Journal: Cell calcium

Article Title: Differential modulation of SK channel subtypes by phosphorylation.

doi: 10.1016/j.ceca.2020.102346

Figure Lengend Snippet: Fig. 1. The apparent Ca2+ sensitivity of SK1 and IK channel subtypes is more decreased than that of the SK3 and SK2 subtypes in immortal ized endothelial cells. Concentration-dependent activation by Ca2+ of the SK channel subtypes heterologously expressed in HEK293 cells (A) and immortalized endothelial cells (B). (C) EC50 values for the activation by Ca2+, recorded from SK channel subtypes expressed in HEK293 cells (HEK) and immortalized endothelial cells (ET). All data are shown as mean ± S.E.M (n = 6-11, **P < 0.01, ***P < 0.001, Two-way ANOVA followed by Tukey’s post hoc tests).

Article Snippet: The proteins were transferred to PVDF membranes and incubated overnight at 4 °C with primary GFP antibody (1:2000; Invitrogen, Lot: 2,015,993), SK1 antibody (1:1000; RayBiotech, Lot: 907,002,018), SK2 antibody (1:200; Alomone Labs, Lot: APC028AN2325), SK3 antibody (1:200; Alomone Labs, Lot: APC025AN1125), IK antibody (1:1000; Santa Cruz, Lot: I2319), mitochondria marker Cytochrome C antibody (1:1000; Novus Biologicals, Lot: AB0115109A-2), ER marker GRP78/HSPA5 (1:4000; Novus Biologicals, Lot: H-1) antibody or plasma membrane marker sodium potassium ATPase alpha 1 (1:4000; Novus Biologicals, Lot: B-5) antibody.

Techniques: Concentration Assay, Activation Assay

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