Journal: Frontiers in Cardiovascular Medicine

Article Title: Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries

doi: 10.3389/fcvm.2022.980628

Figure Lengend Snippet: K + channel-mediated vasodilation underlies conducted dilation of human and porcine RA-IMCAs. (A) Schematic of experimental setup with direction of flow of bradykinin (BK) indicated by carboxyfluorescein (CF). Micrographs showing an isolated, cannulated and pressurized human (B ) and porcine (C) artery. Focal application of BK (and CF, green) to the downstream end of the artery against the direction of superfusion flow caused local and conducted dilation in both human (D) and porcine (E) arteries. The corresponding time course of responses are shown in (F,G) , and , . A bolus of bradykinin was delivered at the point indicated by the arrow, and simultaneous inner diameter measured locally (0 μm) and up to 1,000 μm upstream, positions indicated by arrows in (D,E) . The same human artery used for K Ca 3.1 immunolabel in . Summary graphs show that compared to control ( n = 9, 14) neither L-NAME (100 μM, n = 3, 6) nor Ba 2+ (30 μM, n = 3, 3) affected local or conducted dilation, whereas depolarization to 45 mM KCl abolished conducted dilation ( n = 3, 6) in human (H) and porcine (I) arteries, respectively. Non-parametric mixed effects analysis with Sidak's multiple comparison test; * P < 0.05 vs. control.

Article Snippet: Primary antibodies were as follows: 1:100 rabbit polyclonal anti-rat K Ca 3.1 (aa 350–363; Alomone Laboratories, APC-064); 1:100 mouse monoclonal anti-human K Ca 3.1 (third extracellular loop; Alomone Laboratories, ALM-051); and 1:100 rabbit polyclonal anti-human K Ca 2.3 (aa 2–21, Alomone Laboratories, APC-025).

Techniques: Isolation, Immunolabeling

Journal: Frontiers in Cardiovascular Medicine

Article Title: Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries

doi: 10.3389/fcvm.2022.980628

Figure Lengend Snippet: K + channel expression and hyperpolarization to bradykinin in human and porcine IMCAs. Confocal micrographs of immunolabelling for K Ca 3.1 and K Ca 2.3 in isolated, cannulated and pressurized human (A) and porcine (B) RA-IMCAs with myogenic tone and full dilation to BK. Punctate and diffuse K Ca 3.1 label was evident in the ECs of human and porcine arteries, whereas K Ca 2.3 was less clear in human IMCAs, and highly expressed at EC borders of porcine IMCAs (yellow arrowheads). The elastin was dense in human arteries, with the internal elastic lamina (IEL) seen as longitudinal strings in the porcine arteries. Representative of at least 3 arteries for each label; asterisks indicate corresponding nuclei in upper and lower panels. The pink dashed line in the schematic represents the focal plane. (C) The schematic indicates a sharp microelectrode impaled into a SMC of a porcine RA-IMCA mounted for isometric tension recording. Under control conditions the thromboxane mimetic U46619 (0.6 μM) depolarized and contracted arteries, and BK (10 nM) caused hyperpolarization and relaxation (C) , summarized in (D) . (E) Addition of L-NAME depolarized and contracted porcine left ventricular (LV)-IMCAs. Under these conditions BK (0.1 nM to 100 nM) repolarized and relaxed the artery. Addition of 100 nM acetylcholine (ACh) depolarized and contracted the artery. The asterisk indicates when the electrode came out of the cell. The RMP and tension prior to the addition of L-NAME (100 μM) are indicated by dashed lines. Drugs were added to a static bath at the arrows. RMP, resting membrane potential.

Article Snippet: Primary antibodies were as follows: 1:100 rabbit polyclonal anti-rat K Ca 3.1 (aa 350–363; Alomone Laboratories, APC-064); 1:100 mouse monoclonal anti-human K Ca 3.1 (third extracellular loop; Alomone Laboratories, ALM-051); and 1:100 rabbit polyclonal anti-human K Ca 2.3 (aa 2–21, Alomone Laboratories, APC-025).

