antibodies against total poly (adp-ribose) polymerase (total parp)  (Cell Signaling Technology Inc)

Journal: Experimental and Therapeutic Medicine

Article Title: MiR‑221 and miR‑222 regulate cell cycle progression and affect chemosensitivity in breast cancer by targeting ANXA3

doi: 10.3892/etm.2023.11826

Figure Lengend Snippet: Expression of ANXA3 is associated with chemotherapy sensitivity. (A) Transfection efficiency was assessed according to siANXA3 concentration. (B) Cell viability was evaluated in MDA-MB-231 cells treated with si-ANXA3 and adriamycin at different doses. (C) Percentage of viable MDA-MB-231 cells. Cells were treated with adriamycin (25 nM), miR-221/222 and siANXA3. (D) Cell cycle profile was analyzed using flow cytometry. Fluorescence-activated cell sorting analysis of cells transfected with control or siANXA3. (E) FACS analysis of cells transfected with adriamycin (50 nM) alone or a combination of adriamycin and siANXA3. (F) Expression of two cell cycle regulatory factors, cyclin A and cyclin B1, were evaluated using western blotting after treatment with adriamycin alone and a combination of adriamycin and siANXA3. (G) Flow cytometry and annexin V-FITC/PI labeling were used to examine apoptosis after treatment with adriamycin, siANXA3 and their combination. (H) Expression of apoptotic factors was evaluated by western blotting after treatment with adriamycin alone and a combination of siANXA3. (I) Overall survival was evaluated according to ANXA3 levels using the Kaplan-Meier Plotter software. * P<0.05, ** P<0.01 and *** P<0.001. ANXA3, annexin A3; FACS, fluorescence-activated cell sorting; Adr, adriamycin; SRC, scrambled negative control; siANXA3, small-interfering RNA targeting ANXA3; siCTL, small-interfering RNA negative control; Asy, asynchronous; PARP, poly (ADP-ribose) polymerase; anti-miR-CTL, anti-miR 221/222 negative control; miR, microRNA.

Article Snippet: The primary antibodies were: Anti-annexin A3 (1:1,000; cat. no. ab33068; Abcam); anti-cyclin D1 (1:500; cat. no. sc-20044; Santa Cruz Biotechnology, Inc.); anti-CDK4 (1:500; Santa Cruz Biotechnology, Inc.; cat. no. sc-56277); anti-cyclin B1 (1:500; cat. no. sc-245; Santa Cruz Biotechnology, Inc.); anti-cyclin A (1:500; cat. no. sc-274682; Santa Cruz Biotechnology, Inc.); anti-α tubulin (1:1,000; cat. no. sc-5286; Santa Cruz Biotechnology, Inc.); anti-β-Actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.); apoptosis western blot cocktail (1:250; cat. no. ab136812; Abcam); and total poly ADP ribose polymerase (1:1,000; cat. no. 9542; Cell Signaling Technology, Inc.).

Techniques: Expressing, Transfection, Concentration Assay, Flow Cytometry, Fluorescence, FACS, Western Blot, Labeling, Software, Negative Control, Small Interfering RNA

Journal: Advanced Science

Article Title: A CD10‐OGP Membrane Peptolytic Signaling Axis in Fibroblasts Regulates Lipid Metabolism of Cancer Stem Cells via SCD1

