Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Steady‐state levels of gastric cancer cell lines. Using Western blot assay, steady protein levels of BRCA1, BRCA2 and c‐MET are analysed in primary gastric cancer cell lines HS746T and AGS. Protein levels were normalized against actin

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Low levels of c‐MET partially sensitize GC cell lines in PARP inhibition. A, HS746T/AGS cells, control‐siRNA‐Hs746T/AGS cells and si‐c‐MET Hs746T/AGS cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of c‐MET expression (by Western blot analysis) revealed down‐regulation of the c‐MET receptor in both cell lines (HS746T and AGS); (B) HS746T cells, control‐siRNA‐Hs746T cells and si‐BRCA1/2 Hs746T cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of BRCA1 and BRCA2 expression (by Western blot analysis) revealed down‐regulation of the BRCA1/2 in HS746T cell line; (C) HS746T cells, control‐siRNA‐Hs746T, siBRCA1/2‐Hs746T and siMET/BRCA1/2‐Hs746T cells were cultured with the indicated concentrations of NU1025 (5, 10 and 20 μmol/L) for 48 h for determination of cell viability (MTT metabolic activity assay). Error bars represent SD

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Inhibition, Control, Metabolic Assay, Expressing, Western Blot, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Co‐inhibition of c‐MET (SU11274) and PARP (NU1025) sensitizes GC cells after knockdown BRCA1/2. Knocking down BRCA1 or BRCA2 sensitizes cells to PARP and c‐MET inhibition in HS746T cells expressing low levels of c‐MET (AGS cells, c‐MET knockdown Hs746T cells) to PARP inhibition. A, HS746T cells, control‐siRNA‐Hs746T cells and siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cells were exposed to 5 µmol/L of NU1025 and/or 5 µmol/L of SU11274 for 48 h for determination of cell viability (MTT metabolic activity assay). Results are expressed as percentages. Average values of three experiments ± SD are shown; (B) Western blot analysis of PARP and cl.caspase‐3 in Hs746T‐control‐siRNA, siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cell lines. Cells were cultured with the indicated drugs (5 μmol/L NU1025, 5 μmol/L SU11274 alone or in combination for 24 h of treatment). Protein levels were normalized against actin

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Inhibition, Knockdown, Expressing, Control, Metabolic Assay, Western Blot, Cell Culture

Journal: Molecular cell

Article Title: A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.

doi: 10.1016/j.molcel.2013.01.001

Figure Lengend Snippet: Figure 1. BRCA1 Accumulation at DSB Sites Is Suppressed by 53BP1 in G1 (A) Schematic representation of the Fucci system. (B) Strategy for isolation of G1 and S/G2 cells by fluorescence-activated cell sorting (FACS) of Fucci cells. See Figure S1 for details. (C) BRCA1 is expressed in the G1 and S/G2 pha- ses of cycling cells. Whole-cell extracts were prepared from sorted Fucci cells and probed for BRCA1, cyclin A (S/G2 marker), and tubulin (loading control) by immunoblotting. (D) 53BP1 inhibits BRCA1 foci in G1. HeLa-Fucci cells were first transfected with a nontargeting siRNA (siCTRL) or an siRNA targeting 53BP1 (si53BP1) and then processed for BRCA1 immunofluorescence 1 hr postirradiation (10 Gy). Indicated is the phase of the cell cycle. Scale bar = 5 mm. (E) Quantitation of BRCA1 and g-H2AX foci ac- cording to cell cycle position. HeLa-Fucci cells were transfected with the indicated siRNAs and irradiated with a 10 Gy IR dose. Cells were then fixed and processed for BRCA1 and g-H2AX immunofluorescence. Shown is the quantitation of IR-induced foci in G1 and S/G2 cells. Data are represented as the mean ± SEM (n = 3). (F) 53BP1 inhibits BRCA1 G1 foci in multiple cell lines. U2OS and RPE1 cells were transfected with the indicated siRNAs, fixed 1 hr postirradiation (10 Gy), and processed for BRCA1 and cyclin A immunofluorescence. Shown in the left panel is the quantitation of foci in the cyclin A-negative cells. Data are represented as the mean ± SEM (n = 2). In the right panel are representative micrographs. Scale bar = 5 mm. (G) Top: schematic representation of 53BP1. Bottom: HeLa-Fucci cells were first transfected with a single siRNA targeting 53BP1. After trans- fection, cells were transfected either with an empty vector control (ctrl) or the indicated siRNA- resistant 53BP1 expression vectors. Cells were irradiated (10 Gy dose) and processed for BRCA1 immunofluorescence. Data are represented as the mean ± SEM (n > 3). Representative micrographs are shown in Figure S2.

Article Snippet: The murine retroviral shRNA vectors for 53BP1 (TF515706) and RIF1 (TF502862), as well as the scrambled shRNA vector, were purchased from Origene.

