Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) MFIs from phospho-flow cytometry of the indicated markers are shown. T EX-PROG or T EX-TERM cells from LCMV Cl 13 infected mice 30 d.p.i. were stimulated with PMA ex vivo for 30 mins (all but c-Fos and c-Jun) or 4 hours (c-Fos and c-Jun). (B) Expression of PKC theta and eta by subset from LCMV Cl 13, 30 d. p.i. (C) Validation of PKC theta ( Prkcq ) and PKC eta ( Prkch ) knockouts by flow cytometry and Western blot. (D) Mice were infected with LCMV clone 13, and congenic P14 T cells were negative selected and activated in vitro via platebound anti-CD3/28. After 24 hours, cells were subjected to Cas9-sgRNA electroporation to delete genes of interest, and after a further 24 hours of recovery, electroporated cells were mixed and co-transferred into infected mice (E-F) The frequencies of each congenic population of electroporated cells at the time of co-transfer (E), and the frequencies of cells of each genotype relative to the total CD8 T cell population at the indicated timepoints post-infection (F) (G-H) The frequencies of indicated markers among transferred PD1+ CD8 T cells, 8 d.p.i (I-K) The frequencies of indicated markers (I-J) and TOX MFI (K) in transferred PD1+ cells, 28-30 d.p.i Scatter plots show mean +/- s.e.m. Statistical significance was determined using Student’s t-test (A, I-K) or one way ANOVA (panels B-H), with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Flow Cytometry, Infection, Ex Vivo, Expressing, Western Blot, In Vitro, Electroporation

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Schematic of in vitro exhaustion experiments. P14 T cells were activated in vitro , expanded, and co-cultured with B16-gp33 cells from days 3-7 post-activation. (B) Representative flow plots showing the effect of chronic PMA treatment on T cells in vitro . (C) Cumulative bar graphs of PMA effects on in vitro exhausted cells. (D) nCounter data highlighting PMA-driven gene expression changes. T EX-TERM associated genes are in red, while T EX-PROG associated genes are in blue. Data are shown as log2 fold change. (E) Cartoon schematic of the regulation of PKC kinase activity. (F) Western blots showing PKC protein levels in cultured primary CD8 + T cells after 4 days of PMA treatment. (G) Effects of KO of Prkcq or Prkch on responsiveness to PMA during in vitro exhaustion. (H) Domain architecture of PKC theta. Numbers represent the amino acids defining each domain and the three phosphorylation sites key for PKC kinase activity. (I) A two-round K-to-R mutagenesis screen to identify lysine residues required for PKC activity-induced degradation after 24 hours of PMA treatment. Lysine residues were first mutated in blocks of four to six at a time (left), and then individual lysine residues were mutated within the 409-451 subset (right) (J-K) P14 T cells were transduced with EV or PKC OE vectors, subjected to in vitro exhaustion, and assessed for their capacity to produce cytokines. Scatter plots show mean +/- s.e.m. Statistical significance was determined using Student’s t-test (panel C) or one way ANOVA (panels G, I-K), with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: In Vitro, Cell Culture, Activation Assay, Expressing, Activity Assay, Western Blot, Mutagenesis, Transduction