doi: 10.1002/advs.202101848

Figure Lengend Snippet: CD10 in CAFs sustains cancer stemness and chemoresistance. A–E) Indicated tumor cell lines were cultured alone (−) or cocultured with CD10 + GPR77 + ‐depleted CAFs (CD10 + GPR77 + ‐d) or paired CD10 + GPR77 + CAFs transduced without (−) or with shGFP or shCD10. A) Representative images of mammosphere formation in MCF‐7 cells. Scale bar, 100 µm. B) Representative images of PKH26 and Numb immunofluorescence staining of MCF‐7 cells. Scale bar, 50 µm. C,D) Percentage of ALDH1 + cells in C) MCF‐7 and D) BT‐549 cells detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). E) Percentage of CD44 high CD24 low cells in MCF‐7 cells cocultured with indicated CAFs was detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). F,G) Representative flow cytometry plots for F) MCF‐7 and G) BT‐549 cells treated with docetaxel after being cultured alone (−) or cocultured with the indicated CAFs. The proportion of Annexin V + /Propidium iodide − (early apoptosis) and Annexin V + /Propidium iodide + (late apoptosis) cells is shown. The numerical values indicate Annexin V + percentage. Data are represented as the mean ± SEM of F) n = 4 or G) n = 3 independent experiments. H) Representative immunoblots for cleaved/total caspase‐3 and PARP in SK‐BR3 cells treated with docetaxel after being cultured alone (−) or cocultured with indicated CAFs ( n = 3). I–L) MCF‐7 cells were cocultured with CD10 + GPR77 + CAFs or CD10 + GPR77 + ‐depleted CAFs transduced with lentiviral empty vectors (vector) or CD10‐expressing vectors (CD10 overexpression). I) Representative images of mammosphere formation. Scale bar, 100 µm. J) Percentage of ALDH1 + cells detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). K) Pcercentage of CD44 high CD24 low cells detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). L) After the coculture, MCF‐7 cells were treated with docetaxel. The proportion of Annexin V + /Propidium iodide − and Annexin V + /Propidium iodide + was detected by flow cytometry. The numerical values indicate Annexin V + percentage (mean ± SEM, n = 3).

Article Snippet: The nitrocellulose membranes were blocked and incubated with anti‐caspase‐3 (Cat# 9662, Cell Signaling Technology, 1:1000), anti‐cleaved caspase‐3 (Cat# 9664, Cell Signaling Technology, 1:1000), anti‐total poly (ADP‐ribose) polymerase (PARP) and anti‐cleaved PARP (Cat# 9542, Cell Signaling Technology, 1:1000), CD10 (Cat# ab73409, Abcam, 1:1000), GPR77 (Cat# 342 402, Biolegend), SCD1 (Cat# 2794, Cell Signaling Technology, 1:1000), S6 Ribosomal Protein (Cat# 2317, Cell Signaling Technology, 1:1000), phospho‐S6 Ribosomal Protein (Ser240/244) (Cat# 5364, Cell Signaling Technology, 1:1000), p70 S6 Kinase (Cat# 2708, Cell Signaling Technology, 1:1000), phospho‐p70 S6 Kinase (Thr389) (Cat# 9234, Cell Signaling Technology, 1:1000), I κ B α (Cat# 4814, Cell Signaling Technology, 1:1000), p‐I κ B α (Ser32) (Cat# 2859, Cell Signaling Technology, 1:1000), IKK β (Cat# 8943, Cell Signaling Technology, 1:1000), p‐IKK α / β (Ser176/180) (Cat# 2697, Cell Signaling Technology, 1:1000), SREBP1 (Cat# sc‐365513, Santa Cruz, 1:1000), FASN (Cat# 3180, Cell Signaling Technology, 1:1000), β ‐catenin (Cat# sc‐7963, Santa Cruz, 1:1000), Lamin B1 (Cat# 12987‐1‐AP, Proteintech, 1:5000), β ‐tubulin (Cat# 10068‐1‐AP, Proteintech, 1:2000), or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; Cat# HRP‐60004, Proteintech, 1:10 000).

