Journal: Frontiers in Pharmacology

Article Title: CyPPA, a Positive SK3/SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate ¹

doi: 10.3389/fphar.2012.00011

Figure Lengend Snippet: CyPPA inhibits spontaneous and evoked release of dopamine form TH/SK3 positive cultured rat midbrain neurons . (A) Western blot detection of SK3 in rat midbrain cultures. Extracts of HEK293 cells transiently transfected with rSK1, rSK2, and rSK3 and adult rat brain membranes (RBM) as well as lysates from cultured midbrain neurons were separated on 4–15% SDS-PAGE and immunoblotted with anti-SK3 antibody. A band of approximately 70 kDa was detected in midbrain cultures, RBM lysates, and HEK293 cells transfected with rSK3. This band was absent in lanes loaded with HEK293 cells expressing rSK1 and rSK2. (B) Immunodetection of SK3 subunits in rat midbrain cultures. Cultured midbrain neurons (8 days in vitro ) were double-labeled for SK3 and tyrosine hydroxylase (TH). In TH-positive dopaminergic neurons, the SK3 subunit displayed a primarily somatic–dendritic localization whereas little immunoreactivity was associated with axons (arrow). Scale bar 20 μm. (C) Basal release and stimulated release with 25 mM KCl or inhibited release with 30 μM Cd 2+ and with 1 μM TTX. (D) Basal release and stimulated release with 3 μM haloperidol and inhibited release with 10 μM quinpirole. Blue bars represent inhibited release with increasing concentrations of CyPPA (* P < 0.05, ** P < 0.01). The data presented are representative of three independent experiments.

Article Snippet: Neurobasal media and B-27 were obtained from Invitrogen (Nærum, Denmark) and 3 H-DA was acquired from PerkinElmer Life and Analytical Sciences (Boston, MA, USA) The primary antibodies used were a rabbit polyclonal anti-SK3 antibody (1:100, APC-025) from Alomone Labs (Jerusalem, Israel) and a monoclonal mouse anti-tyrosine hydroxylase antibody (1:150, clone LNC1) from Millipore (Copenhagen, Denmark).

Techniques: Cell Culture, Western Blot, Transfection, SDS Page, Expressing, Immunodetection, In Vitro, Labeling

Journal: Cancer Discovery

Article Title: Integrated Multiomic Profiling Identifies the Epigenetic Regulator PRC2 as a Therapeutic Target to Counteract Leukemia Immune Escape and Relapse

doi: 10.1158/2159-8290.CD-21-0980

Figure Lengend Snippet: EZH2 inhibitors reestablish functional recognition of relapsed leukemia by CD4 + T cells. A, Outline of the experimental layout to test the functional effects of pharmacologic inhibition of PRC2 in relapsed leukemia on its CD4 + T cell–mediated recognition. CD4 + T cells, either primed against AML blasts collected from UPN01 at diagnosis or selected for being alloreactive to HLA-DPB1*04:01, were tested against HLA class II–positive AML blasts at diagnosis or against their HLA class II–negative relapsed counterpart, cultured in medium alone or pretreated for 7 days with EPZ-6438 or IFNγ. B and C, CD4 + T cells purified from a healthy individual and primed against UPN01 leukemia cells collected at diagnosis were tested against diagnosis and relapse target cells from the same patient by the IFNγ ELISpot assay ( B ; showing for each condition the number of IFNγ spots detected from one out of three replicates) or by the CellTrace Violet (CTV)–dilution assay ( C ; highlighting for each condition the percentage of CD4 + T cells that upon proliferation diluted the vital dye). D, Representative FACS plots showing CD107a degranulation of CD4 + cells from a healthy donor primed against UPN01 AML at diagnosis and tested against the diagnosis (red) and relapse (blue) AML cells from the same patient, pretreated or not with EPZ-6438 or with IFNγ. FSC-H, forward scatter height. E and F, Dot plots summarizing the results of functional experiments performed using CD4 + T cells from three different healthy individuals against AML cells from UPN01 ( E ) or UPN05 ( F ). Purified CD4 + T cells were primed against AML blasts collected at diagnosis and tested against their original stimulator (red dots) or leukemia cells collected from the same patient at relapse (blue dots), pretreated or not with EPZ-6438 or with IFNγ. For each panel, the left-side plot shows CD4 + T-cell degranulation and the right-side plot shows the corresponding results in terms of target cell death (subtracting the spontaneous cell death detected in target cells). Ann V–pos, Annexin V–positive. G–J, Third-party CD4 + T cells alloreactive to HLA-DPB1*04:01 were tested against diagnosis and relapse target cells from UPN01 ( G and H ) or UPN03 ( I and J ), by a CD137 expression assay ( G and I ; showing for each condition the percentage of CD4 + T cells that upregulated CD137 in response to the target), by the CTV-dilution assay ( H ; highlighting for each condition the percentage of CD4 + T cells that upon proliferation diluted the vital dye), and by a cytotoxicity assay ( J ; highlighting for each condition the percentage of target cells staining positive for Annexin V upon subtraction of spontaneous target cell death, shown as the white histogram profile). K, Outline of the in vivo experiment to test the functional effects of pharmacologic inhibition of PRC2 on reestablishing CD4 + T cell–mediated recognition of relapsed leukemia. Mice were engrafted with UPN01 AML at diagnosis or relapse, treated with EPZ-6438 or vehicle alone, and infused with CD4 + T cells primed against the diagnosis AML blasts. b.i.d., twice a day. L, Absolute counts of human AML blasts recovered from the spleen of the mice at the end of the in vivo experiment. The P value was calculated by a two-sided Mann–Whitney test at a 95% confidence interval. EZH2i, EZH2 inhibitor; n.s., not significant.

