Journal: British journal of pharmacology

Article Title: BBIT20 inhibits homologous DNA repair with disruption of the BRCA1–BARD1 interaction in breast and ovarian cancer

doi: 10.1111/bph.15506

Figure Lengend Snippet: Antiproliferative effect of BBIT20, cisplatin and olaparib, in a panel of human breast cancer, ovarian cancer and non-malignant cells

Article Snippet: A total of 2.5 × 10 6 IGROV-1 cells (expressing heterozygous mutBRCA1) were implanted subcutaneously (in PBS/Matrigel 1:1; Corning, Enzifarma, Porto, Portugal) in the right flank of female mice with 6 to 8 weeks.

Techniques:

Journal: Cell Metabolism

Article Title: PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers

doi: 10.1016/j.cmet.2018.09.002

Figure Lengend Snippet: High-OXPHOS Ovarian Cancer Cells Rely on the TCA Cycle (A) Representative WB of five ETC proteins in OCCLs. Actin is internal control. OXPHOS status was defined from quantification in (B). (B) Mean of five ETC protein levels quantified from WB as in (A) and normalized to actin. Data are means ± SEM (n = 3 independent experiments). p value from Student’s t test. (C) Representative electron microscopy (e.m.) pictures from OCCLs. Scale bars, 0.5 μm and 0.25 μm (low and high magnification). (D) Scatterplot showing mitochondrial area per unit of cell surface (in μm 2 ) assessed using e.m. of OCCLs listed in (A). Data are means ± SEM (n ≥ 8 e.m. pictures per cell line). p value from Student’s t test. (E) Specific MFI of Mitotracker Deep Red dye in low- (IGROV1, SKOV3, OVCAR8) and high- (OC314, CAOV3, OVCAR4) OXPHOS OCCLs. Data are means ± SEM (n = 3 independent experiments per cell line). p values from Student’s t test. (F) Representative OCR pattern as a function of time (in min), normalized to total protein levels. Oligomycin (Oligo), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone (Rot), and antimycin A (Anti A) were added to measure basal OCR, ATP content, maximal OCR, and non-mitochondrial OCR. N = 14 OCCLs listed in (A). Data are means of four replicates per cell line. (G and H) Basal or maximal OCR (G) and mitochondrial ATP content (H) normalized to total protein levels. N = 14 OCCLs listed in (A). Data are means ± SEM (n ≥ 3 independent experiments). p values from Student’s t test. (I) Correlations between basal (left) or maximal (right) OCR and mean of ETC protein levels. p values are from Spearman test. (J and K) Basal EACR (J) and OCR (K) in presence of 10 mM glucose (Glc) or 2 mM glutamine (Gln), in control conditions (no Glc no Gln or in presence of both 10 mM Glc and 2 mM Gln) in low- (IGROV1, SKOV3, OVCAR8, OV7) and high- (OC314, CAOV3, OVCAR4) OXPHOS OCCLs. Each dot is the mean value for each cell line (n ≥ 3 independent experiments). Bar plots show means ± SEM for each OXPHOS subgroup. p values from Student’s t test. (L) Consumption of [ 13 C]-glutamine (left) or [ 13 C]-glucose (right) in low- (IGROV1) and high- (OC314) OXPHOS cells after 24 hr of incubation in the corresponding medium. Data are means ± SEM (n = 3 replicates per cell line). (M) Schematic representation of [ 13 C]-glutamine- (purple dots) or [ 13 C]-glucose-derived carbons (green dots). Bar plots show distribution of isotopologues (M0 to M6 according to labeled carbons) for each metabolite in low- (IGROV1, L) and high- (OC314, H) OXPHOS cells after 24 hr of incubation with -Glc- (10 mM 13 C-glucose + 2 mM glutamine) or -Gln- (2 mM 13 C-glutamine + 10 mM glucose). Data are shown as means (n = 3 replicates per cell line). (N) Percentage (%) of OCR inhibition 30 min after etomoxir treatment (40 μM) in presence of 10 mM glucose and 2 mM glutamine in low- (IGROV1, SKOV3, OVCAR8, OV7) and high- (OC314, CAOV3, OVCAR4) OXPHOS OCCLs. Data are means ± SEM (n ≥ 3 independent experiments). p values are from Student’s t test. AcCoA, acetyl coenzyme A; aKG, alpha ketoglutarate; CID, carbon isotopologue distribution; Cit, citrate; Glc, glucose; G6P, glucose 6-phosphate; Glu, glutamate; Gln, glutamine; Mal, malate; Pyr, pyruvate; Suc, succinate; 3PG, 3-phosphoglycerate. See also Figure S2 .