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Timeline of adoptive transfer experiments of P14 T cells into mice infected with LCMV Cl 13. Overexpression of PKC theta variants or an EV control were introduced using a retroviral (RV) vector. (B-C) MFI of PKC theta among all transferred GFP+ (B) and GFP+ T TERM cells (C). (D-F) Numbers of total GFP+ cells of the indicated genotypes, in total (D), or in SLAMF6 + TIM3 - (E) or SLAMF6 - TIM3 + (F) subsets. (G) Frequency GFP+ P14 T cells expressing CXCR6 in LCMV Cl 13. (H-I) Normalized MFI of the indicated markers in LCMV Cl 13. (J) Timeline of adoptive transfer experiments in B16gp33 tumor-bearing mice. (K-L) Tumor volumes (K) and masses at endpoint (L) of tumors by P14 genotype. (M) IFNγ producing functionality of P14 T cells infiltrating B16gp33 tumors by genotype. Scatter plots show mean +/- s.e.m. Statistical significance was determined using one way ANOVA where applicable, with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Adoptive Transfer Assay, Infection, Over Expression, Control, Retroviral, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Schematic of preparation of primary T cells for global phosphoproteomics. (B) Cartoon highlighting and simplifying major known pathways downstream of PKC. (C) Select, top-ranking PKC theta-specific or PKC eta-specific phosphopeptides identified from the anti-CD3 stimulation condition. Phosphorylated residues are highlighted in bold and followed with an asterisk (*). The gene name of the protein from which the peptide originates is shown to the left of each peptide. (D) GO terms associated with the top 100 PKC theta- vs eta-specific downstream phosphoproteins. (E) Volcano plot of Kinase Library analysis of kinases active downstream of PKC theta or eta after anti-CD3 stimulation. Re-stimulated sgPrkch or sgPrkcq samples were normalized to unstimulated controls of the matching genetic background, and the normalized phosphoproteomes were subsequently compared to one another. The kinases active in each condition (plotted) are inferred by identifying enrichment of ideal peptide motifs for each kinase in the entire proteomics dataset. (F) Phospho-flow cytometry of the indicated markers within in vitro activated CD8 T cells, stimulated for 30 minutes with PMA, in the indicated genetic backgrounds. (G) Fraction of top 100 PKC eta-specific peptides that score highly as CK1 family targets according to Kinase Library peptide motifs. (H) Schematic of in vitro exhaustion experiments used to screen inhibitors of kinases downstream of PKC theta and eta. (I) Screening effects of inhibitors of kinase families putatively downstream of the PKCs for markers of T EX-PROG differentiation. Scatter plots show mean +/- s.e.m. Statistical significance was determined using one way ANOVA where applicable, with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Flow Cytometry, In Vitro

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Cartoon of PKC theta signaling through the CARMA1-BCL10-MALT1 complex. (B) Western blot showing Regnase-1 levels after 30 minutes of PMA or vehicle treatment. T cells were collected after in vitro activation and 5 days of expansion. (C) Cartoon schematic of PKC eta proximity labeling strategy and conditions via TurboID. The TurboID-PKC eta fusion protein was transduced into primary T cells, and the expanded transductants were treated and collected 6-7 days post-activation. (D) Venn diagram of raw TurboID statistics. (E) Scatterplot of comparing the log2 fold enrichment of candidate PKC eta interacting proteins in anti-CD3-stimulated vs unstimulated conditions. Dashed lines and colors indicate protein subsets based on enrichment thresholds. Enrichment value was determined by dividing the peak area of candidate proteins by the mean peak area of negative control conditions. Only proteins with p adj < 0.01 are plotted. (F) Changes between the subcellular localizations and cellular functions of top candidate PKC eta interactors between stimulated and unstimulated conditions.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Western Blot, In Vitro, Activation Assay, Labeling, Negative Control

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Timeline of casein kinase I gene deletions in LCMV Cl 13 adoptive transfer experiments. (B) Frequency of the indicated genotype transferred P14 T cells among all CD8 + T cells .(C-E) Representative data (C), frequencies (C-D), and MFIs (E) of markers of differentiation and functionality of P14 T cells of the indicated genotypes. (F) Timeline of Csnk1g2 deletion in B16gp33 adoptive transfer experiments. (G) Masses of B16gp33 tumors at sacrifice timepoint by indicated genotype. (H) MFI of PD-1 in TTERM P14 T cells infiltrating B16gp33 tumors. (I) (Left) UMAP of integrated single cell RNA-seq datasets of CD8 + T cells isolated from human cancer biopsies (GSE146771, GSE99254, GSE98638), with cell subsets indicated by color. (Middle, right) Violin plots of PRKCQ and PRKCH expression by T cell subset. (J) Schematic of in vitro exhaustion experiments used to test the effects of CK1 inhibition and perturbations of PKC signaling on human CAR-T cells. (K) TNF production of in vitro exhausted human CAR-T cells by the indicated genotypes and treatments. Scatter plots show mean +/- s.e.m. Statistical significance was determined using Student’s t-test (panels C-E, G-H) or one way ANOVA (panels B, K), with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Adoptive Transfer Assay, RNA Sequencing Assay, Isolation, Expressing, In Vitro, Inhibition

