Journal: Cancer research communications

Article Title: Na + /H + -exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells

doi: 10.1158/2767-9764.crc-22-0270

Figure Lengend Snippet: (A) Liver weight of mice with doxycycline (DOX)-repressible c-Myc-driven HCC fed with regular or sodium bicarbonate (NaHCO3)-containing drinking water and treated with anti-PD1 antibody (iPD1) or isotype control (IgG). Each dot represents an individual mouse liver weight in the different groups, all determined upon euthanasia at twelve weeks after birth (N = 10, 43, 43, 30, and 31 animals for H2O + DOX, H2O + IgG, NaHCO3 + IgG, H2O + iPD1, and NaHCO3 + iPD1 groups, respectively; unpaired t-test). (B) Representative microscopic images of the mTORC1 target phospho-S6 ribosomal protein (pS6)-stained tumor slices. Scale: 100 μm (inset: 40 μm). (C) Quantification of pS6-positive objects in images in B (N = 5 whole-tumor scans from different animals, one-way ANOVA with multiple comparisons). * p < 0.05, *** p < 0.001.

Article Snippet: Cells were then stained with PE-Cy7-conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 min on ice before analysis.

Techniques: Control, Staining

Journal: Cancer research communications

Article Title: Na + /H + -exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells

doi: 10.1158/2767-9764.crc-22-0270

Figure Lengend Snippet: (A) In vitro cytotoxicity of Torin1 (mTOR inhibitor)-treated NK-92 cells against the human melanoma cell line WM3629, relative to the untreated cells (E-T ratio = 3:1, N = 6 wells, one-way ANOVA with multiple comparisons versus untreated). (B) Representative immunoblots of the mTORC1 phosphorylation substrates phospho-p70 S6 kinase (pS6K) and phospho-S6 ribosomal protein (pS6) in constitutively active RHEB (RHEBN153T, hereafter referred to as RHEB)-overexpressing or empty vector (EV) control NK-92 cells treated with pH-controlled media for 6 or 24 hours. Endogenous RHEB is shown as the dim, lower band in the images, where the bright, upper band represents the overexpressed RHEB. (C) In vitro cytotoxicity of RHEB-expressing or EV control NK-92 cells against human melanoma cell lines WM3629 and WM4237 in pH-controlled media (E-T ratio = 3:1, N = 4 wells for each cell line, unpaired t-test). (D) Degranulation of RHEB-overexpressing or EV control NK-92 cells towards the human leukemia cell line K562 in pH-controlled media (E-T ratio = 1:2, N = 3 wells, unpaired t-test). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then stained with PE-Cy7-conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 min on ice before analysis.

Techniques: In Vitro, Western Blot, Phospho-proteomics, Plasmid Preparation, Control, Expressing

Journal: Cancer research communications

Article Title: Na + /H + -exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells

doi: 10.1158/2767-9764.crc-22-0270

Figure Lengend Snippet: (A) Representative immunoblots assessing mTORC1 substrates pS6K and pS6 in NK-92 cells expressing constitutively active NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. (B) Degranulation of trametinib (MEK inhibitor)-treated NK-92 cells against K562 cells, relative to untreated cells (E-T ratio = 1:2, N = 3 wells, one-way ANOVA with multiple comparisons versus untreated). (C) Representative immunoblots assessing phospho-p44/42 MAPK (pERK1/2) in NK-92 cells expressing NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. (D) Percentage of NHE1-, NHE1-E262I-, or EV-expressing NK-92 cells positive for intracellular pERK1/2 overtime after interacting with K562 cells (E-T ratio = 1:1, N = 3 wells, two-way ANOVA with multiple comparisons). * p < 0.05, *** p < 0.001.

Article Snippet: Cells were then stained with PE-Cy7-conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 min on ice before analysis.

