Journal: Cancer Science

Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl − /H + transporter inhibition in human breast cancer cells

doi: 10.1111/cas.13715

Figure Lengend Snippet: Effects of siRNA‐mediated inhibition of ClC‐3, ClC‐7, and chloride intracellular channel protein 1 (CLIC)1 on expression levels of human epidermal growth factor receptor 2 (HER2) transcripts in MDA‐MB‐453 cells. A, Real‐time PCR assay for HER2 in control siRNA (si‐cont)‐, ANO1 siRNA (si‐ANO1)‐, ClC‐3 siRNA (si‐ClC‐3)‐, ClC‐7 siRNA (si‐ClC‐7)‐, and CLIC1 siRNA (si‐CLIC1)‐transfected MDA‐MB‐453 cells for 72 h. Expression levels were expressed as a ratio to β‐actin (ACTB). B‐D, Protein lysates of si‐cont‐, si‐ANO1‐, si‐ClC‐3‐, si‐ClC‐7‐, and si‐CLIC1‐transfected MDA‐MB‐453 cells were probed by immunoblotting with anti‐HER2 and anti‐ACTB antibodies on the same filter (B, C). Summarized results were obtained as described in Section from HER2 and ACTB band signals (D). After compensation, the HER2 signal in the si‐cont group was expressed as 1.0 (n = 4 for each). Results are expressed as means ± SEM. ** P < .01 vs si‐cont. ANO, anoctamine

Article Snippet: Commercially available siRNA oligonucleotides against ClC‐3, ClC‐4, ClC‐5, ClC‐7, CLIC1, ANO1, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Thermo Fisher Scientific.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

Journal: Cancer Science

Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl − /H + transporter inhibition in human breast cancer cells

doi: 10.1111/cas.13715

Figure Lengend Snippet: Cellular distribution of ClC‐3 in MDA‐MB‐453 cells. (A‐C) Immunocytochemical staining of MDA‐MB‐453 cells for ClC‐3. A, Confocal images of a cell labeled with anti‐ClC‐3 and Alexa Fluor 488 anti‐rabbit IgG antibodies. B, a fluorescent image merged with a transparent image. C, the profile of fluorescence intensity on the white dotted line in (B) is shown in “green”. The plasma membrane is shown in ‘red’. D‐H, ClC‐3 fluorescent images in control siRNA (si‐cont) (D)‐, ClC‐3 siRNA (si‐ClC‐3) (E)‐, ClC‐4 siRNA (si‐ClC‐4) (F)‐, and ClC‐5 siRNA (si‐ClC‐5)‐transfected MDA‐MB‐453 cells for 72 h. Mean fluorescent intensity in the si‐cont group was expressed as 1.0 (n = 24 for each); summarized data are shown in (H). Results are expressed as means ± SEM. ** P < .01 vs si‐cont

Article Snippet: Commercially available siRNA oligonucleotides against ClC‐3, ClC‐4, ClC‐5, ClC‐7, CLIC1, ANO1, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Thermo Fisher Scientific.

Techniques: Staining, Labeling, Fluorescence, Transfection

Journal: Cancer Science

Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl − /H + transporter inhibition in human breast cancer cells

doi: 10.1111/cas.13715

Figure Lengend Snippet: Effects of siRNA‐mediated inhibition of ClC‐3 for 72 h on the expression levels of phosphorylated AKT (P‐AKT) and phosphorylated STAT3 (P‐STAT3) proteins in MDA‐MB‐453 cells. A, B, Protein lysates of control siRNA (si‐cont) and ClC‐3 siRNA (si‐ClC‐3)‐transfected MDA‐MB‐453 cells were probed by immunoblotting with anti‐P‐AKT, anti‐AKT, and anti‐ACTB (A) or anti‐P‐STAT3, STAT3, and ACTB (B). After compensation, the ratio of P‐AKT to AKT or P‐STAT3 to STAT3 in si‐cont was expressed as 1.0 (n = 6 for each). Results are expressed as means ± SEM. ** P < .01 vs si?cont. ACTB, β‐actin; STAT, signal transducer and activator of transcription

Article Snippet: Commercially available siRNA oligonucleotides against ClC‐3, ClC‐4, ClC‐5, ClC‐7, CLIC1, ANO1, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Thermo Fisher Scientific.

Techniques: Inhibition, Expressing, Transfection, Western Blot

Journal: Cancer Science

Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl − /H + transporter inhibition in human breast cancer cells

doi: 10.1111/cas.13715

Figure Lengend Snippet: Distribution of ClC and chloride intracellular channel protein (CLIC) subtype transcripts and effects of siRNA‐mediated inhibition of YMB‐1 cells. A, B, Real‐time PCR assay for 7 ClC subtypes (ClC‐1‐ClC‐7) (A) and 6 CLIC subtypes (CLIC1‐CLIC6) in YMB‐1 cells (n = 3 for each). C, Real‐time PCR assay for human epidermal growth factor receptor 2 (HER2) in control siRNA (si‐cont)‐, ClC‐3 siRNA (si‐ClC‐3)‐, ClC‐7 siRNA (si‐ClC‐7)‐, and CLIC1 siRNA (si‐CLIC1)‐transfected YMB‐1 cells for 72 h. Expression levels are expressed as a ratio to β‐actin (ACTB). D, Protein lysates of si‐cont‐, si‐ClC‐3‐, si‐ClC‐7‐, si‐ANO1‐, and si‐CLIC1‐transfected YMB‐1 cells were probed by immunoblotting with anti‐HER2 and anti‐ACTB antibodies on the same filter (Figure A,B). After compensation, the HER2 signal in the si‐cont group was expressed as 1.0 (n = 4 for each). Results are expressed as means ± SEM. ** P < .01 vs si‐cont

Article Snippet: Commercially available siRNA oligonucleotides against ClC‐3, ClC‐4, ClC‐5, ClC‐7, CLIC1, ANO1, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Thermo Fisher Scientific.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Transfection, Expressing, Western Blot

Journal: Cancer Science

Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC‐3 Cl − /H + transporter inhibition in human breast cancer cells

doi: 10.1111/cas.13715

Figure Lengend Snippet: Effects of si RNA ‐mediated inhibition of ClC‐3 and ANO 1 on expression levels of human epidermal growth factor receptor 2 ( HER 2)‐related transcriptional factor transcripts in MDA ‐ MB ‐453 and YMB ‐1 cells, respectively. Real‐time PCR assay for AP ‐2α (A, H), AP ‐2β (B, I), AP ‐2γ (C, J), STAT 3 (D, K), EBP 1 (E, L), MBP ‐1 (F, M), and HDAC 6 (G, N) in control si RNA (si‐cont) and ClC‐3 si RNA (si‐ClC‐3)‐transfected MDA ‐ MB ‐453 cells for 72 h (A‐G) and ANO 1 si RNA (si‐ ANO 1)‐transfected YMB ‐1 cells for 72 h (H‐N), respectively. Expression levels were expressed as a ratio to ACTB (n = 4 for each). Results are expressed as means ± SEM . ANO , anoctamine; AP , activator protein; EBP , ErbB3‐binding protein; HDAC , histone deacetylase; MBP ‐1, major basic protein 1; STAT , signal transducer and activator of transcription

Article Snippet: Commercially available siRNA oligonucleotides against ClC‐3, ClC‐4, ClC‐5, ClC‐7, CLIC1, ANO1, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Thermo Fisher Scientific.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Transfection, Binding Assay, Histone Deacetylase Assay