Journal: Molecular Biology of the Cell

Article Title: The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

doi: 10.1091/mbc.e13-06-0333

Figure Lengend Snippet: FIGURE 3: Bcr is a novel interaction partner of the Par-Tiam1 complex. (A) Schematic depicting the role of the Par-Tiam1 complex in polarized cell migration. Tiam1-mediated Rac1 activation promotes cytoskeletal remodeling important for protrusion formation, whereas PKCζ regulates the reorientation of the centrosome. (B) Bcr interacts with Par3 in COS7 cells. Lysates from COS7 cells expressing Par3 in the presence or absence of Flag-tagged Bcr or Tiam1 (positive control) were immunoprecipitated (IP) with an α-Flag antibody, and then immunoblotted with an α-Par3 antibody. Lysates were also immunoblotted with α-Par3 and α-Flag antibodies to demonstrate protein expression. N = 3. (C) Bcr interacts with Par3 in astrocytes. Lysates from cultured rat cortical astrocytes (DIV21) were immunoprecipitated with control immunoglobulin G (IgG; nonimmune: NI) or α-Bcr antibodies, and then immunoblotted with the α-Par3 or α-Bcr antibody. N = 3. (D) Bcr interacts with PKCζ in COS7 cells. Lysates from COS7 cells expressing Tiam1 (positive control) or Bcr alone or in combination with Flag-PKCζ were immunoprecipitated with α-Flag antibody, and then immunoblotted with α-Tiam1 or α-Bcr antibodies. Lysates were also blotted with α-Tiam1, α-Bcr, or α-Flag antibodies to confirm protein expression. N = 3. (E) Bcr interacts with PKCζ in cortical astrocytes. Lysates from cultured rat cortical astrocytes (DIV21) were immunoprecipitated with control IgG (NI) or α-Bcr antibodies, and then immunoblotted with α-PKCζ or α-Bcr antibodies. N = 3. (F) Bcr and Tiam1 require PKCζ to interact with Par6 in COS7 cells. Flag-tagged Tiam1 or Bcr was coexpressed alone or in combination with myc-Par6 in the absence or presence of Flag-PKCζ. Tiam1 and Bcr were immunoprecipitated with α-Tiam1 or α-Bcr antibodies, respectively, and then immunoblotted with anti-myc antibody to assess Par6 association. Lysates were blotted with α-Flag antibody to confirm protein expression. N = 3. (G) Domain structures of full-length Bcr and Abr as well as truncated Bcr constructs used in

Article Snippet: PKCζ pseudosubstrate inhibitor was from Santa Cruz Biotechnology.

Techniques: Migration, Activation Assay, Expressing, Positive Control, Immunoprecipitation, Cell Culture, Control, Construct

Journal: Molecular Biology of the Cell

Article Title: The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

doi: 10.1091/mbc.e13-06-0333

Figure Lengend Snippet: FIGURE 4: Bcr negatively regulates PKCζ signaling by facilitating its degradation. (A) Western blot analysis of lysates obtained from WT and Bcr−/− mouse cortical astrocytes. Bcr-deficient astrocytes showed an increase in total PKCζ levels. Lysates were also immunoblotted with α-GAPDH antibodies for a loading control. (B) Quantification of PKCζ levels. N = 3. (C) Representative images showing the localization of PKCζ in WT and Bcr−/− astrocytes migrating in a scratch assay. PKCζ is localized to the leading edge (white arrows) in WT cells, whereas PKCζ immunostaining was increased and more diffuse in Bcr−/− cells. Dashed black line shows scratch location. Scale bar: 10 μm. (D) Western blot analysis of lysates obtained from WT and Bcr−/− mouse cortical astrocytes. Bcr-deficient astrocytes showed an increase in p-GSK-3β levels. Lysates were also blotted with total GSK-3β antibodies for a loading control. (E) Quantification of p-GSK-3β levels. N = 3. (F) Western blot analysis of lysates obtained from WT and Bcr−/− mouse cortical astrocytes. Bcr-deficient astrocytes showed an increase in β-catenin levels. Lysates were also blotted with α-GAPDH antibodies for a loading control. (G) Quantification of β-catenin levels. N = 3. (H) Western blot analysis of lysates from COS7 cells expressing myc (control) or myc-Bcr and treated with DMSO (control) or 10 μM of the proteasomal inhibitor MG132. Bcr overexpression reduces PKCζ levels, which is blocked by treating cells with MG132. Lysates were blotted with α-GAPDH antibodies for a loading control. (I) Quantification of PKCζ levels from (H). N = 3. (J) The ability of Bcr to reduce PKCζ levels depends on its Rac-GAP activity. Lysates from COS7 cells expressing myc (control) or myc-tagged Bcr, GAP-dead Bcr (BcrGD), constitutively active Rac (RacV12), or RacV12 plus myc-Bcr were immunoblotted with an α-PKCζ antibody. Lysates were