Techniques: Isolation, FACS, Marker, Control, Western Blot, Transfection, Quantitation Assay, Irradiation, Plasmid Preparation, Expressing

Journal: Molecular cell

Article Title: A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.

doi: 10.1016/j.molcel.2013.01.001

Figure Lengend Snippet: Figure 2. RIF1 Is an Effector of 53BP1 in the Inhibition of BRCA1 in G1 (A) RIF1 inhibits BRCA1 foci in G1. HeLa-Fucci cells were transfected with the indicated siRNAs. At 1 hr postirradiation (10 Gy), cells were pro- cessed for BRCA1 immunofluorescence. Shown is the quantitation of BRCA1 IR-induced foci in G1 cells. Data are represented as the mean ± SEM (n = 3). Representative micrographs are shown in Figure S3C. (B) Immunoblot analysis of the experiment shown in (A). (C) U2OS cells were transfected with the indicated siRNAs. Cells were transfected with either a GFP (control) or an siRNA-resistant GFP-RIF1 expres- sion vector 24 hr later. At 1 hr postirradiation (10 Gy), cells were processed for BRCA1 and cyclin A immunofluorescence. Shown is the quantitation of BRCA1 foci in the cyclin A-negative (i.e., G1) cells. Data are represented as the mean ± SEM (n = 3). Representative micrographs are shown in Figure S3D. (D) U2OS cells were transfected with si53BP1 #1 and either an empty vector control (ctrl) or the indicated siRNA-resistant 53BP1 expression vectors. At 1 hr postirradiation (10 Gy), cells were processed for RIF1 immunofluorescence. Shown are representative micrographs. Scale bar = 5 mm. (E) Quantitation of the experiments shown in (D). Data are represented as the mean ± SEM (n = 3). (F) ATM inhibits RIF1 foci. U2OS cells were treated either with DMSO or various concentrations of the ATM inhibitor KU-55933 prior to irradiation. At 1 hr postirradiation (10 Gy), cells were processed for 53BP1 and RIF1 immunofluorescence. Data are represented as the mean ± SEM (n = 3). (G) U2OS cells were transfected with the indicated siRNAs. Cells were irradiated (10 Gy) at 48 hr and processed for 53BP1 immunofluorescence 1 hr postirradiation. Data are represented as the mean ± SEM (n = 3). Representative micrographs are shown in Figure S3E.

Article Snippet: The murine retroviral shRNA vectors for 53BP1 (TF515706) and RIF1 (TF502862), as well as the scrambled shRNA vector, were purchased from Origene.

Techniques: Inhibition, Transfection, Quantitation Assay, Western Blot, Control, Plasmid Preparation, Expressing, Irradiation

Journal: Nature communications

Article Title: S100A9-CXCL12 activation in BRCA1-mutant breast cancer promotes an immunosuppressive microenvironment associated with resistance to immunotherapy.

doi: 10.1038/s41467-022-29151-5

Figure Lengend Snippet: Fig. 3 S100a9 gene is regulated by both Brca1 and S100a9. a Expression of S100a9 and S100a8 in Brca1 MT (G600) and WT (B477) mammary epithelial cell lines. b Expression of S100a9 and S100a8 in B477 cells with the expression of shBrca1 at different concentrations. c Expression of S100a9 and S100a8 in B477 cells in which the mBrca1 gene was overexpressed. d Expression of S100A9 and S100A8 in MDA-MB-231 (231) control and 231 cells with the expression of shBRCA1(knock down BRCA1). e Luciferase activity assay of mouse S100a9 promoter after 72 h transfection with PGL vector only, S100a9 promotor, S100a9 promotor with Brca1 cDNA, and S100a9 promotor with S100a9 cDNA in B477 and G600 cells. f S100A9 proteins in B477 mouse mammary epithelial cells and 231 cells with the expression of shBrca1 or shBRCA1, respectively by IF. g BRCA1 and S100A9 protein levels in B477 mouse WT mammary epithelial cells and 231 cells without or with the expression of shBRCA1(shBr/shBR). The data are expressed as means ± SD (a–e) and P values determined by unpaired two-tailed Student’s t test (a, c, d) and by one-way ANOVA followed by Tukey’s multiple comparisons (b) or two-way ANOVA (e). The experiments were independently repeated three times with similar results (a–g). Scale bar: white color, 20 μM. Source data are provided as a Source data file.

Article Snippet: Mouse Brca1 cDNA (Cat# MG212086) was purchased from Origene.