Article Snippet: IGROV1 , D. Lallemand and J.S. Brugge Lab , N/A.

Techniques: Control, Electron Microscopy, Incubation, Derivative Assay, Labeling, Inhibition

Journal: Cell Metabolism

Article Title: PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers

doi: 10.1016/j.cmet.2018.09.002

Figure Lengend Snippet: High-OXPHOS HGSOCs Exhibit Features of Oxidative Stress (A) Left: PCA on ETC protein levels (N = 127 HGSOCs; low-OXPHOS, blue, N = 53; high-OXPHOS, red, N = 74). Right: same PCA representation showing HRD (high-LST, green, N = 37) and HRP (low-LST, blue, N = 18) HGSOCs. Unavailable data (NA) are in gray. Bottom: contingency table showing the repartition of low- and high-OXPHOS HGSOCs in HRP and HRD subgroups. p values from Fisher’s exact test. (B) Same as in (A) on TCGA data (N = 169 HGSOCs; low-OXPHOS = 90; high-OXPHOS = 79; HRP = 68; HRD = 97). (C) Gene set enrichment analysis (GSEA) of NFE2L2-target genes in high-OXPHOS HGSOCs (top, Curie; bottom, TCGA). p value from false discovery rate (FDR). (D) Levels of antioxidant enzymes in HGSOCs (top, Curie; bottom, TCGA). Proteomic data are normalized as in Figure 1 E. Medians are shown. p values from Mann-Whitney test. (E) Carbonylation scores (carbonylated/total protein levels) in HGSOCs. N = 40. Medians are shown. p values from Mann-Whitney test. (F) sn-2 lysophospholipid abundance (metabolomics data) in HGSOCs. N = 45. p values from Mann-Whitney test. (G) Same as in (F) for gamma-glutamyl intermediates from metabolomic data. N = 45. p values from Student’s t test. (H) Specific MFI using CellROX probe in low- (IGROV1, SKOV3, OVCAR8) and high- (OC314, CAOV3, OVCAR4) OXPHOS OCCLs. Data are means ± SEM (n ≥ 3 independent experiments). p values from Student’s t test. (I) Left: same as in (H) using Bodipy C11 probe. Right: representative views of Bodipy C11 immunofluorescence (IF). (J) Same as in (H) using RhoNox-M (RhoM) probe normalized to the lysosomal content, assessed by lystrocker probe. (K) ETC mRNA levels (ATP5A, UQCRC2, SDHB, COXII, and NDUFB8) in high-OXPHOS OCCLs (CAOV3, OC314, and OVCAR4) treated (red dotted bar) or not (red empty bar) with NAC (5 mM) during 48 hr. Data (fold change normalized to untreated) are means ± SEM (n = 3 independent experiments). p values from one-sample t test. (L) Basal OCR in high-OXPHOS cells (CAOV3, OC314, OVCAR4) upon NAC treatment (5 mM, 48 hr), normalized to the mean of untreated condition for each cell line. Data are means ± SEM (n = 3 independent experiments). p values from Student’s t test. See also Figure S3 and and .

Article Snippet: IGROV1 , D. Lallemand and J.S. Brugge Lab , N/A.

