Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Unperturbed B cell development in Sam68 complete KO mice. A . left panel- representative images of the spleen from WT, Het (+/-) and KO (-/-) mice. Right panel- quantification of absolute splenic cell number from these mice ( n = 10–13, 4–5 independent sampling). Statistical analysis: One-way ANOVA, multiple comparison test. ** p < 0.01. B . left panel- flow cytometric analysis of B cell subpopulations in bone marrow (Hardy fractions A-F), spleen, lymph node, peritoneal cavity and T cell subpopulations in thymus from WT, Het and KO mice. Right panel- quantification of the different cell populations as depicted in the flow cytometry. For the BM B cells, % of cells are calculated as follows: Fraction A- CD43 + B220 + CD24 − BP1 − , Fraction B- CD43 + B220 + CD24 + BP1 − , Fraction C- CD43 + B220 + CD24 lo BP1 + , Fraction C´- CD43 + B220 + CD24 hi BP1 + , Fraction D -CD43 − B220 lo IgD − IgM − , Fraction E -CD43 − B220 lo IgD − IgM + and Fraction F -CD43 − B220 hi . Bars represent mean ± SEM ( n = 3 for each genotype). DP- double positive, DN- double negative

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Sampling, Comparison, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Impaired T-dependent immune response in Sam68 KO mice against SRBC. A . Scheme showing the experimental timeline for T-dependent SRBC immunization. B . Flow cytometric analysis of B cell activation and GC reaction in SRBC treated WT and Sam68 KO mice 10 days post immunization. C . Histogram showing expression of GL7 on the activated (CD95 + CD38 − ) B cells in the spleen of SRBC treated WT and KO mice. D . Quantification of CD38 − CD95 + activated B cells in the spleen of SRBC treated WT and KO mice. Bars represent mean ± SEM. Statistical analysis- unpaired t-test. E . Quantification of anti-SRBC IgG1, IgG2b and IgG3 in the serum of WT (black) and Sam68 KO (red) mice at day10 after immunization by flow cytometry. Statistical analysis: Two-way ANOVA, multiple comparison test ( n = 5–6, two independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001. F . Flow cytometric analysis of T-follicular helper (TfH, CD4 + CXCR5 + PD1 + ) cells in the spleen of WT and Sam68 KO mice 10 days after SRBC immunization. G. Histogram showing the expression of BCL6 in the TfH cells in WT and Sam68 KO mice. H - I . Quantification of CD4 + T cells (H) and TfH cells (I) in the spleen of immunized WT and Sam68 KO mice. Bars represent mean ± SEM. Statistical analysis- unpaired t-test

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Activation Assay, Expressing, Flow Cytometry, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Impaired T-dependent immune response and memory response in Sam68 complete KO mice against TNP-Ova. A. Scheme showing the experimental timeline for primary immune response using TNP-Ova. B. Quantification of total serum IgG in WT (black) and KO (red) mice at day0 (unfilled) and day10 (filled) after TNPOva immunization. The amount of serum IgG is normalized with respect to the IgG at day0. Bars represent mean ± SEM (n=5-8, total two independent experiments). Statistical analysis: One-way ANOVA, multiple comparison test. C-D- Quantification of anti-TNP IgG (C) and anti-TNP IgM (D) in the serum of TNP-Ova treated WT (black) and KO (red) mice. Statistical analysis: Two-way ANOVA, multiple comparison test. E. Flow cytometric analysis of B cell activation and GC reaction in TNP-Ova treated WT and KO mice. F. Histogram showing expression of GL7 on the activated (CD95+ CD38-) B cells in the spleen of TNP-Ova treated WT and KO mice. G. Quantification of GL7+ cells in the spleen of control and TNP-Ova treated WT (black unfilled and shaded respectively) and KO (red unfilled and shaded respectively) mice. H. Scheme showing the experimental timeline for secondary immune response using TNP-Ova. I-J. Quantification of total serum IgG (I) and anti-TNP IgG (J) in WT (black) and Sam68 KO (red) mice at different time point after TNP-Ova immunization. The amount of serum IgG is normalized with respect to the IgG at day0. Statistical analysis: Two-way ANOVA, multiple comparison test (n=5-7, two independent experiments). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Comparison, Activation Assay, Expressing, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Analysis of SRBC induced GC response in competitive bone marrow chimeric Rag −/− γc −/− mice. A . Flow cytometric analysis showing the percentage of CD45.1 + WT and CD45.2 + Sam68 KO B cells in total spleen (CD19 + , left most panel) and in GC cells (CD38 − CD95 + GL7 + , right most panel). B . Quantification of the percentage of WT and KO B cells in blood, spleen and GC ( n = 9, total three independent experiments). C . Flow cytometric analysis showing the percentage of CD45.1 + WT and CD45.2 + WT B cells in blood and GC. D . Quantification of the percentage of CD45.1 + WT and CD45.2. + WT B cells in blood and GC ( n = ). Bars represent mean ± SEM. Statistical analysis: One-way ANOVA, Holm-Sidak’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Sam68 regulates Traf4 expression in B cells through mir29. A - B . Real time quantitative PCR (RT-qPCR) analysis measuring expression of miR29a, b and c (A) and Traf4 (B) in WT (black bars) and Sam68 KO (red bars) splenic B cells at different time points after CD40 stimulation. Bars represent mean ± SEM ( n = 3–4 independent experiments). Statistical analysis: Two-way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001. C . Flow cytometric analysis showing the electroporation efficiency of empty vector (EV) and pR miR29a in WT B cells as detected by DsRed/mRFP expression. % of DsRed or mRFP. + cells are indicated in the figure. D - E . RT-qPCR analysis measuring expression of miR29a (D) and Traf4 (E) in WT B cells electroporated either with EV or pR miR29a (29a OE). Bars represent mean ± SEM ( n = 3). Statistical analysis: one sample t-test. * p < 0.05. F . Representative western blot showing Traf4 expression in WT B cells overexpressing miR29a (29a OE) compared to EV expressing cells. Traf4 over-expressing WT B cells (Traf4 OE) are used as positive control. Lower band- endogenous Traf4, upper band- 3X FLAG tagged overexpressed Traf4 (see methods and Supplementary Fig. for details). Gapdh is used as loading control. G. Quantification of the band intensities of the western blot shown in F. Bars represent mean ± SEM ( n = 3). Statistical analysis: One way ANOVA, multiple comparison test. * p < 0.05. H. Flow cytometric analysis showing the retroviral transduction efficiency of EV and mTraf4 in WT and Sam68 KO B cells as detected by DsRed expression. % of DsRed + cells are indicated in the figure. I. Proliferation analysis of WT and Sam68 KO B cells overexpressing either EV (upper panel) or Traf4 protein (bottom panel) 48 h post stimulation with IL4 + anti-CD40 antibody. Green histograms indicate the different generation of cells as modeled by proliferation modeling in Flowjo. J. Quantification of proliferation parameters for WT and KO cells overexpressing EV (open bars) or Traf4 (T4, dotted bars). Bars represent mean ± SEM ( n = 3–4). Statistical analysis: One way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Electroporation, Plasmid Preparation, Western Blot, Positive Control, Control, Retroviral, Transduction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Sam68 KO B cells show proliferation defect upon Anti-CD40 antibody stimulation in vitro. A . Schematic representation of the in vitro splenic B cell proliferation assay. B . Flow cytometric analysis of splenic B cell proliferation 96 h after treatment with IL4 + LPS (upper panel) and IL4 + anti-CD40 antibody (bottom panel) for WT and Sam68 KO mice. C . Quantification of proliferating B cells in response to IL4, IL4 + LPS and IL4 + anti-CD40 antibody in culture derived from WT (black) and KO (red) mice spleen ( n = 3). D . Quantification of WT and Sam68 KO B cells class switched to IgG1 in response to IL4, IL4 + LPS and IL4 + anti-CD40 antibody. Statistical analysis: Two-way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: In Vitro, Proliferation Assay, Derivative Assay, Comparison