Article Snippet: PKCζ pseudosubstrate inhibitor was from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Wound Healing Assay, Immunostaining, Expressing, Over Expression, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

doi: 10.1091/mbc.e13-06-0333

Figure Lengend Snippet: FIGURE 5: Bcr regulates polarity by restricting PKCζ function. (A) PKCζ inhibitor can partially rescue protrusion formation in Bcr−/− cortical mouse astrocytes. Representative images showing WT and Bcr−/− astrocytes treated overnight with PBS (control) or 10 μM of PKCζ pseudosubstrate inhibitor and then fixed and immunostained for acetylated tubulin. Dashed white line represents the scratch. Scale bar: 10 μm. n = ∼100; N = 3. (B) PKCζ inhibitor can partially rescue centrosome reorientation in Bcr−/− cortical mouse astrocytes. Quantification of polarized centrosomes in WT and Bcr−/− cortical mouse astrocytes treated overnight with PBS (control) or 10 μM of PKCζ pseudosubstrate inhibitor. n = ∼100; N = 3. (C) Overexpression of Bcr, but not Abr, negatively regulates PKCζ levels. Western blot analysis was performed on lysates from COS7 cells expressing control (empty vector) or myc-tagged Tiam1, Bcr, or Abr. Lysates were immunoblotted with α-PKCζ antibodies to assess PKCζ levels and α-GAPDH for a loading control. N = 3. (D) Quantification of PKCζ levels from (C). N = 3. (E) Bcr and Abr overexpression reduces Pak phosphorylation in COS7 cells. Western blot analysis of lysates from COS7 cells expressing control (myc) or myc-tagged Tiam1, Tiam1 and Bcr, or Tiam1 and Abr, immunoblotted for PKCζ. Lysates were blotted with an α-GAPDH antibody for a loading control. (F) Quantification of PKCζ levels from (E). N = 3. (G) Expression of Bcr, but not Abr, rescues protrusion defects in Bcr−/− cortical mouse astrocytes. WT astrocytes were transfected with eGFP as a positive control. Bcr−/− astrocytes were transfected with eGFP alone or in combination with Bcr or Abr expression plasmids, and then astrocytes were subjected to scratch assays. Cells were then fixed 18 h postscratch and immunostained for acetylated tubulin (red). Yellow dashed line represents scratch. Scale bar: 10 μm. (H) Quantification of the protrusion assay. n = ∼100; N = 3. (I) Expression of Bcr, but not Abr, can rescue centrosome reorientation defects in Bcr−/− cortical mouse astrocytes. Quantification of polarized centrosomes in WT astrocytes transfected with eGFP (positive control) or Bcr−/− cortical mouse astrocytes transfected with eGFP, eGFP and Bcr, or eGFP and Abr. n = ∼100; N = 3. Data are shown ± SEM.

Article Snippet: PKCζ pseudosubstrate inhibitor was from Santa Cruz Biotechnology.

Techniques: Control, Over Expression, Western Blot, Expressing, Plasmid Preparation, Phospho-proteomics, Transfection, Positive Control

Journal: Cell

Article Title: PKC? regulates ATF2 availability to alter mitochondrial permeability following genotoxic stress