Techniques: Cell Culture, Immunofluorescence, Staining, Flow Cytometry, Western Blot, Transduction, Plasmid Preparation, Expressing, Over Expression

Journal: Advanced Science

Article Title: A CD10‐OGP Membrane Peptolytic Signaling Axis in Fibroblasts Regulates Lipid Metabolism of Cancer Stem Cells via SCD1

doi: 10.1002/advs.202101848

Figure Lengend Snippet: CD10 supports CSCs by cleavage of OGP. A,B) Indicated tumor cell lines after prolonged mammosphere culture were treated with 5 × 10 −9 m OGP, OGP (L2A), or OGP (Y10A). Representative images of mammosphere formation in A) BT‐474 mammo cells and B) MCF‐7 mammo cells. Scale bar, 100 µm. C) MCF‐7 and D) BT‐549 cells pretreated with 100 × 10 −9 m biotinylated OGP, due to an undetectable signal of fluorescence‐labeled peptide at a lower concentration, [ ¹²⁴ , ¹²⁵ , ¹²⁶ , ¹²⁷ , ¹²⁸ , ¹²⁹ ] without (−) or with 10 × 10 −6 m non‐biotinylated OGP (L2A) or OGP (Y10A) were incubated with neutravidin‐Texas red. The cell surface‐bound biotinylated OGP was evaluated by fluorescence microscopy. Scale bar, 50 µm. E–J) Indicated cell lines were cultured alone (−) or cocultured with the indicated CAFs in the presence of 500 × 10 −9 m OGP (L2A) or OGP (Y10A). Unlabeled peptides were used at 100 times more than the physiological concentration for the competitive binding assay as previously reported. [ ¹³⁰ , ¹³¹ ] E,F) Percentage of ALDH1 + cells in E) MCF‐7 and F) BT‐549 cell (mean ± SEM, n = 4). G,H) Representative immunofluorescent images of G) PKH26 and H) Numb in MCF‐7 cells. Scale bar, 50 µm. I,J) Representative images of mammosphere formation in I) MCF‐7 and J) BT‐474 cells. Scale bar, 100 µm. K,L) After coculture with (E–J) cancer cells were treated with docetaxel. Apoptosis of tumor cells was determined after 12 h. K) Percentage of apoptotic MCF‐7 and BT‐549 cells evaluated by flow cytometry (mean ± SEM, n = 4). L) Apoptosis of SK‐BR3 cells determined by western blotting for cleaved caspase‐3 and PARP ( n = 3).

Article Snippet: The nitrocellulose membranes were blocked and incubated with anti‐caspase‐3 (Cat# 9662, Cell Signaling Technology, 1:1000), anti‐cleaved caspase‐3 (Cat# 9664, Cell Signaling Technology, 1:1000), anti‐total poly (ADP‐ribose) polymerase (PARP) and anti‐cleaved PARP (Cat# 9542, Cell Signaling Technology, 1:1000), CD10 (Cat# ab73409, Abcam, 1:1000), GPR77 (Cat# 342 402, Biolegend), SCD1 (Cat# 2794, Cell Signaling Technology, 1:1000), S6 Ribosomal Protein (Cat# 2317, Cell Signaling Technology, 1:1000), phospho‐S6 Ribosomal Protein (Ser240/244) (Cat# 5364, Cell Signaling Technology, 1:1000), p70 S6 Kinase (Cat# 2708, Cell Signaling Technology, 1:1000), phospho‐p70 S6 Kinase (Thr389) (Cat# 9234, Cell Signaling Technology, 1:1000), I κ B α (Cat# 4814, Cell Signaling Technology, 1:1000), p‐I κ B α (Ser32) (Cat# 2859, Cell Signaling Technology, 1:1000), IKK β (Cat# 8943, Cell Signaling Technology, 1:1000), p‐IKK α / β (Ser176/180) (Cat# 2697, Cell Signaling Technology, 1:1000), SREBP1 (Cat# sc‐365513, Santa Cruz, 1:1000), FASN (Cat# 3180, Cell Signaling Technology, 1:1000), β ‐catenin (Cat# sc‐7963, Santa Cruz, 1:1000), Lamin B1 (Cat# 12987‐1‐AP, Proteintech, 1:5000), β ‐tubulin (Cat# 10068‐1‐AP, Proteintech, 1:2000), or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; Cat# HRP‐60004, Proteintech, 1:10 000).

Techniques: Fluorescence, Labeling, Concentration Assay, Incubation, Microscopy, Cell Culture, Competitive Binding Assay, Flow Cytometry, Western Blot