Article Snippet: The complete list of antibodies we used is as follows: CD3 Alexa700 (clone OKT3, cat. #317340, lot #B229089), CD8 APC-H7 (clone SK1, cat. #344714, lot #B341619), CD14 APC-Cy7 (clone M5E2, cat. #301820, lot #B229089), mouse CD45 PerCP-Cy5.5 (clone 30-F11, cat. #103132, lot #B165130), human CD45 PE-Cy7 (clone HI30, cat. #304016, lot #B264588), CD33 BV510 (clone WM53, cat. #303422, lot #B244960), CD45RA Alexa700 (clone HI100, cat. #304120, lot #B257456), CD62 L PerCP/Cy5.5 (clone DREG-56, cat. #304824, lot #B268797), CD69 APC (clone FN50, cat. #310910, lot #B302800), CD95 PE/Cy7 5 (clone DX2, cat. #305622, lot #B251981), HLA-ABC Pacific Blue (clone W6/32, cat. #311418, lot #B191431), HLA-DR FITC (clone L243, cat. #307604, lot #B275368), LAG3 BV605 (clone 11C3C65, cat. #369324, lot #B98854), PD-1 FITC (clone EH12.2H7, cat. #329904, lot #329904), PD-L1 PE (clone 29E2A3, cat. #329706, lot #B282353), and TIM3 BV421 (clone F38-2E2, cat. #345008, lot #B310545) from BioLegend; CD3 BUV737 (clone SK7, cat. #565466, lot #9164571), CD4 BUV395 (clone SK3, cat. #563550, lot #9084515), and CD34 BV605 (clone 8G12, cat. #745247, lot #B256713) from BD Biosciences; B7-H3 APC (clone 85504, cat. #FAB1027A, lot #AAPJ0213061) from R&D Systems; CD25 PE (clone REA570, cat. #563550, lot #5200406623) from Miltenyi Biotec GmbH; and KLRG1 PE-eFluor 610 (clone 13F12F2, cat. #563550, lot #4351541) from e-Biosciences/Thermo Fisher.

Techniques: Functional Assay, Inhibition, Cell Culture, Purification, Enzyme-linked Immunospot, Dilution Assay, Expressing, Cytotoxicity Assay, Staining, In Vivo, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Expression of a Diverse Array of Ca 2+ -Activated K + Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney

doi: 10.1371/journal.pone.0155006

Figure Lengend Snippet: Listing of ion channels evaluated by qPCR and the forward and reverse primers employed in the study for each channel.

Article Snippet: Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Expression of a Diverse Array of Ca 2+ -Activated K + Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney

doi: 10.1371/journal.pone.0155006

Figure Lengend Snippet: A. qPCR analysis of mRNA expression levels of marker channels relative to the AQP2 water channel. The mechanosensitive TRPV4 channel mRNA levels and ROMK mRNA levels are relatively high while a second mechanosensitive TRP channel, TRPV2, are very low. For comparison, mRNA expression levels for all channels are also given, relative to AQP2 mRNA levels, in the accompanying . B. qPCR mRNA expression levels of KCa channels relative to the ROMK channel. It shows that SK1, SK3, IK1 are expressed at relative high mRNA levels, BKα at moderate mRNA levels, and SK2 at low mRNA levels. C. mRNA expression of BK channel subunits relative to BKα mRNA levels. It shows high relative mRNA levels for BKβ1 and BKβ2, but relatively low levels for BKβ4. D. Immunoblots of mCCDcl1 cells for the key channels showing appropriate protein bands for TRPV4 (98 kD), SK1 (64 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD).

Article Snippet: Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight.

Techniques: Expressing, Marker, Western Blot

Journal: PLoS ONE

Article Title: Expression of a Diverse Array of Ca 2+ -Activated K + Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney

doi: 10.1371/journal.pone.0155006

Figure Lengend Snippet: A. qPCR anlysis of mRNA expression levels of marker channels relative to the AQP2 water channel. As in mCCDcl1 cells, the mechanosensitive TRPV4 channel mRNA levels and ROMK mRNA levels are relatively high while mRNA levels for a second mechanosensitve TRP channel, TRPV2, are very low. For comparison mRNA expression levels for all channels are also given, relative to AQP2 mRNA levels, in the accompanying . B. qPCR analysis of mRNA expression levels of KCa channels in mouse kidney relative to the ROMK channel. It shows that SK1, SK2, and SK3 are expressed at relative high mRNA levels while IK1 and BKα are expressed at moderate mRNA levels. C. mRNA expression of BK channel subunits relative to BKα mRNA levels. The results show high relative mRNA levels for BKβ4, moderate mRNA levels for BKβ2, and low mRNA levels for BKβ4.