doi: 10.1016/j.cell.2012.01.016

Figure Lengend Snippet: A) SCC9 cells were transfected with empty vector or constitutively active PKCε (caPKCε), and subject to immunoblot analysis with indicated antibodies. Densitometric ratios of bands for (−caPKCε, +caPKCε) are as follows: pS729/β-actin (0.193, 0.636); pT52/Total ATF2 (0.215, 0.662). B) Control or ATF2-knocked down LU1205 cells were subject to immunoblot analysis with indicated antibodies. C) Indicated cell lines were subject to (10 μM, ETO, overnight) etoposide treatment and subject to immunoblot analysis with indicated antibodies. Densitometric band ratios for pT52/Total ATF2 (−ETO, +ETO) are as follows: SCC9 (1, 0.4), UACC903 (1, 0.7), 501Mel (1, 0.9), LU1205 (1, 1). Densitometric band ratios for pS729/Total PKCε (−ETO, +ETO) are as follows: SCC9 (1, 0.7), UACC903 (1, 0.7), 501Mel (1, 0.97), LU1205 (1, 1). D) Control or ETO-treated coverslip-grown LU1205, 501Mel or UACC903 cells were immunofluorescently stained with indicated antibodies. E) 501Mel cells treated with 10 μM PKCε translocation inhibitor (PKCε-i) alone or in the presence or absence of 5 μM etoposide (ETO, overnight) were stained with Annexin-V and subject to FACS analysis. n = 10,000 cells per replicate; three replicates per condition were performed. •: p = 0.0009; #: p = 0.0032

Article Snippet: MitoTracker Red CMXRos, PKCε translocation inhibitor, Gö6850, and Leptomycin B were purchased from Invitrogen (CA, USA), Santa Cruz Biotechnologies, (CA, USA), EMD Chemicals (NJ, USA), and Sigma (USA), respectively.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Staining, Translocation Assay

Journal: Cell

Article Title: PKC? regulates ATF2 availability to alter mitochondrial permeability following genotoxic stress

doi: 10.1016/j.cell.2012.01.016

Figure Lengend Snippet: Coverslip-grown SCC9 cells were immunofluorescently stained with indicated antibodies after: A) overnight treatment with 1 or 5 μM PKCε translocation inhibitor (PKCε-i); B) overnight treatment with 20 nM scrambled control (siSC) or PKCε-targeted (siPKCε) siRNA; C) transfection with HIS-tagged constitutively active PKCε (caPKCε, red) and treatment with 5 μM etoposide (ETO, overnight). D) SCC9 cells treated overnight with ETO or 5 μM PKCε translocation inhibitor (PKCε-i) were lysed and subject to immunoblot analysis with indicated antibodies. E) In vitro PKCε kinase assay with Millipore control peptide (CTL), purified GST-ATF2 50–100aa WT (WT), GST-ATF2 50–100aa WT + PKCε catalytic inhibitor, Gö6850 (WT+Gö) (3) or GST-ATF2 50–100aa T52A (52A) as substrate. *: non-specific bands. Coomassie-stained gel (left) and autoradiograph (right) are displayed as indicated. F) Coverslip-grown SCC9 cells were transfected with HA-tagged wild-type ATF2 (WT), ATF2T52A or ATF2T52E, and subsequently immunofluorescently stained with indicated antibodies. G) SCC9 control or ATF2 knocked-down cells (shATF2) were transfected with Jun2 promoter-luciferase construct and either empty vector (EV) or caPKCε were assayed for luciferase activity before and after overnight ETO or Gö6850 (2 μM) treatment.•: p = 0.0034; #: p = 0.0157. H) SCC9 cells stably knocked-down for ATF2 with a 3′-UTR-targeted shRNA were reconstituted with Jun2 promoter-luciferase construct and ATF2WT, ATF2T52A or ATF2T52E and were subsequently assayed for luciferase activity. *: p = 0.0047. Scale bars represent 10 μm.

Article Snippet: MitoTracker Red CMXRos, PKCε translocation inhibitor, Gö6850, and Leptomycin B were purchased from Invitrogen (CA, USA), Santa Cruz Biotechnologies, (CA, USA), EMD Chemicals (NJ, USA), and Sigma (USA), respectively.

