Journal: bioRxiv

Article Title: Multidimensional characterization of cellular ecosystems in Hodgkin lymphoma

doi: 10.1101/2025.03.18.643177

Figure Lengend Snippet: (A) Cohort and study design overview. We analyzed whole exome and targeted sequencing data from 114 CHL fresh frozen samples, including 103 patients treated with ABVD-based regimens. Subsequently, we developed tissue microarrays of the cohort and applied whole transcriptome sequencing of CD30+ HRS cells using Nanostring GeoMx technology as well as multiplexed analysis using imaging mass cytometry. Key findings were validated using an independent validation cohort (n=293). (B) Oncoplot showing the most recurrently mutated genes within the CHL samples in the discovery cohort, with clinical annotations. (C) Significantly amplified (red) or deleted (blue) regions across our in-house CHL cohort. (D) Pairwise (Fisher’s exact test) comparison of the incidence of mutations within thymic vs non-thymic CHL cases. The size of the point indicating the strength of association. (E) Frequency of STAT6 mutations in CHL according to age group (YOUNG: < 45 years, OLD: 45 years or older) at diagnosis. YOUNG: < 45 years. Fisher’s exact test was used to determine the significance of differences in observed mutational frequencies. (F) Progression-free survival (PFS) according to mutation and copy number status of STAT6 in young CHL patients (age < 45). P -values were calculated using a log rank test.

Article Snippet: Immunoblotting was performed as previously described( Alig et al ., 2023 ) using the following primary antibodies: STAT1 Rabbit mAb (D1K9Y), STAT3 Mouse mAb (124H6), STAT6 Rabbit mAb (D3H4), AKT(pan) Rabbit mAb (C67E7), Phospho-STAT1 (Y701) Rabbit mAb (58D6), Phospho-STAT3 (Y705) Rabbit mAb (D3A7), Phospho-STAT5 (Y694) Rabbit mAb (C11C5), Phospho-STAT6 (Y641) Rabbit mAb (C11A12), Phospho-AKT (S473) Rabbit mAb (193H12), GAPDH Rabbit mAb (14C10) (all from Cell Signaling Technology, dilution 1:1000), STAT5 Rabbit polyclonalAb (C-17) (dilution 1:5000), CSF2RB (IL-3/IL-5/GM-CSFRβ Mouse mAb (A-3) (dilution 1:100) (Santa Cruz Biotechnology), followed by secondary staining using anti-Mouse (dilution 1:10000; Promega, catalog no. W4021) or Rabbit IgG (H+L) HRP conjugate (dilution 1:5000; Promega, catalog no. W4011), and detected using Amersham ECL Detection Reagents (Cytiva, catalog no. RPN3004).

Techniques: Sequencing, Imaging, Mass Cytometry, Amplification, Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: TRAF3 regulates STAT6 activation and T-helper cell differentiation by modulating the phosphatase PTP1B

doi: 10.1016/j.jbc.2024.107737

Figure Lengend Snippet: Impact of TRAF3 on STAT6 activation in T cells. Primary mouse CD4 T cells or HT28.11 cells were left unstimulated, stimulated through TCR–CD28, or cultured with IL-4 for the indicated number of minutes/hours. Cells were lysed, and indicated proteins were quantified by Western blotting. A , representative Western blots of phospho-STAT6 Y641 and total STAT6. B , quantification of independent experiments, including blot shown in ( A ). Each symbol represents one biological replicate. C , representative Western blots of phospho-STAT6 Y641 and total STAT6 in primary mouse CD4 T cells stimulated with IL-4. D , quantification of three independent experiments, including the blots shown in ( C ). Each symbol represents one biological replicate. E , representative Western blots of phospho-STAT6 Y641 and total STAT6 in HT28.11 cells stimulated with IL-4. F , quantification of six independent replicates, including the blots shown in ( E ). ∗∗∗∗ p < 0.0001 by Sidak multiple comparisons test. Error bars represent SEM. IL-4, interleukin 4; STAT6, signal transducer and activator of transcription 6; TCR; T-cell receptor; TRAF3, tumor necrosis factor receptor–associated factor 3.

Article Snippet: Rabbit antibeta actin (catalog no.: 4967), rabbit anti–phospho Akt T308 (C31E5E; catalog no.: 2965), rabbit anti–phospho Akt S473 (D9E; catalog no.: 4060), rabbit anti-JAK1 (6G4; catalog no.: 3344), rabbit anti-PTP1B (catalog no.: 5311), rabbit anti–phosphoSTAT1 Y701 (58D6; catalog no.: 9167), rabbit anti-STAT1 (catalog no.: 9172), rabbit anti–phosphoSTAT6 Y641 (D8S9Y; catalog no.: 56554), and rabbit anti-STAT6 (D3H4; catalog no.: 5397) were purchased from Cell Signaling Technologies.

