Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Effects of α 1 -AR blockade in the VTA on the acquisition of cocaine-evoked CPP. (A,F) The experimental time-line and schedule of intra-VTA microinfusions during the acquisition of CPP induced by one (A) or two (F) cocaine (20 mg/kg, i.p.) conditionings. (B) Graphical representation of the two methods of calculating conditioned responses in the CPP paradigm. CPP score is defined as a difference between time spent (t 2 ) in the cocaine-paired chamber minus time spent in (t 3 ) control chamber (saline-paired) during post-conditioning (post-test). Delta score is defined as a difference between time spent (t 2 ) in the cocaine-paired chamber during post-conditioning minus time spent (t 1 ) in the same chamber but during pre-conditioning (pre-test). (C,G) There were no pre-existing differences in the time spent in the cocaine- and saline-paired chambers during pre-test between future vehicle (Veh)- and prazosin (Praz)-treated subjects. (D,E) Intra-VTA microinfusion of prazosin attenuated the acquisition of CPP induced by one ( D for CPP score and E for delta score) and two ( H for CPP score and I for delta score) cocaine (20 mg/kg, i.p.) conditionings. (J) Localization of histologically verified cannula placements. Data are presented as individual data points as well as the mean and SEM. ** p < 0.01, *** p < 0.001, Newman–Keuls post-hoc test vs. Praz 0 μg.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques:

Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Effects of α 1 -AR blockade in the VTA on cocaine reinforcement in the cocaine self-administration paradigm. (A) Rats, after intravenous catheter and intra-VTA cannula implantation surgeries and recovery were trained to self-administer cocaine (∼0.5 mg/kg/inf.) for 8 days. On day 9, intra-VTA vehicle (Veh) or prazosin (Praz) microinfusions were performed immediately prior to subsequent cocaine self-administration session. (B–D) There were no pre-existing differences during cocaine self-administration training in the number of (B) active and (C) inactive lever presses as well as in (D) the number of cocaine infusions between future vehicle (Veh)- and prazosin (Praz)-treated subjects. (E) Intra-VTA microinfusion of prazosin (Praz 1 μg/side) on day 9 had no effect on the numbers of active and inactive lever responses and on (F) the number of cocaine infusions. (G) Intra-VTA α 1 -AR blockade had no effects on the numbers of active lever presses over time. Similarly, prazosin treatment on day 9 had no effects on (H) the numbers of active and inactive lever presses and (I) cocaine infusions, 24 h after treatment (on day 10). (J) Finally, prazosin microinfusion on day 9 had no effects on active lever responding during time-out. (K) Localization of histologically verified cannula placements. Data are presented as individual data points as well as the mean and SEM. Act, active lever; Inact, inactive lever.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques:

Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Effects of α 1 -AR blockade in the VTA on cocaine-evoked 50-kHz USVs. α 1 -AR blockade in the VTA with 1 μg/side prazosin (Praz) had no effect on the number of appetitive USVs after (A) saline (i.p.) or cocaine administration at (B) 10 mg/kg, i.p. or (C) 20 mg/kg dose. (D) Intra-VTA prazosin infusion had no effect on the cocaine-induced increase in the number of appetitive USVs and (E) did not modulate selected categories USVs after cocaine (20 mg/kg, i.p.) administration. (F) Localization of histologically verified cannula placements. Data are presented as individual data points as well as the mean and SEM. *** p < 0.001, Newman–Keuls post-hoc test vs. Cocaine 0 mg/kg.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques:

Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Localization of α 1 -AR-expressing cells in the anterior parts of the VTA of TH-cre + rats. (A) Representative image of the EYFP- and α 1 -AR-expressing cells. (B–E) Magnified cells with α 1 -AR expression (red), EYFP (green), DAPI (blue), and merged channels showing α 1 -AR-immunoreactive and EYFP-expressing cells (white arrows) in the medial part of the anterior VTA. (F–I) Magnified cells with α 1 -AR (red), EYFP (green), DAPI (blue), and merged channels showing co-localized α 1 -AR-positive and EYFP-expressing cells (white arrows) in the lateral part of the anterior VTA. (J–Q) α 1 -AR expression in astrocytes (S100B) and GAD67-positive GABAergic interneurons. (J–M) Magnified images of astrocytes (red), α 1 -AR immunostaining (green), DAPI (blue), and composite images showing co-localization (white arrows) between α 1 -AR and S100B expression. (N–Q) Magnified images of GAD67-positive interneurons (red), α 1 -AR expression (green), DAPI (blue), and composite images showing co-localization between α 1 -AR and GAD67 immunoreactivity (white arrows). PBP, parabrachial pigmented nucleus; RLi, rostral linear nucleus.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques: Expressing, Immunostaining

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: Cell Reports

Article Title: Human Labor Pain Is Influenced by the Voltage-Gated Potassium Channel K V 6.4 Subunit

doi: 10.1016/j.celrep.2020.107941

Figure Lengend Snippet:

Article Snippet: Anti-K V 2.1 rabbit polyclonal , Alomone , Cat# APC-012; RRID: AB_2040162.

Techniques: Recombinant, Transfection, Software, Transferring