Journal: International Journal of Molecular Sciences

Article Title: Down-Regulation of Ca 2+ -Activated K + Channel K Ca 1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

doi: 10.3390/ijms17122083

Figure Lengend Snippet: Gene and protein expression of K Ca 1.1 in human breast cancer cell lines and effects of its pharmacological and/or siRNA-mediated blockade on the viability and K Ca 1.1 activity in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in seven human breast cancer cell lines ( n = 3 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Expression of K Ca 1.1 proteins (about 130 kDa) in MDA-MB-453, YMB-1, and MCF-7 cells. Protein lysates of the examined cells were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( C , D ) Effects of the treatment with the K Ca 1.1 blocker, paxilline (10 μM) for 72 h ( C ) and the transfection with K Ca 1.1 siRNA for 96 h ( D ) on the viability in MDA-MB-453 cells. Cell viability in the vehicle-treated or control siRNA-transfected group is arbitrary expressed as 1.0, and the data are shown as “relative cell viability” ( n = 5 for each); ( E ) Current-voltage relationship for the current amplitude at the end of the depolarization pulse in MDA-MB-453 cells following treatment with 1 µM paxilline (see ). Results are expressed as means ± SEM. * p < 0.05; ** p < 0.01 vs. the vehicle control or control siRNA.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

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

Journal: International Journal of Molecular Sciences

Article Title: Down-Regulation of Ca 2+ -Activated K + Channel K Ca 1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

doi: 10.3390/ijms17122083

Figure Lengend Snippet: Down-regulation of K Ca 1.1 transcripts and proteins by the treatment with VDR agonists for 72 h in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in vehicle-, 1 μM calcitriol-, and 1 μM calcipotriol-treated MDA-MB-453 cells ( n = 4 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Band patterns on agarose gels for the PCR products of K Ca 1.1 exons (exon 1–4, 5–14, 15–23, and 24–30) in vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells. A DNA molecular weight marker is indicated on the right of the gel; ( C ) Protein lysates of vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( D ) Summarized results are obtained as the optical density of K Ca 1.1 and ACTB band signals in C . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in the vehicle control was expressed as 1.0 (dotted line, n = 3 for each); ( E ) Effects of the treatment with 1 µM calcitriol or 1 µM calcipotriol on the cell surface expression of K Ca 1.1 proteins by a flow cytometric analysis. Non-permeabilized MDA-MB-453 cells were stained with an Alexa Fluor @ 488-conjugated anti-K Ca 1.1 antibody (extracellular). Data were expressed as the relative cell population of K Ca 1.1-positive cells to those in the vehicle control (1.0) ( n = 4 for each). Results are expressed as means ± SEM. ** p < 0.01 vs. the vehicle control.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Molecular Weight, Marker, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Down-Regulation of Ca 2+ -Activated K + Channel K Ca 1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

doi: 10.3390/ijms17122083

Figure Lengend Snippet: Inhibitory effects of K Ca 1.1 activities (1 µM paxilline-induced depolarization responses) in MDA-MB-453 cells treated with VDR agonists for 72 h and effects of co-treatment with calcitriol (1 µM) and 1 μM paxilline on the viability of MDA-MB-453 cells. ( A ) Measurement of paxilline-induced depolarization responses in vehicle (black symbol)-, calcitriol (red symbol)-, and calcipotriol (blue symbol)-treated MDA-MB-453 cells. The fluorescence intensity of DiBAC 4 (3) before the application of paxilline at 0 s is expressed as 1.0. The time courses of changes in the relative fluorescence intensity of DiBAC 4 (3) are shown; ( B ) Summarized data are shown as the paxilline-induced ∆ relative fluorescence intensity of DiBAC 4 (3) in vehicle-, calcitriol-, and calcipotriol-treated MDA-MB-453 cells. Cells were obtained from four different batches. Numbers used for the experiments are shown in parentheses; ( C ) Effects of the treatment with calcitriol (1 µM) alone, paxilline (10 µM) alone, and calcitriol plus paxilline for 72 h on the viability in MDA-MB-453 cells. Cell viability in the vehicle-treated is arbitrary expressed as 1.0, and the data are shown as “relative cell viability” ( n = 5 for each). Results are expressed as means ± SEM. **: p < 0.01 vs. the vehicle control.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Down-Regulation of Ca 2+ -Activated K + Channel K Ca 1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

doi: 10.3390/ijms17122083

Figure Lengend Snippet: Effects of the proteasome inhibitor, MG132 (100 nM) on VDR agonist-induced K Ca 1.1 protein degradation and K Ca 1.1 activity in MDA-MB-453 cells. ( A ) Protein lysates from MDA-MB-453 cells after drug treatments were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( B ) Summarized results are obtained as the optical density of K Ca 1.1 and ACTB band signals in A . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in the vehicle control was expressed as 1.0 (dotted line, n = 3 for each); ( C ) Summarized data are shown as the paxilline-induced ∆ relative fluorescence intensity of DiBAC 4 (3) in vehicle-, calcitriol-, and calcipotriol-treated MDA-MB-453 cells. Cells were obtained from three different batches. Numbers used for the experiments are shown in parentheses. Results are expressed as means ± SEM.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Activity Assay, Western Blot, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Down-Regulation of Ca 2+ -Activated K + Channel K Ca 1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

doi: 10.3390/ijms17122083

Figure Lengend Snippet: Effects of the pharmacological and siRNA-mediated blockade of HDACs on expression levels of K Ca 1.1 transcripts in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in MDA-MB-453 cells treated with the following HDAC inhibitors for 48 h ( n = 4 for each): vorinostat (suberanilohydroxamic acid), a pan-HDAC inhibitor; AATB (4-(acetylamino)- N -[2-amino-5-(2-thienyl)phenyl]-benzamide), a HDAC1 (30 nM) and HDAC2 (300 nM) inhibitor; T247 (N-(2-aminophenyl)-4-[1-(2-thiophen-3-ylethyl)-1H-[1], [2], [3]triazol-4-yl]benzamide), a selective HDAC3 inhibitor; and NCT-14b (( S )- S -7-(adamant-1-ylamino)-6-(tert-butoxycarbonyl)-7-oxoheptyl-2-methylpropanethioate), a selective HDAC6 inhibitor ; ( B ) Real-time PCR assay for K Ca 1.1 in MDA-MB-453 cells transfected with control siRNA (si-ctrl) and siRNAs specific for HDAC2 and HDAC3 (siHDAC2, siHDAC3) for 48 h ( n = 4 for each). Expression levels were expressed as a ratio to ACTB. Results are expressed as means ± SEM ( n = 4 for each). * p < 0.05; ** p < 0.01 vs. the vehicle control or control siRNA-transfected group.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Down-Regulation of Ca 2+ -Activated K + Channel K Ca 1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

doi: 10.3390/ijms17122083

Figure Lengend Snippet: Effects of treatments with VD agonists on expression levels of HDAC2 transcripts and proteins in MDA-MB-453 cells. ( A ) Real-time PCR assay for HDAC2 in VD agonist-treated MDA-MB-453 cells for 72 h ( n = 4 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Protein lysates of VD agonist-treated MDA-MB-453 cells were probed by immunoblotting with anti-HDAC2 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( C ) Summarized results are obtained as the optical density of HDAC2 and ACTB band signals in B . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the HDAC2 signal in the vehicle control was expressed as 1.0 (dotted line, n = 4 for each). Results are expressed as means ± SEM ( n = 4 for each). ** p < 0.01 vs. the vehicle control.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

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