Techniques: Expressing, Isolation

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Comparison of K Ca 1.1 expression and activity between LNCaP cells cultured as 2D monolayers and 3D spheroids. ( A ): Phenotypic properties of LNCaP cells cultured in ultra-low attachment PrimeSurface 96U plates (upper panel: on day 0; lower panel: on day 7). Brightfield images were obtained with the Axio Observer Z1 microscope system (Carl Zeiss, Oberkochen, Germany). Bars show 500 μm. ( B ): Time course of the voltage-sensitive fluorescent dye imaging of K Ca 1.1 activator (1 μM NS1619)-induced hyperpolarizing responses in isolated cells from ‘2D’ monolayers and ‘3D’ spheroids of LNCaP. The fluorescent intensity of DiBAC 4 (3) before the application of NS1619 is expressed as 1.0. Images were measured every 5 s. ( C ): Summarized results of NS1619-induced hyperpolarizing responses in cells isolated from at least three different batches in each group. Cell numbers used in experiments are shown in parentheses. The values for fluorescent intensity were obtained by measuring the average for 1 min (12 images). ( D ): K Ca 1.1 protein expression in the lipid-raft-enriched protein lysates of both groups. Blots were probed with anti-K Ca 1.1 (approximately 100 kDa, upper panel) and anti-ACTB (43 kDa, lower panel) antibodies. ( E ): Summarized results were obtained as the optical density of K Ca 1.1 and ACTB band signals. 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 ‘2D’ was expressed as 1.0 ( n = 4 for each). ( F ): Real-time PCR examination of K Ca 1.1 in both groups ( n = 4 for each). Expression levels were shown as a ratio to ACTB. *: p < 0.05 vs. ‘2D’.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Microscopy, Imaging, Isolation, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Effects of chemotherapy agents on the viability of 2D- and 3D-cultured LNCaP cells and the effects of a pretreatment with a K Ca 1.1 inhibitor on chemoresistance acquired by 3D-cultured LNCaP cells. ( A – D ): Effects of the treatment with 100 nM DTX, 100 nM PTX, 1 μM DOX, and 10 μM CIS for 48 h on the viability of ‘2D’- and ‘3D’-cultured LNCaP cells using the WST-1 assay ( n = 5 for each). Cell viability additing 0.1% dimethylsulfoxide, DMSO in DTX, PTX, and DOX and water in CIS instead of chemotherapy agents was expressed as 1.0. ( E – H ): Effects of the treatment with chemotherapy agents for 48 h on the viability of vehicle- and 10 μM PAX-pretreated (for 24 h), 3D-cultured LNCaP cells ( n = 5 for each). **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Cell Culture, WST-1 Assay

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Effects of antiandrogens on the viability of 2D- and 3D-cultured LNCaP cells and the effects of a K Ca 1.1 inhibitor on the antiandrogen resistance acquired by 3D-cultured LNCaP cells. ( A , B ): Effects of the treatment with 10 μM BCT and 10 μM EZT for 48 h on the viability of ‘2D’- and ‘3D’-cultured LNCaP cells using the WST-1 assay ( n = 5 for each). Cell viability additing 0.1% DMSO instead of antiandrogens was expressed as 1.0. ( C , D ): Effects of the treatment with antiandrogens for 48 h on the viability of vehicle- and 10 μM PAX-pretreated (for 24 h), 3D-cultured LNCaP cells ( n = 5 for each). **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Cell Culture, WST-1 Assay

