Journal: medRxiv

Article Title: The PKN1- TRAF1 signaling axis as a potential new target for chronic lymphocytic leukemia

doi: 10.1101/2021.03.22.21253655

Figure Lengend Snippet: (A) Base line expression of TRAF1 in shCTL, shTRAF1 and shPKN1 RAJI cell lines, representative of 7 independent experiments. (B) shCTL RAJI cells were treated with the indicated inhibitor at doses ranging from 0.1nM-10μM for 24h. TRAF1 expression was measured by intracellular flow cytometry after treatment. dMFI refers to the MFI for the TRAF1 stain minus the FMO (background) control. To calculate the IC50 for PF-941222, AT7867, ipatasertib and uprosertib where TRAF1 expression was not maximally reduced, TRAF1 was assumed to reach 0 dMFI. N.D. = not determined. (C). Effect of OTSST167 on TRAF1 levels and death of RAJI cells measured by activated caspase 3. (D) Baseline expression of TRAF1 and phosphorylation of signaling intermediates pS6, pNF-κBp65 and pERK in shCTL, shPKN1 and shTRAF1 RAJI cell lines (top). shCTL RAJI cells were treated with OTSSP167 or XL-228 at the indicated concentration for 24h. TRAF1, pS6, pNF-κBp65 and pERK were measured by flow cytometry (middle, bottom). Data are representative of 2 independent experiments.

Article Snippet: Cells were then stained with anti-CD19 BV605, anti-CD5 PE-Cy7, anti-human pNFκBp65-eFluor 660 (clone B33B4WP), anti-pS6 PE (clone cupk43k) and anti-pERK PerCP-eF710 (clone MILAN8R) from eBioscience for 1h at room temperature.

Techniques: Expressing, Flow Cytometry, Staining, Concentration Assay

Journal: medRxiv

Article Title: The PKN1- TRAF1 signaling axis as a potential new target for chronic lymphocytic leukemia

doi: 10.1101/2021.03.22.21253655

Figure Lengend Snippet: Phosphorylated signaling intermediates pS6, pNF-κBp65 and pERK were measured in live CLL cells after treatment with DMSO control or (A) OTSSP167 or (B) XL-228 for 24h at the indicated concentrations. In each panel, line graphs (left) show mean dMFI ± SD, n=6 CLL patient samples (2 independent experiments pooled), and histograms (right) from a representative donor depict the MFI of pS6, pNF-κBp65 or pERK in CLL cells after treatment with DMSO or the indicated concentration of inhibitor. The same 6 CLL samples were used here as in and .

Article Snippet: Cells were then stained with anti-CD19 BV605, anti-CD5 PE-Cy7, anti-human pNFκBp65-eFluor 660 (clone B33B4WP), anti-pS6 PE (clone cupk43k) and anti-pERK PerCP-eF710 (clone MILAN8R) from eBioscience for 1h at room temperature.

Techniques: Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: PTEN drives Th17 cell differentiation by preventing IL-2 production

doi: 10.1084/jem.20170523

Figure Lengend Snippet: Pten deficiency in Th17 cells induces phosphorylation of STAT5. (A–F) pAkt (S473) (A), pAkt (T308) (B), pS6 (C), pSTAT3 (D), pSTAT5 (E), and IL-6R (F) expression was measured by flow cytometry. Mean fluorescence intensity of each experiment was measured and pooled from three independent experiments (right). Naive CD4 + T cells from WT ( Pten fl/fl ; red line) and cKO ( Pten fl/fl Il17a cre ; blue line) mice were cultured under Th17-polarizing conditions for 3 d. (G) Naive CD4 + T cells from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice were cultured under Th17-polarizing conditions for 3 d in the presence or absence of a STAT5 inhibitor (10 µM). The percentage and number of IL-17A + and FOXP3 + cells were measured by flow cytometry. Data were pooled from three individual experiments (right). (H) Cells were cultured under the same conditions described in (G). Il17a, Rorc, Il23r, and Foxp3 mRNA levels were measured by qRT-PCR, and data were normalized to Gapdh expression. Data were pooled from three independent experiments. Flow cytometry data represent three independent experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. n.s., not significant.

Article Snippet: Cells were stimulated with mouse recombinant IL-6 (100 ng/ml, eBioscience) for 15 min at 37°C or with PMA (40 nM) for 15 min at 37°C and then harvested, fixed, and permeabilized in IC Fixation Buffer (eBioscience), and stained with FITC-conjugated anti-pSTAT3 (Y705, 557814, BD Bioscience), FITC-conjugated anti-pSTAT5 (Y694, 11–9010-41, eBioscience), PE-conjugated anti-pAkt (T308, 558275, BD Bioscience), PE-conjugated anti-pAkt (S473, 560378, BD Bioscience), and PE-conjugated anti-pS6 (S235/S236, 12–9007-41, eBioscience).

Techniques: Expressing, Flow Cytometry, Fluorescence, Cell Culture, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Heterozygous splice mutation in PIK3R1 causes human immunodeficiency with lymphoproliferation due to dominant activation of PI3K

doi: 10.1084/jem.20141759

Figure Lengend Snippet: PI3K signaling is constitutively active in patient immune cells. (A) Signaling diagram showing the major molecules downstream of the p110δ/p85α PI3K complex. PDK1: phosphoinositide-dependent kinase-1; mTORC: mechanistic target of rapamycin complex; GSK3: glycogen synthase kinase 3; S6K: ribosomal protein S6 kinase; 4EBP1: eukaryotic translation initiation factor 4E-binding protein 1; FOXO: forkhead box O. (B) Immunoblot for AKT phosphorylated at threonine 308, total AKT, and actin (top), and flow cytometric analysis of AKT phosphorylation at serine 473 (bottom) in T cell blasts from healthy relatives or control subjects (Ctrl) and the indicated patients. (C) Flow cytometric analysis of the phosphorylation of S6 at serine residues 235/236 and serine residues 240/244 in T cell blasts from healthy relatives or control subjects (Ctrl) and the indicated patients (stained in same experiment as in B, bottom). (D) Immunoblot for phosphorylation of FOXO protein threonines (T) and GSK3 protein serines (S), total protein, or β-tubulin loading control, as indicated. Four (B and C) or three (D) independent experiments on the two indicated patients and controls were performed.

Article Snippet: For phosflow analyses, anti-pAKT S473 Alexa Fluor 647 (D9E; Cell Signaling Technology), anti-pAKT T308 Alexa Fluor 488 (C31E5E; Cell Signaling Technology), anti-pS6 S235/236 PE (N7-548; BD), and/or anti-pS6 S240/244 Alexa Fluor 647 (D68F8; Cell Signaling Technology) were used.

Techniques: Binding Assay, Western Blot, Staining