-actin, gp-91 siRNA and negative control scrambled siRNA were purchased from Santa Cruz Biotechnology Inc.

Techniques: Western Blot, Transfection, Viability Assay, Negative Control, Control

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: RNA sequencing reveals that tomatidine modulates ATF4-dependent ER stress genes in pancreatic cancer cells Human and murine PDAC cell lines were treated for 40 h with 6.4 μg/mL tomatidine, RNA was isolated, and RNA sequencing was performed to analyze differences in gene regulation. N = 3 biological separate experiments. (A–D) (A) Heatmap of all genes and how they change in tomatidine-treated vs. untreated cells for Panc1 cells (FDR<0.05). Volcano plot highlighting ER stress-related genes in (B) Panc1 and (C) MT5 cells. (Upregulated genes on the right of the central axis and vice versa with higher fold change as we go away from the origin on y axis) (D) IPA upstream analysis of ATF4-related genes in Panc1 cells. (E) Heatmap elucidating targeting of ATF4-related genes in treated vs. untreated Panc1 cells. (Fold change: +2.5 to −1.5; FDR<0.03). (F) Top 10 pathways focusing on UPR, ATF4, and ER stress via Reactome analysis of the RNA-sequencing data of treated vs. untreated samples for Panc1 cells. (FDR< 0.03).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: RNA Sequencing, Isolation

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: ATF4 expression in PDAC (A) KM plotter was utilized to examine ATF4 expression in human PDAC specimens examining high vs. low expression compared to overall survival. (B) Data distribution elucidating significance of the overall survival curve prepared by KM Plotter. (C and D) (C) scRNA-seq datasets of pancreatic cancer tissue from metastatic patients were obtained from NIH dbGAP (accession phs002045.v1.p1) and (D) analyzed for ATF4 expression. (See also Table S1 ). (E) Pancreatic tumor tissue from KPC mice were stained by multiplex IF and imaged using Akoya Vectra Polaris and the Phenochart software to analyze ATF4 in multiple cellular compartments. Markers used to determine ATF4 (white), epithelial cells (CK19; red), stroma (αSMA; orange and PDGFRβ; Green), CD8 T+ cells (yellow), and macrophages (F4/80; turquoise). Scale bar = 100 μm.

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Staining, Multiplex Assay, Software

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine inhibits ATF4-dependent signaling in PDAC (A and B) (A) MiaPaca-2 tumor cells were treated with tomatidine for 72 h and cell lysates were immunoblotted for ATF4, 4EBP1, and phospho-4EBP1(p-4EBP1) protein expression and (B) p-4EBP1/4EBP1 levels quantified by densitometry. (C) Immunofluorescence (IF) was performed on vehicle (DMSO) or tomatidine-treated Panc1 cells to track ATF4 (FITC-Green) translocation from nucleus (DAPI-Blue) to cytoplasm. Scale bar = 50 μm. (D–G) (D) Nuclear to cytoplasmic translocation was quantified. Panc1 cells were treated with vehicle (DMSO) or tomatidine for 40 h and ATF4 transcriptional activity was analyzed by chromatin immunoprecipitation (ChIP) qPCR evaluating binding of ATF4 to the downstream promoter regions of (E) eIF4EBP1 (F) CHOP- B site and (G) ASNS. Data are reported as the means + SEMs. n = 3 or more independent biological replicates (4B, One-way ANOVA with Tukey’s test for pairwise comparisons was used to analyze the data; 4D-G, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: In vivo tomatidine treatment inhibits pancreatic tumor growth (A–C) (A) MT5 tumor-bearing C57BL/6 mice (5 mice/group) were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle control (40% HPBCD) and monitored for tumor growth. (n = 5/group). RNA isolated from the tumor tissues of MT5 tumor-bearing C57BL/6 mice treated with vehicle or 5 mg/kg daily i.p. injections of tomatidine were assessed for (B) ATF4 and (C) eIF4EBP1 expression via qPCR. Data are reported as the means + SEMs. n = 5 mice per group. (5A, mixed between-within subjects ANOVA shows a significant interaction between days of treatment and group; 5B-C, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: In Vivo, Control, Isolation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine mediated inhibition of ATF4 signaling can increase sensitivity to ferroptotic cell death in PDAC (A) Panc-1 tumor cells were treated with tomatidine (6.4 μg/mL) and analyzed by RNA sequencing. Ingenuity pathway analysis of the regulated genes suggested ferroptosis as a top hit for tomatidine-treated cells. (B–D) (B) Pancreatic cancer cells were treated with vehicle (DMSO), erastin (to induce ferroptosis), ferrostatin-1 (to inhibit ferroptosis), tomatidine, or in different combinations and lipid peroxidation of (C) Panc-1 and (D) MiaPaca-2 was analyzed by flow cytometry using Bodipy-11. (E) Panc-1 and MiaPaca-2 cells treated with vehicle (DMSO) or tomatidine and lysates collected after 24 h were immunoblot for GPX4 expression. (F) Panc1 cells were plated overnight and treated with tomatidine for 6 h and then assayed by Seahorse assay to analyze mitochondrial fitness. (G) Schematic showing how tomatidine can regulate ATF4-dependent signaling to induce ferroptosis in pancreatic cancer. Data are reported as the means + SEMs. n = 3 or more independent biological replicates. (7C-D, Two-tailed independent student’s test was used to analyze the data; 7F, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05, ∗∗p < 0.007).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Inhibition, RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet:

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Control, Recombinant, Viability Assay, Luciferase, ChIP-qPCR, Gene Expression, Software