Techniques: Staining, Translocation Assay, Control, Transfection, Western Blot, In Vitro, Kinase Assay, Purification, Autoradiography, Luciferase, Construct, Plasmid Preparation, Activity Assay, Stable Transfection, shRNA

Journal: Cell

Article Title: PKC? regulates ATF2 availability to alter mitochondrial permeability following genotoxic stress

doi: 10.1016/j.cell.2012.01.016

Figure Lengend Snippet: A) SCC9 cells were transfected with empty vector (EV) or HA-tagged ATF2 (W), ATF2T52A (A) or ATF2T52E (E) in the presence or absence of 5 μM etoposide (ETO, overnight), crosslinked and lysed for whole cell lysate (input) or immunoprecipitated for VDAC1 and subject to immunoblot analysis with indicated antibodies. B) SCC9 cells were treated with ETO (E) or PKCε translocation inhibitor (i, 5 μM) or both (i-E), lysed for whole cell lysate (input) or immunoprecipitated for VDAC1 and subject to immunoblot analysis with indicated antibodies. C) SCC9 cells transfected with EV, W, A or E in the presence (*, red bars) or absence (grey bars) of 5 μM etoposide overnight were pulse labeled with tetramethylrhodamine ethyl ester (TMRE) or nonylacridine orange (NaO), and subsequently analyzed by FACS analysis. Histogram bars represent ratios of TMRE/NaO uptake. See Figure S4 for individual TMRE and NaO values. N = 10,000 cells per replicate; 3 replicates per condition were performed. •: p < 0.0001; #: p = 0.01 D) Coverslip-grown SCC9 cells transfected with EV, W, A or E were immunostained for cytochrome C and MitoTracker, and analyzed for cytochrome C release by fluorescence microscopy (n>100 cells per replicate; 3 replicates per condition were performed).•: p = 0.0008 E) Right, SCC9 cells treated with PKCε-i (5μM) alone or in the presence of ETO (overnight) were stained with Annexin-V and propidium iodide (PI) and subject to FACS analysis. •: p < 0.0001; #: p = 0.6018. Representative FACS plots (left) and corresponding Annexin V-negative and Annexin V-positive (AV−, AV+) percentages are as follows: control (97%, 3%); PKCε-i (92%, 8%); ETO (89.5%, 11.5%); and ETO+PKCε-i (87%, 13%). Averaged value histograms (right) are displayed as indicated. F) SCC9 cells were transfected with EV, W, A or E in the presence (*, red bars) or absence (grey bars) of ETO. Cells were stained with Annexin-V and subject to FACS analysis. n = 10,000 cells per replicate; three replicates per condition were performed. •: p = 0.0001; #: p<0.0001

Article Snippet: MitoTracker Red CMXRos, PKCε translocation inhibitor, Gö6850, and Leptomycin B were purchased from Invitrogen (CA, USA), Santa Cruz Biotechnologies, (CA, USA), EMD Chemicals (NJ, USA), and Sigma (USA), respectively.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Translocation Assay, Labeling, Fluorescence, Microscopy, Staining, Control

Journal: Cell

Article Title: PKC? regulates ATF2 availability to alter mitochondrial permeability following genotoxic stress

doi: 10.1016/j.cell.2012.01.016

Figure Lengend Snippet: A) Primary keratinocytes (NHEK) and melanocytes (HEM), squamous carcinoma (M7, P9, SCC9) and melanoma (501Mel, LU1205, WM793 and UACC903) cell lines were subject to immunoblot analysis with indicated antibodies. Histogram represents ratio A/B, where A = densitometric ratio values of pT52/total ATF2 bands, and B = densitometric ratio values of pS729/total PKCε bands. B) Box plots showing distribution of PKCε in metastatic (left) and primary (right) specimens. PKCε levels are denoted on the Y-axis (P < 0.0001, t-statistic – 4.257). The mean +/− one standard deviation is depicted by the horizontal bars and is higher in the metastatic specimens. C) Kaplan Meier survival curves showing significantly shorter survival in higher PKCε expressers in primary tumors. D) Alignment of amino acid sequences flanking Thr52 in ATF2, as compared to consensus sequence used as PKCε inhibitor peptide. E) Schematic representation of PKCε-mediated regulation of the subcellular localization, and therefore, the oncogene or tumor suppressor activities of ATF2.

Article Snippet: MitoTracker Red CMXRos, PKCε translocation inhibitor, Gö6850, and Leptomycin B were purchased from Invitrogen (CA, USA), Santa Cruz Biotechnologies, (CA, USA), EMD Chemicals (NJ, USA), and Sigma (USA), respectively.