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Expression of MRP transcripts in both 2D- and 3D-cultured LNCaP cells and the effects of K Ca 1.1 blockade with PAX on their expression in 3D-cultured LNCaP cells. (A–D): Real-time PCR examination of MRP1 ( A ), MRP3 ( B ), MRP4 ( C ), and MRP5 ( D ) in ‘2D’ monolayers and ‘3D’ spheroids of LNCaP cells ( n = 4 for each). ( E – H ): Real-time PCR examination of MRP transcripts in vehicle- and PAX-treated, 3D-cultured LNCaP cells ( n = 4 for each). Expression levels were shown as a ratio to ACTB. **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Regulation of K Ca 1.1 protein degradation through the FBXW7 ubiquitin E3 ligase in LNCaP cells. ( A , B ) Protein expression of FBXW7 in whole cell protein lysates of ‘2D’- and ‘3D’-cultured LNCaP cells. Blots were probed with anti-FBXW7 (approximately 70 kDa) and anti-ACTB (43 kDa) antibodies ( A ). Summarized results ( B ) were obtained as the optical densities of FBXW7 and ACTB band signals ( n = 4 for each). After compensation for the optical densities of the protein band signals with that of the ACTB signal, the optical density in ‘2D’ was expressed as 1.0 ( n = 4 for each). ( C , D ): Protein expression of K Ca 1.1 in control siRNA (si-Cont)- and FBXW7 siRNA (si-FBXW7)-transfected (for 72 h), 2D-cultured LNCaP cells. Blots were probed with anti-K Ca 1.1 (approximately 100 kDa) and anti-ACTB (43 kDa) antibodies ( C ). Summarized results ( D ) were obtained as the optical densities of K Ca 1.1 and ACTB band signals. The optical density in ‘si-Cont’ was expressed as 1.0 ( n = 4 for each). **: p < 0.01 vs. ‘2D’; # : p < 0.05 vs. ‘si-Cont’.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Cell Culture, Transfection

Journal: Cancer Science

Article Title: Ca 2+ ‐activated K + channel K Ca 1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

doi: 10.1111/cas.15046

Figure Lengend Snippet: Comparison of Ca 2+ ‐activated K + channel K Ca 1.1 expression and activity between human OS MG‐63 cells cultured as 2D monolayers and 3D spheroids. A, Phenotypic properties of MG‐63 cells cultured with ultra‐low attachment PrimeSurface 96 U plates (upper panel: day 0, lower panel: day 4). Brightfield images were obtained with the Axio Observer Z1 microscope system (Carl Zeiss). Bars show 50 μm. B, Time course of the voltage‐sensitive fluorescent dye imaging of K Ca 1.1 activator (1 μmol/L NS1619)‐induced hyperpolarizing responses in isolated cells from 2D monolayers (“2D”) and 3D spheroids (“3D”) of MG‐63. The fluorescence intensity of DiBAC 4 (3) before the application of NS1619 is expressed as 1.0. Images were measured every 5 s. C, Summarized results of NS1619‐induced hyperpolarizing responses in cells isolated from at least 3 different batches in each group. Cell numbers used in experiments are shown in parentheses. The values for fluorescence intensity were obtained by measuring the average for 1 min (12 images). D, K Ca 1.1 protein expression in the lipid raft‐enriched protein lysates of both groups. Blots were probed with anti‐K Ca 1.1 (approximately 100 kDa, upper panel) and anti‐ACTB (43 kDa, lower panel) antibodies. E, Summarized results were obtained as the optical density of K Ca 1.1 and ACTB band signals. 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 “2D” was expressed as 1.0 (n = 4 for each). F, Fixed, non‐permeabilized MG‐63 cells were stained with an Alexa 488‐fused anti‐K Ca 1.1 (extracellular) antibody, and mean fluorescence intensities were measured using flow cytometry. Their values in “2D” were expressed as 1.0 (n = 4 for each). G, Real‐time PCR examination of the K Ca 1.1 transcript in both groups (n = 4 for each). Expression levels were shown as a ratio compared with ACTB. Results are expressed as means ± SEM. ** P < .01 vs 2D

Article Snippet: Fixed and non‐permeabilized cells were stained with an anti‐K Ca 1.1 polyclonal antibody (rabbit; extracellular, APC‐151, Alomone Labs) followed by an Alexa Fluor 488‐conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