Techniques: Western Blot, Expressing

Journal: Cancer Immunology Research

Article Title: Intracellular K + Limits T-cell Exhaustion and Preserves Antitumor Function

doi: 10.1158/2326-6066.CIR-23-0319

Figure Lengend Snippet: The Na + /K + ATPase restrains tonic signaling and CD8 + T-cell dysfunction. A, Summary bar graphs quantifying fluorescence of the voltage-sensitive indicator DiSBAC 2 3, reflecting the transmembrane electrochemical gradient ( V m ) of CD8 + T cells in standard RPMI-1640 (Vehicle) or isotonic hyperkalemic conditions (K + 20 mmol/L) captured by flow cytometry following CRISPR-Cas9–mediated disruption (Scramble, sgRNA Atp1a1 ) or transduction with retroviral particles. OE, overexpression; DN, Dominant negative. B and C, Summary quantification of cytoplasmic Ca 2+ as interval Fluo-3/FuraRed fluorescence following TCR cross-linking ( B ) or ionomycin induced store-operated calcium flux ( C ) as indicated, captured by flow cytometry. Iono, Ionomycin. n = 3 technical replicates depicted, representative of two independent experiments. D, Representative Phosflow cytometry plots and summary quantification of live, singlet, CD8 + T-cell populations in the presence or absence of soluble TCR cross-linking stimulation for five minutes. E, Concatenated single-cell overlay and colorimetric density tSNE-based depiction of the indicated proteins captured by flow cytometry. F, Representative flow cytometry plots and summary quantification for the indicated markers in ex vivo expanded CD8 + T cells. G, Representative and summary quantification of cytokines following acute TCR restimulation of ex vivo expanded CD8 + T cells. Error bars represent standard deviation. *, P < 0.05; **, P < 0.005; ****, P < 0.001, two-tailed Student t tests ( A, D, F, and G ). ****, P < 0.001 for two-way ANOVA ( B and C ).

Article Snippet: All antibodies were diluted in 1:200 for staining, except for phosflow antibodies (pS6 S235/6 PE-Cy7, pAKT S473 AF488, pERK T202/Y204 Pacific Blue (Cell Signaling Technology), and pCD3 Y142 PE (BD Biosciences), which were diluted in 1:400 pCD3ζ Y142 PE (BD Biosciences).

Techniques: Fluorescence, Flow Cytometry, CRISPR, Disruption, Transduction, Retroviral, Over Expression, Dominant Negative Mutation, Cytometry, Ex Vivo, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Adiponectin Limits IFN-γ and IL-17 Producing CD4 T Cells in Obesity by Restraining Cell Intrinsic Glycolysis

doi: 10.3389/fimmu.2019.02555

Figure Lengend Snippet: Obesity increases Th17 cell glycolysis and adiponectin dampens Th17- glycolysis in an AMPK dependent manner. (A) Plasma adiponectin levels in normal control diet (NCD) and high fat diet (HFD) fed mice. Western blot analysis of phospho-AMPK was determined. CD4+ T cells from NCD and HFD fed mice were stimulated with anti-CD3/CD28 for 15 min and expression of phospho-AMPK was measured (B) . (C) MFI of pS6 on CD4+ T cells in NCD and HFD mice following 30 min culture with anti-CD3/CD28. (D–F) Relative messenger RNA expression of glycolytic enzymes hk1 (D) , ldh-a (E) , and pkm (F) in purified Th17 cells in NCD and HFD mice. (G,H) Extracellular acidification rate was measured by seahorse in differentiated Th17 cells from HFD mice and the response to adiponectin and compound C were recorded. (I) Naïve CD4+ T cells were differentiated into Th17 cells in the presence of adiponectin and frequencies of IL-17+ Th17 cells were measured. Two-tailed non-parametric Mann–Whitney U -test was performed for statistical analysis.

Article Snippet: CD4-PE-Cy7 and pS6-PE antibodies were purchased from eBioscience and all the other antibodies were procured from Biolegend (Fell, Germany).

Techniques: Western Blot, Expressing, RNA Expression, Purification, Two Tailed Test, MANN-WHITNEY

Journal: Blood

Article Title: Aberrant STAT5 and PI3K/mTOR pathway signaling occurs in human CRLF2 -rearranged B-precursor acute lymphoblastic leukemia