Techniques: Western Blot, Standard Deviation, Sequencing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression

doi: 10.1007/s00262-020-02492-0

Figure Lengend Snippet: PKCζ is necessary for C2 ceramide-induced inhibition of Akt phosphorylation and up-regulation of endogenous ceramide. a, b 2 × 106 B16F10 cells were either treated with PKCζ pseudo-substrate inhibitor (10 µM) for 1 h followed by C2 ceramide (20 µM) treatment for 24 h or transfected with empty vector (EV) or over-expressed PKCζ vector (PKCζ OV) as described in “Materials and methods”. Protein levels of PKCζ, p-Akt, Akt, and GAPDH were measured by western blotting. The expressions were normalized with GAPDH level. c B16F10 cells were treated with C2 ceramide. Cell lysates were immunoprecipitated after 24 h of incubation with anti-Akt antibody, and the changes in expression of PKCζ and Akt were analyzed by western blotting. d, e B16F10 cells were either treated with PKCζ pseudo-substrate inhibitor (10 µM) for 1 h followed by C2 ceramide (20 µM) treatment for 24 h or transfected with overexpressed PKCζ vector (PKCζ OV) as described in “Materials and methods”. Ceramide expression was measured by flow cytometry and confocal microscopy. Each experiment was performed thrice. Images were from one of the three representative experiments. Quantified data were represented as mean ± SD. *P < 0.05, **P < 0.001 vs untreated

Article Snippet: C2 ceramide, C6 ceramide, C8 ceramide, C16 ceramide, and PKCζ pseudo-substrate inhibitor were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: Inhibition, Phospho-proteomics, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Incubation, Expressing, Flow Cytometry, Confocal Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression

doi: 10.1007/s00262-020-02492-0

Figure Lengend Snippet: C2 ceramide represses the recruitment of M2 macrophages and maneuvers its repolarization in PKCζ dependent manner in vitro. 2 × 106 B16F10 cells were treated with or without PKCζ pseudo-substrate inhibitor (10 µM) for 1 h followed by C2 ceramide (20 µM) treatment for 24 h. 2 × 106 RAW264.7 cells were co-cultured with conditioned medium (CM) from B16F10 cells in transwell chambers as described in “Materials and methods”. CM of B16F10 cells used in control was left untreated. a Migration of treated RAW264.7 cells was assessed. b Expressions of CD206, CD163 and GAPDH mRNAs were analyzed by RT-PCR. The expressions were normalized with GAPDH level. c, d The treated RAW264.7 cell-free supernatants were assessed either for TNF-α, IFN-γ, IL-12p70, IL-10, and TGF-β by ELISA after 24 h or for nitrite generation after 48 h. e, f MHC-II and IFN-γ receptor expressions on treated RAW264.7 cells were analyzed by flow cytometry. Each experiment was performed thrice. Images were from one of the three representative experiments. Quantified data were represented as mean ± SD. *P < 0.05, **P < 0.001 vs untreated

Article Snippet: C2 ceramide, C6 ceramide, C8 ceramide, C16 ceramide, and PKCζ pseudo-substrate inhibitor were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: In Vitro, Cell Culture, Control, Migration, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression

doi: 10.1007/s00262-020-02492-0

Figure Lengend Snippet: Effect of C2 ceramide on A375 human melanoma cells. a 2 × 106 A375 cells were treated with C2 ceramide (dose: 0, 5, 10, 20, 30, 40, 50 µM) for 24 h and cell viability was analyzed by MTT assay. b A375 cell proliferation was evaluated after C2 ceramide (20 µM, 24 h) treatment by (3H)-thymidine incorporation assay. c The apoptosis of A375 melanoma cells was detected with Annexin-V FITC and PI staining by flow cytometry. d The cell-free supernatants, harvested from melanoma cells with or without C2 ceramide treatment, were assessed for TNF-α, IFN-γ, IL-12p70, IL-10, and TGF-β level by ELISA. e, f 2 × 106 A375 cells were either treated with PKCζ pseudo-substrate inhibitor (10 µM) for 1 h followed by C2 ceramide (20 µM) treatment for 24 h transfected with empty vector (EV) or overexpressed PKCζ vector (PKCζ OV) as described in “Materials and methods”. Protein levels of PKCζ, p-Akt, Akt, and GAPDH were measured by western blotting. The expressions were normalized with GAPDH level. g A375 cells were either treated with PKCζ pseudo-substrate inhibitor (10 µM) for 1 h followed by C2 ceramide (20 µM) for 24 h or transfected with over-expressed PKCζ vector (PKCζ OV). Ceramide expression was measured by flow cytometry. h 2 × 106 A375 cells were treated with or without PKCζ pseudo-substrate inhibitor (10 µM) for 1 h followed by C2 ceramide (20 µM) treatment for 24 h. 2 × 106 THP-1 cells were co-cultured with conditioned medium (CM) from A375 in transwell chambers as described in “Materials and methods”. CM of A375 cells used in control was left untreated. Migration of treated THP-1 cells was assessed. i, j MHC-II and IFN-γ receptor expressions on treated THP-1 cells were analyzed by flow cytometry. Each experiment was performed thrice. Images were from one of the three representative experiments. Quantified data were represented as mean ± SD. *P < 0.05, **P < 0.001 vs untreated