Techniques: Expressing, Activity Assay, Cell Culture, Microscopy, Imaging, Isolation, Fluorescence, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Ca 2+ ‐activated K + channel K Ca 1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

doi: 10.1111/cas.15046

Figure Lengend Snippet: Comparison of K Ca 1.1 expression and activity between human CS SW‐1353 cells cultured as 2D monolayers and 3D spheroids. A, Phenotypic properties of SW‐1353 cells cultured with ultra‐low attachment PrimeSurface 96 U plates (upper panel: day 0, lower panel: day 4). Bars show 50 μm. B, Time course of the voltage‐sensitive fluorescent dye imaging of NS1619‐induced hyperpolarizing responses in isolated cells from the 2D monolayers (“2D”) and 3D spheroids (“3D”) of SW‐1353. The fluorescence intensity of DiBAC 4 (3) before the application of NS1619 was expressed as 1.0. Images were measured every 5 s. C, Summarized results of NS1619‐induced hyperpolarizing responses in cells isolated from at least 3 different batches in each group. Cell numbers used in experiments are shown in parentheses. The values for fluorescence intensity were obtained by measuring the average for 1 min (12 images). D, K Ca 1.1 protein expression in the lipid raft‐enriched protein lysates of both groups. Blots were probed with anti‐K Ca 1.1 (approximately 100 kDa, upper panel) and anti‐ACTB (43 kDa, lower panel) antibodies. E, Summarized results were obtained as the optical density of K Ca 1.1 and ACTB band signals. 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 “2D” was expressed as 1.0 (n = 4 for each). F, Real‐time PCR examination of the K Ca 1.1 transcript in both groups (n = 4 for each). Expression levels were shown as a ratio to ACTB. Results are expressed as means ± SEM

Article Snippet: Fixed and non‐permeabilized cells were stained with an anti‐K Ca 1.1 polyclonal antibody (rabbit; extracellular, APC‐151, Alomone Labs) followed by an Alexa Fluor 488‐conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

Techniques: Expressing, Activity Assay, Cell Culture, Imaging, Isolation, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Ca 2+ ‐activated K + channel K Ca 1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

doi: 10.1111/cas.15046

Figure Lengend Snippet: Effects of the siRNA‐mediated inhibition of FBXW7 on expression levels of the K Ca 1.1 protein in 2D‐cultured MG‐63 cells. A, Real‐time PCR examination of FBXW7 transcripts in control siRNA (si‐cont)‐ and FBXW7 siRNA (si‐FBXW7)‐transfected 2D monolayers of MG‐63 cells (n = 4 for each). Expression levels are shown as a ratio compared with ACTB. Protein expression of K Ca 1.1 in si‐cont and si‐FBXW7 groups. Blots were probed with anti‐K Ca 1.1 (approximately 100 kDa) and anti‐ACTB (43 kDa) antibodies (B). Summarized results were obtained as the optical density of K Ca 1.1 band signals (C). After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the optical density in “si‐cont” was expressed as 1.0 (n = 4 for each). Results are expressed as means ± SEM. ** P < .01 vs si‐cont

Article Snippet: Fixed and non‐permeabilized cells were stained with an anti‐K Ca 1.1 polyclonal antibody (rabbit; extracellular, APC‐151, Alomone Labs) followed by an Alexa Fluor 488‐conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

Techniques: Inhibition, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Transfection

Journal: Cancer Science

Article Title: Ca 2+ ‐activated K + channel K Ca 1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

doi: 10.1111/cas.15046

Figure Lengend Snippet: Effects of the treatment with the K Ca 1.1 inhibitor, PAX on chemoresistance acquired in MG‐63 and SW‐1353 spheroids. Effects of the treatment with 100 nmol/L PAC, 1 μmol/L DOX, and 10 μmol/L CIS for 48 h on cell viability in vehicle‐ and PAX‐treated MG‐63 (A‐C) and SW‐1353 (D‐F) spheroids. The viability in the untreated cells with PAX was expressed as 1.0. Results are expressed as means ± SEM. ** P < .01 vs vehicle control

Article Snippet: Fixed and non‐permeabilized cells were stained with an anti‐K Ca 1.1 polyclonal antibody (rabbit; extracellular, APC‐151, Alomone Labs) followed by an Alexa Fluor 488‐conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

Techniques:

Journal: Cancer Science

Article Title: Ca 2+ ‐activated K + channel K Ca 1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

doi: 10.1111/cas.15046

Figure Lengend Snippet: Decreased expression of MRP1 by the inhibition of K Ca 1.1 in sarcoma spheroids. Real‐time PCR examination of MRP1 transcripts in vehicle‐ and PAX‐treated MG‐63 (A) and SW‐1353 (D) spheroids (n = 4 for each). Expression levels are shown as a ratio compared with ACTB. Protein expression of MRP1 in the lipid raft‐enriched protein lysates of both groups. Blots were probed with anti‐MRP1 (approximately 250 kDa) and anti‐ACTB (43 kDa) antibodies (B, E). Summarized results were obtained as the optical density of MRP1 (C, F) band signals. After compensation for the optical density of the MRP1 protein band signal with that of the ACTB signal, optical density in the vehicle control was expressed as 1.0 (n = 4 for each). Results are expressed as means ± SEM. ** P < .01 vs vehicle control

Article Snippet: Fixed and non‐permeabilized cells were stained with an anti‐K Ca 1.1 polyclonal antibody (rabbit; extracellular, APC‐151, Alomone Labs) followed by an Alexa Fluor 488‐conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

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

Journal: Cancer Science

Article Title: Ca 2+ ‐activated K + channel K Ca 1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

doi: 10.1111/cas.15046

Figure Lengend Snippet: Decreased expression of FBXW7 and increased protein expression of K Ca 1.1 by SIRT1 inhibition in 2D‐cultured MG‐63 cells. Real‐time PCR examination of FBXW7 (A) and K Ca 1.1 transcripts (D) in vehicle‐, vorinostat (10 μmol/L)‐, and NCO‐01 (50 μmol/L)‐treated, 2D‐cultured MG‐63 monolayers. Expression levels were shown as a ratio to ACTB. Protein expression of K Ca 1.1 in protein lysates of the vehicle‐, vorinostat (10 μmol/L)‐, and NCO‐01 (50 μmol/L)‐treated 2D monolayers of MG‐63 cells. Blots were probed with anti‐K Ca 1.1 (approximately 100 kDa) and anti‐ACTB (43 kDa) antibodies (B). Summarized results were obtained as the optical density of K Ca 1.1 band signal (C). After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, optical density in the vehicle control was expressed as 1.0 (n = 4 for each). Results are expressed as means ± SEM. * P < .05, ** P < .01 vs vehicle control

Article Snippet: Fixed and non‐permeabilized cells were stained with an anti‐K Ca 1.1 polyclonal antibody (rabbit; extracellular, APC‐151, Alomone Labs) followed by an Alexa Fluor 488‐conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

Techniques: Expressing, Inhibition, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Ca 2+ ‐activated K + channel K Ca 1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

doi: 10.1111/cas.15046

Figure Lengend Snippet: Increased expression of NRF2 transcripts decreases by inhibition with K Ca 1.1, and effects of NRF2 and HSF1 inhibitors on the expression of MRP1 transcripts in sarcoma spheroids. Real‐time PCR examination of NRF2 and HSF1 in 2D‐ and 3D‐cultured MG‐63 (A, C) and SW‐1353 cells (G, I) and in vehicle‐ and PAX (10 μmol/L)‐treated (24 h) MG‐63 (B, D) and SW‐1353 spheroids (H, J). Real‐time PCR examination of MRP1 in vehicle‐ and ML385 (10 μmol/L)‐treated (for 24 h) MG‐63 (E), and SW‐1353 (K) spheroids and in vehicle‐ and KRIBB11 (10 μmol/L)‐treated (for 24 h) MG‐63 (F) and SW‐1353 spheroids (L). Expression levels are shown as a ratio compared with ACTB. Results are expressed as means ± SEM. ** P < .01 vs “2D” or the vehicle control

Article Snippet: Fixed and non‐permeabilized cells were stained with an anti‐K Ca 1.1 polyclonal antibody (rabbit; extracellular, APC‐151, Alomone Labs) followed by an Alexa Fluor 488‐conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

Techniques: Expressing, Inhibition, Real-time Polymerase Chain Reaction, Cell Culture

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