doi: 10.1182/blood-2011-12-389932

Figure Lengend Snippet: Expanded analysis of primary CRLF2r ALL samples demonstrates inhibition of JAK/STAT and PI3K pathway signaling. Analysis of JAK/STAT, PI3K/mTOR, and MAPK pathway STIs in 29 CRLF2wt (A) and CRLF2r (B) primary human leukemias. Data are displayed as whisker plots of 25th to 75th percentiles with means (central bars) and ranges (whiskers). The dashed horizontal line represents normalized basal phosphorylation for each phosphoprotein. Pink bars indicate TSLP; striped red, ruxolitinib, solid red, ruxolitinib + TSLP; striped green, rapamycin; solid green, rapamycin + TSLP; striped blue, PI103; solid blue, PI103 + TSLP; striped purple, PP242; solid purple, PP242 + TSLP; striped gray, PD901; solid gray, PD901 + TSLP. Minimal effects of TSLP stimulation or STIs were observed for the CRLF2wt samples. TSLP stimulation induced pSTAT5, pAkt, pS6, p4EBP1, peIF4E, and pERK in the CRLF2r samples (P < .05), and incubation with ruxolitinib resulted in continued inhibition of all aforementioned phosphoproteins (P < .05). PI103 inhibited TSLP-induced pAkt, pS6, and pERK (P < .05). PP242 abrogated TSLP-induced pAkt, pS6, and peIF4E (P < .05). PD901 inhibited TSLP-induced pS6, peIF4E, and pERK (P < .05).

Article Snippet: 37 Antibodies were from BD Biosciences (CD3-Ax700 or CD3-Pacific Blue; CD10-PE-Cy7, CD19-Ax700 or CD19-APC-Cy7; CD127-Ax647, pSTAT5 Y694 -Ax647, pERK T42/44 -Ax647, pAkt S473 -Ax488 or -V450; pS6 S235/236 -Ax488 or -V450; and isotype controls), Cell Signaling Technologies (unconjugated pS6 S235/23 and p4EBP1 T37/46 ), Invitrogen (unconjugated peIF4E S209 and Pacific Blue–conjugated secondary donkey anti–rabbit IgG), EBioscience (TSLPR-PE or -PerCP-eFluor-710 clone 1A6, CD127-FITC, CD132-PE), and Jackson ImmunoResearch (FITC-conjugated secondary donkey anti–rabbit IgG and goat anti–rat IgG).

Techniques: Inhibition, Whisker Assay, Incubation

Journal: Cell reports

Article Title: mTOR Overcomes Multiple Metabolic Restrictions to Enable HIV-1 Reverse Transcription and Intracellular Transport

doi: 10.1016/j.celrep.2020.107810

Figure Lengend Snippet: (A) Cells from HIV-1+ participants have increased mTOR signaling, compared to cells from healthy controls. PBMCs from viremic HIV-1+ research participants and healthy controls (HC) were surface phenotyped to identify CD4 T cells and analyzed without any ex vivo stimulation for the expression of the canonical phosphorylated target downstream of mTOR, phosphorylated ribosomal protein S6 (p-S6), by multiparameter flow cytometry. Each group had cells from 4 independent donors. Means and SDs of data are shown. (B) mTOR inhibition decreased p-S6 in cells from HIV-1+ participants. (C) mTOR inhibition decreased p-S6 in cells from both viremic and ART-treated HIV-1+ participants. (B and C) PBMCs from viremic HIV-infected (n = 13) (B) and ART-suppressed HIV-infected (n = 14) (C) study participants were treated with either mTORi or DMSO vehicle for 8 h ex vivo and analyzed for mTOR activity (p-S6) by flow cytometry. A representative histogram is presented for PBMCs obtained from a HIV-infected donor cultured in the absence and presence of mTORi. Data from PBMCs for all of the participants are plotted in (C). The means and SDs of data are shown. Significant differences were determined by 2-tailed Student’s t tests: *p < 0.05, **p < 0.01, and ***p < 0.005.

Article Snippet: The following antibodies were used: CD3-BV510 (BD Biosciences, #563109), CD4-PE-Cy7 (Biolegend, #344612), CD71-AF700 (BD Biosciences, #563769), Bcl-2-PE (BD Biosciences, # 340651), pStat5-PE-CF594 (pY694) (BD Biosciences, #562501), and pS6-AF488 (Cell Signaling Technology, #5018).

Techniques: Ex Vivo, Expressing, Flow Cytometry, Inhibition, Infection, Activity Assay, Cell Culture

Journal: Cell reports

Article Title: mTOR Overcomes Multiple Metabolic Restrictions to Enable HIV-1 Reverse Transcription and Intracellular Transport

doi: 10.1016/j.celrep.2020.107810

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used: CD3-BV510 (BD Biosciences, #563109), CD4-PE-Cy7 (Biolegend, #344612), CD71-AF700 (BD Biosciences, #563769), Bcl-2-PE (BD Biosciences, # 340651), pStat5-PE-CF594 (pY694) (BD Biosciences, #562501), and pS6-AF488 (Cell Signaling Technology, #5018).

Techniques: Recombinant, ATP Assay, Plasmid Preparation, Expressing, Software