Techniques: Expressing, Control, Knockdown, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Nature communications

Article Title: S100A9-CXCL12 activation in BRCA1-mutant breast cancer promotes an immunosuppressive microenvironment associated with resistance to immunotherapy.

doi: 10.1038/s41467-022-29151-5

Figure Lengend Snippet: Fig. 4 Tumor permissive microenvironment in Brca1 MT mammary glands. a S100a9/S100a8 mRNA expression in the subpopulations of luminal and stromal cells of WT 4-month mammary gland (WTV4MG) and MT 4-months-old virgin mammary gland (MTV4MG) (n = 3 mice). b Protein level of S100a9 in both WT (B477) and MT (G600) mammary epithelial cell lines and tumor tissues by Western blots (n = 3 individual experiment-up and n = 3 mice-down). c Co-staining of S100a9 (red) and CK18 (green) with antibodies on WTV4MG, MTV4MG, WTV6MG, and MTV6MG tissues (n = 3 pairs in each group, Scale bar: 20 μM). d The S100a9 and Arg1 positive cell populations by FACS analysis from the blood and mammary tissues of both WT and MT mice at 4-month and 6-month, respectively (FACS gating strategies see in Supplementary Fig. 8c, n = 3 mice/ group). e Co-staining with S100a9 (red) and CD206 (green) antibodies (left panel) and co-staining with S100a9 (red) and CK18 (green) antibodies (right panel) on tumor-adjacent tissues by IF (40X confocal microscope, Scale bar: 20 μM.) (n = 3 mice and 3 individual experiment). f Secreted S100a9 proteins (left) from both tumor cell and MDSC cells in tumor-adjacent mammary gland (n = 3 mice) and present in the supernatant of cultured cancer cells (right) (n = 3 individual experiment, Scale bar: 10 μM). g Protein levels of S100a9, TGF-β, and Il-10 in mammary gland tissues of both WT and Brca1 MT mice at 4-month (n = 3 mice). h Protein levels of S100a9, TGF-β, and IL-10 in mammary tissues of both WT and Brca1 MT mice at 6-month (n = 3 mice). The data are expressed as means ± SD (a) and P values determined by unpaired two-tailed Student’s t test. The experiments were independently repeated three times with similar results (a, b). Source data are provided as a Source data file.

Article Snippet: Mouse Brca1 cDNA (Cat# MG212086) was purchased from Origene.

Techniques: Expressing, Western Blot, Staining, Microscopy, Cell Culture, Two Tailed Test

Journal: Nature communications

Article Title: S100A9-CXCL12 activation in BRCA1-mutant breast cancer promotes an immunosuppressive microenvironment associated with resistance to immunotherapy.

doi: 10.1038/s41467-022-29151-5

Figure Lengend Snippet: Fig. 8 Schematic for the mechanisms by which BRCA1 deficiency in breast cancer formation and improves responses to anti–PD1 antibody. In BRCA1 deficiency epithelial cells, S100A9 expression level constantly increases in early stages and secrets out to recruit and activate MDSCs, which creates an immunosuppression microenvironment by inhibit expansion and activation of T cells. This process can be further enhanced by CXCL12 that positively regulated by S100A9 and form a positive feedback loop. And this immunosuppression microenvironment is beneficial for tumor growth. The inhibitors of S100A9 and CXCL12, Tasquinimod and AMD3465, combine with anti-PD1 antibody can rescue the immunosuppression microenvironment and repress tumor growth. Green arrows indicate a decrease; red arrows indicate an increase.

Article Snippet: Mouse Brca1 cDNA (Cat# MG212086) was purchased from Origene.

Techniques: Expressing, Activation Assay

Journal: Biology of reproduction

Article Title: Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells.

doi: 10.1095/biolreprod.107.063107

Figure Lengend Snippet: FIG. 1. Purification and functional char- acterization of dimeric TRF. A) Chromato- graphic profile of highly purified human TRF. The TRF solution was loaded onto an ultragel Aca44 column as described in Materials and Methods. OD, optical density. B) Each fraction was analyzed using native gel electrophoresis and immunoblotting. VLMB, very low-mobility band; LMB, low- mobility band; HMB, high-mobility band. C) Sensorgrams of interaction between high- and low-mobility purified fractions of hTRF and the monoclonal anti-hTRF anti- body. The interaction between both forms of hTRF and the monoclonal antibody is indicated by a dashed line. After reinjection of the monoclonal antibody, only the accessible epitope on the dimeric form generated a secondary signal (dark line), in contrast to the monomeric form (light line). The arrows directed to the top indicate the beginning of injection of the anti-hTRF antibody, and those directed toward the bottom indicate the end of the injections. The principle of discrimination between monomeric versus dimeric TRF by SPR is schematized on the right. RU, resonance units.

Article Snippet: Monoclonal mouse antibodies raised against the N-terminal domain of human TRF were from AbD Serotec (Cergy Saint-Christophe, France).

Techniques: Purification, Functional Assay, Nucleic Acid Electrophoresis, Western Blot, Generated, Injection