Techniques: Activation Assay, Cell Culture, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: TRAF3 regulates STAT6 activation and T-helper cell differentiation by modulating the phosphatase PTP1B

doi: 10.1016/j.jbc.2024.107737

Figure Lengend Snippet: Impact of TRAF3 on IL-4-mediated STAT6 activation in T cells stimulated through TCR–CD28. Primary mouse CD4 T cells or HT28.11 cells were stimulated as indicated, then lysed, and the indicated proteins were quantified by Western blotting. A , representative Western blots of phospho-STAT6 Y641 and total STAT6 in primary mouse CD4 T cells left untreated or treated with combinations of the following: TCR–CD28 (24 h), IL-4 (during last 15 min of TCR–CD28 stimulation), and an IL-4 neutralizing antibody (24 h). B , quantification of four biological replicates, including the blots in ( A ). C , representative Western blots of phospho-STAT6 Y641 and total STAT6 in HT28.11 cells left untreated or treated with the indicated combinations of the following: TCR–CD28, 2 h; IL-4, 15 min. D , quantification of six independent replicates, including the blots in ( C ). E , STAT6 activation ratio (detailed in text) from six independent replicates, including the blots in ( C ). ∗ p < 0.05, ∗∗ p < 0.001 by Fisher multiple comparisons test ( B and D ) or unpaired t test ( E ). Error bars represent SEM. IL-4, interleukin 4; STAT6, signal transducer and activator of transcription 6; TCR; T-cell receptor; TRAF3, tumor necrosis factor receptor–associated factor 3.

Article Snippet: Rabbit antibeta actin (catalog no.: 4967), rabbit anti–phospho Akt T308 (C31E5E; catalog no.: 2965), rabbit anti–phospho Akt S473 (D9E; catalog no.: 4060), rabbit anti-JAK1 (6G4; catalog no.: 3344), rabbit anti-PTP1B (catalog no.: 5311), rabbit anti–phosphoSTAT1 Y701 (58D6; catalog no.: 9167), rabbit anti-STAT1 (catalog no.: 9172), rabbit anti–phosphoSTAT6 Y641 (D8S9Y; catalog no.: 56554), and rabbit anti-STAT6 (D3H4; catalog no.: 5397) were purchased from Cell Signaling Technologies.

Techniques: Activation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: TRAF3 regulates STAT6 activation and T-helper cell differentiation by modulating the phosphatase PTP1B

doi: 10.1016/j.jbc.2024.107737

Figure Lengend Snippet: Role of PTP1B in TRAF3-mediated regulation of STAT6 activation. A – C , cells of the human CD4 T-cell line HT28.11 were stimulated through TCR–CD28 and/or with IL-4, then JAK1 was immunoprecipitated, and lysates were analyzed by Western blotting. A , representative Western blots of JAK1, PTP1B, and TRAF3 coimmunoprecipitating with JAK1 (“IP:JAK1,” top ) or in whole cell lysates (“input,” bottom ) from five independent replicates. B , quantification of PTP1B coimmunoprecipitating with JAK1 from four independent replicates including the blots in ( A ) ( top ). C , quantification of TRAF3 coimmunoprecipitating with JAK1 from four independent replicates including the blots in ( A ) ( top ); only WT HT28.11 samples are quantified because TRAF3 was undetectable in crTRAF3 −/− samples. D – G , cells of the human CD4 T-cell line HT28.11 were treated with the PTP1B inhibitor TCS-401 or an equal volume of dimethyl sulfoxide (DMSO) for 2 h, stimulated through TCR–CD28 and/or with IL-4, then lysed for quantification of proteins of interest by Western blotting. D , representative Western blots of phospho-STAT6 Y641 following IL-4 stimulation for the indicated number of minutes with or without TCS-401, as indicated. E , quantification of five independent replicates, including the blots shown in ( D ). F , representative Western blots of phospho-STAT6 Y641 following TCR–CD28 and/or IL-4 stimulation, ±TCS-401/DMSO, as indicated. G , quantification of four independent replicates, including the blots shown in ( F ). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by Tukey multiple comparisons test. Each graphed symbol represents an individual biological replicate, and error bars represent SEM. IL-4, interleukin 4; STAT6, signal transducer and activator of transcription 6; TCR, T-cell receptor; TRAF3, tumor necrosis factor receptor–associated factor 3.

Article Snippet: Rabbit antibeta actin (catalog no.: 4967), rabbit anti–phospho Akt T308 (C31E5E; catalog no.: 2965), rabbit anti–phospho Akt S473 (D9E; catalog no.: 4060), rabbit anti-JAK1 (6G4; catalog no.: 3344), rabbit anti-PTP1B (catalog no.: 5311), rabbit anti–phosphoSTAT1 Y701 (58D6; catalog no.: 9167), rabbit anti-STAT1 (catalog no.: 9172), rabbit anti–phosphoSTAT6 Y641 (D8S9Y; catalog no.: 56554), and rabbit anti-STAT6 (D3H4; catalog no.: 5397) were purchased from Cell Signaling Technologies.

Techniques: Activation Assay, Immunoprecipitation, Western Blot