Article Snippet: Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight.

Techniques: Expressing, Marker

Journal: PLoS ONE

Article Title: Expression of a Diverse Array of Ca 2+ -Activated K + Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney

doi: 10.1371/journal.pone.0155006

Figure Lengend Snippet: Results show immunostaining for the ion channel of interest and for nuclei (DAPI). A. AQP2 staining of cells (green, 488 nm) shows prominent staining along the plasma membrane with low staining within the cytosol. B. SK1 staining of cells (green, 488 nm) shows clear staining along the plasma membrane borders and modest staining within the cytosol. C. SK3 staining of cells (red, 594 nm) shows significant staining along the cell borders and within the cytosol. Modest staining of the nuclei is apparent (SK3, Merged). D. IK1 staining (green, 488 nm) is modest along the plasma membrane borders and within the cytosol. E. BKα staining of cells (green, 488 nm) with prominent staining of the cell borders and low staining of the cytosol and nuclei. F. TRPV4 staining of cells (green, 488 nm) with prominent staining of the cell plasma membrane and modest staining of the cytosol and nuclei. All fluorescence images were obtained at 100x (oil) with a Nikon A1R Confocal Laser Microscope.

Article Snippet: Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight.

Techniques: Immunostaining, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Expression of a Diverse Array of Ca 2+ -Activated K + Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney

doi: 10.1371/journal.pone.0155006

Figure Lengend Snippet: Results show staining for the ion channel of interest and for AQP2 (Red, 594 nM), a marker of CNT and CCD in the cortex. A bifurcation of AQP2-positive tubules was used to identify CNT segments (above the bifurcation) and CCD segments (single tubule below the bifurcation). A. SK1 staining of tubule segments (green, 488 nm) shows strong staining of many cells along both the CNT and CCD (SK1, labels CNT and CCD). Merged images with AQP2 staining verify the CNT and CCD sites of localization. B. SK3 staining of tubule segments (green, 488 nm) shows prominent staining of most cells along both the CNT and CCD (SK3, labels CNT and CCD). Merged images with AQP2 verify the CNT and CCD sites of localization. C. IK1 staining (red, 594 nm) is strong in most cells of both the CNT and CCD (IK1, labels CNT and CCD) as verified in the Merged image. D. BKα staining (green, 488 nm) is prominent in most cells of both the CCD (BKα, CCD) and along the CNT (data not shown) as verified in the Merged images. E. TRPV4 staining (green, 488 nm) is apparent in most cells of the CCD (TRPV4, CCD) and in the CNT (data not shown) as verified in the Merged images. All fluorescence images were obtained at 40x (oil) with a Zeiss Axioskop 40 microscope using a AxioCam MRm CCD camera.

Article Snippet: Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight.

Techniques: Staining, Marker, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Expression of a Diverse Array of Ca 2+ -Activated K + Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney

doi: 10.1371/journal.pone.0155006

Figure Lengend Snippet: AQP2 staining was used to identify PC, the AQP2-positive cells, and IC, the APQ2-negative cells. Bar graphs give the mean ± SEM for the normalized intensities for each KCa channel for both PC and IC. The number of PC and IC cells analyzed, n, is indicated on the bar graphs. The images of CCD show representative immunofluorescence examples for each KCa channel and AQP2 (40X) where one or two PC (AQP2-positive) and IC (AQP2-negative) cells are labelled as “PC” or “IC.” The outer border of the tubule in each image is indicated by the dashed white line (indicating the basal side or anti-luminal side of tubular cells). “L” identifies the tubular lumen. The immunofluorescence intensity was determined for each KCa channel using ImageJ and normalized to the intensity levels of AQP2 expression in PC (see ). Each example gives staining for AQP2 (red), the KCa channel (green), and the merged image (AQP2, KCa channel) that includes DAPI staining to identify nuclei. A. Expression of SK1 in PC and IC showing dominant relative expression in IC over PC (P<0.001). SK1 expression was apparent within the cytosol and along the luminal border of both PC and IC as apparent in the representative images. Light staining along the basolateral border is also apparent in some cells. B. Expression of SK3 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). SK3 expression was apparent within the cytosol with light expression along the basal aspect of some cells and more dominant expression along the luminal border of PC. C. Expression of IK1 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). IK1 expression was apparent within the cytosol, especially of IC, but also apparent along the luminal border of PC with light staining along the basal aspect of the cells. D. Expression of BKα in PC and IC showing strong staining along the luminal border of both PC and IC. However, the intensity of staining between PC and IC was found to vary depending on the primary antibody used for the immunostaining. In our hands, the Alomone APC-151 BKα antibody staining was most apparent in PC over IC (P<0.001, compare bar graph labeled APC-151 with APC-107). In contrast, the Alomone APC-107 BKα antibody staining was most apparent in IC over PC. Since the antibodies were made to different epitopes of the BKα channel, such variations can be anticipated (see text).

Article Snippet: Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight.

Techniques: Staining, Immunofluorescence, Expressing, Immunostaining, Labeling