Article Snippet: C2 ceramide, C6 ceramide, C8 ceramide, C16 ceramide, and PKCζ pseudo-substrate inhibitor were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: MTT Assay, Thymidine Incorporation Assay, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Cell Culture, Control, Migration

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression

doi: 10.1007/s00262-020-02492-0

Figure Lengend Snippet: C2 ceramide restricts tumor progression via PKCζ-mediated repolarization of TAM towards M1 phenotype. a, b Control and lentiviral PKCζ-silenced B16F10 tumor-bearing mice (n = 4 mice/group) were left untreated or treated with liposome-encapsulated C2 ceramide as narrated in “Materials and methods”. Tumor growth and bodyweight of mice were monitored at 2-day interval. At day 21, animals were killed and the final tumor volume and body weight were taken. c, d For histopathological observation, liver and kidney sections of mice were stained with H&E (original magnification: × 200). e Single-cell suspension was generated from enzymatic digestion of the tumors from different experimental mouse groups. Tumor-associated macrophages (F4/80+/CD68+) were purified as mentioned in “Materials and methods”. The proportion of CD11b+/CD206+ M2-type TAMs was assessed from the purified TAMs by flow cytometry. f, g Isolated TAMs (1 × 105 cells) from experimental mouse groups were harvested in 96-well plate and subsequently assessed either for TNF-α, IFN-γ, IL-12p70, IL-10, and TGF-β level by ELISA after 24 h or for nitrite generation after 48 h. h, i MHC-II and IFN-γ receptor expressions on isolated TAMs were analyzed by flow cytometry. Each experiment was performed thrice. Images were from one of the three representative experiments. Quantified data were represented as mean ± SD. *P < 0.05, **P < 0.001 vs untreated

Article Snippet: C2 ceramide, C6 ceramide, C8 ceramide, C16 ceramide, and PKCζ pseudo-substrate inhibitor were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: Control, Staining, Suspension, Generated, Purification, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression

doi: 10.1007/s00262-020-02492-0

Figure Lengend Snippet: Repolarization of tumor microenvironment by C2 ceramide-induced PKCζ. a, b Single-cell suspension was generated from enzymatic digestion of the tumors from different experimental mouse groups. The level of VEGF was measured by ELISA from the tumor cell suspension representing TME. Expressions of VEGF, VEGFR1, VEGFR2, HIF1α, and GAPDH mRNAs were analyzed by RT-PCR. The expressions were normalized with GAPDH level. c–e Cryo-sectioned tumor samples were analyzed by immunofluorescence staining to detect the expressions of VEGF, HIF1α, VEGFR1, VEGFR2, ceramide and IFN-γ receptor (original magnification: × 100). f Tumors from different experimental mouse groups were assessed for population of CD8+ T cells, CD4+IFNγ+ T cells, Tregs (CD4+CD25+Foxp3+), MDSCs (CD11b+Gr1+) and mature DCs (CD11c+CD86+) by flow cytometry. g Expressions of Perforin and Granzyme B were analyzed from purified tumor-induced activated CD8+ cells (CD69+/CD8+) by flow cytometry. Each experiment was performed thrice. Images were from one of the three representative experiments. Quantified data were represented as mean ± SD. *P < 0.05, **P < 0.001 vs untreated

Article Snippet: C2 ceramide, C6 ceramide, C8 ceramide, C16 ceramide, and PKCζ pseudo-substrate inhibitor were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: Suspension, Generated, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Flow Cytometry, Purification