Techniques: MANN-WHITNEY, Immunofluorescence

Journal: Cell Metabolism

Article Title: PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers

doi: 10.1016/j.cmet.2018.09.002

Figure Lengend Snippet: PML Is a Key Actor in High-OXPHOS Ovarian Cancer (A) Representative views of PML IHC in HGSOCs, with PML-NBs (arrows). Scale bars, 50 μm and 10 μm (low and high magnification). (B) PML Hscore (left) and number of PML-NBs per cell (right) in HGSOCs. N = 71. Medians are shown. p values from Mann-Whitney test. (C) PML protein levels in HGSOCs from TCGA cohort (N = 169). Medians are shown. p values from Mann-Whitney test. (D) Representative views of PML IF in OCCLs, with PML-NB (high magnification). Scale bars, 50 μm and 10 μm (low and high magnification). (E) Number of PML-NBs per cell in low- (IGROV1, SKOV3, OVCAR8) and high- (CAOV3, OC314, OVCAR4) OXPHOS OCCLs. Data are means ± SEM (n = 3 independent experiments). p values from Student’s t test. (F) Representative pattern of OCR as a function of time (min) normalized to total protein levels. High-OXPHOS OCCLs (CAOV3, OC314, and OVCAR4) were transfected with non-targeting small interfering RNA (siRNA) (siCTRL) or with two different siRNA targeting PML (siPML#1 and siPML#2). (G) Basal (left) and maximal (right) OCR following PML silencing in low- (IGROV1, SKOV3, OVCAR8) and high- (OC314, CAOV3, OVCAR4) OXPHOS OCCLs. Each dot is the mean value for each cell line (n ≥ 3 independent experiments). Bar plots show means ± SEM for each OXPHOS subgroup. p values from paired t test. (H) ETC mRNA levels (ATP5A, UQCRC2, SDHB, COXII, and NDUFB8) in high-OXPHOS OCCLs (CAOV3, OC314, and OVCAR4) transfected with non-targeting siRNA (siCTRL) or with two siRNA targeting PML (siPML#1 and #2). Data (fold change normalized to non-targeting siRNA) are means ± SEM (n ≥ 3 independent experiments). p values from one-sample t test. (I) Basal OCR in high-OXPHOS OCCLs (CAOV3, OC314, and OVCAR4) transfected with shCTRL, shPML#1 or shPML#2 in presence of 10 mM glucose and 2 mM glutamine (left), 10 mM glucose (middle), or 2 mM glutamine (right). Each dot is the mean value for each cell line (n = 3 independent experiments). Bar plots show means ± SEM of the three cell lines per condition. p values from Student’s t test. (J) Same as in (I). Bar plot shows percent (%) of OCR inhibition, 30 min after etomoxir treatment (40 μM) in presence of 10 mM glucose and 2 mM glutamine. (K) Representative views of PML IF in high-OXPHOS OCCLs (CAOV3) following NAC treatment (5 mM, 48 hr). High-magnification views show PML-NBs. Scale bars, 50 μm and 10 μm (low and high magnification). (L) Representative WB showing acetylated PGC-1α after PGC-1α immunoprecipitation (top) and total PGC-1α protein (bottom) from high-OXPHOS OCCLs (CAOV3, OC314, and OVCAR4) transfected with siCTRL, siPML#1, or siPML#2. (M) Ratio of acetylated PGC-1α to total PGC-1α protein levels upon PML silencing, as shown in (L). Data are means ± SEM of fold changes normalized to siCTRL (n = 3 independent experiments). p values from one sample t test. (N) Basal OCR following PGC-1α silencing (siPGC-1α#1 and #2) normalized to siCTRL in high-OXPHOS OCCL (CAOV3, OC314, and OVCAR4). Data are means ± SEM (n = 3 independent experiments). p values from one-sample t test. (O) As in (H) after transfection with siCTRL, siPGC-1α#1, or siPGC-1α#2. Data (fold change normalized to non-targeting siRNA) are means ± SEM (n = 3 independent experiments). p values are from one-sample t test. (P) Basal OCR in low-OXPHOS cells (IGROV1, SKOV3, and OVCAR8) upon rosiglitazone treatment (10 μM, 48 hr) normalized to the mean of untreated condition for each cell line. Data are means ± SEM (n = 2 independent experiments). p values from one-sample t test. (Q) As in (H) in cells in low-OXPHOS OCCLs (IGROV1, SKOV3, and OVCAR8) treated (stripped bar) or not (empty bar) with rosiglitazone (10 μM, 48 hr). Data (fold change normalized to untreated) are means ± SEM (n = 2 independent experiments). p values from one-sample t test. See also Figure S4 .

Article Snippet: IGROV1 , D. Lallemand and J.S. Brugge Lab , N/A.

Techniques: MANN-WHITNEY, Transfection, Small Interfering RNA, Inhibition, Immunoprecipitation

Journal: Cell Metabolism

Article Title: PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers

doi: 10.1016/j.cmet.2018.09.002

Figure Lengend Snippet:

Article Snippet: IGROV1 , D. Lallemand and J.S. Brugge Lab , N/A.

Techniques: Virus, Derivative Assay, Recombinant, Protease Inhibitor, Western Blot, Staining, Membrane, Electron Microscopy, SYBR Green Assay, Bicinchoninic Acid Protein Assay, DNA Profiling, Reverse Transcription, Software, Imaging, Cell Culture