Journal: International Journal of Molecular Sciences

Article Title: Proinflammatory Cytokine Preconditioning Enhances the Therapeutic Potency of Different Types of MSCs in Inflammation

doi: 10.3390/ijms27094090

Figure Lengend Snippet: The effects of naïve and Cytomix-stimulated MSCs on T cell proliferation. The gating strategy is shown in . ( A – C ) BM-MSCs, iMSCs WT and iMSCs B2M KO inhibition on the expansion of CD3+, CD3+CD4+ and CD3+CD8+ T cells via CM. Cytomix enhanced the three types of MSCs’ inhibition on the EI of T cells. ( D – F ) All types of MSCs significantly inhibited the division of CD3+, CD3+CD4+ and CD3+CD8+ T cells when co-cultured with PBMCs, in a dose-dependent manner. ( G – I ) All of the MSCs, naïve or Cytomix stimulated, significantly reduced the EI of CD3+, CD3+CD4+ or CD3+CD8+ T cells, at the lowest ratio of MSCs, with iMSCs WT both naïve and Cytomix-stimulated presenting the strongest inhibition. ( J ) Co-culturing all types of MSCs, naïve or Cytomix-stimulated, increased the production of G-CSF in the media, with a dose-dependent effect. ( K ) All types of MSCs significantly decreased the level of IFN-γ at the ratios of 1:20 and 1:10, while the three types of naïve MSCs significantly decreased it at 1:50. ( L ) Naïve and Cytomix-stimulated iMSC WTs significantly increased the level of TNF-α at the ratios of 1:50, 1:20 and 1:10, while Cytomix-stimulated iMSC B2M KO significantly increased it at the ratio of 1:50 and 1:20. ( M ) Naïve BM-MSCs significantly increased the level of IL-10 production at the ratios of 1:20 and 1:10. Representative results of four independent experiments are shown. Results are shown as mean ± SD ( n = 3 in CM; n = 4 in co-culture, each group), and analysed by two-way ANOVA with Šídák’s multiple comparisons test to compare naïve vs. Cytomix in each type of MSCs (no significance in all of the settings), or Tukey’s multiple comparisons test to compare different ratios of MSCs to PBMCs in each MSC group, or Dunnett’s multiple comparisons test to compare different MSC groups vs. vehicle in the setting of the same ratio. */**/***/**** represent comparisons of each type of MSC vs. vehicle; #/## represent comparisons of different ratios of each cell type with different levels of p < 0.05/0.01. EI: expansion index; DP: division percentage.

Article Snippet: The PBMCs were stained with anti-human CD3-APC-Vio770, CD4-APC and CD8-PE-Vio770 antibodies (Cat# 130-113-136, 130-113-222, 130-110-680; Miltenyi Biotec) for 30 min on ice in the dark.

Techniques: Inhibition, Cell Culture, Co-Culture Assay

Journal: bioRxiv

Article Title: A targetable dependency on nonsense-mediated decay for proteostasis and immune control in small cell lung cancer

doi: 10.64898/2026.03.31.715503

Figure Lengend Snippet: A) DNA Sanger sequencing of the amplified B2M locus in representative H841 CRISPR/Cas9 WT and B2M-KO clones aligned to the reference sequence (NCBI, NG_012920.2) with sgRNA and the identified insertion mutation in H841 B2M-KO cells highlighted. B-D) Pharmacological NMD inhibition with either KVS0001 or SMG1i-11j compounds in healthy PBMC donors upon T cell artificial activation. ( B ) Proportion of viable CD45+ cells (left) and T cells (right) within PBMC populations after 4 days treatment with a T cell artificial activation cocktail (Act = CD3+CD28+IL-2) vs unstimulated conditions (Naive). ( C ) Proliferation of CD4+ (left) and CD8+ (right) T cells derived from T cell counts expressed as fold change (FC) for activated (Act) conditions relative to the unstimulated (Naive) control. ( D ) Proportion of T cells (CD3+), B cells (CD19+), NK cells (CD56+) and Myeloid cells (CD11b+) within PBMC populations (CD45+) from healthy donors directly after thawing (d-1), at the beginning of stimulation (CD3+CD28+IL-2) (d0) 4 days post-stimulation (d4). Graphs represent mean + SEM (n = 7). ***P<0,001; **P<0.01; *P<0.05; ns=non-significant (One-way ANOVA). E) Representative flow cytometry dot plots for the staining of CD45 vs CD56 (for NK cells) and CD8 vs CD4 (both for T cells) following 4 days co-culture of healthy donor PBMCs with WT H841 tumor cells. F) In vivo tumor growth in immunocompetent C57BL/6J mice transplanted with murine RP1380 TetO-shCTRL fed with normal or doxycycline-containing diet (n ≥ 4). G) Flow cytometry immunophenotyping of RP1380 TetO-shCTRL tumors harvested at the end of experiments shown in panel F (n ≥ 4). H-I) MHC-I surface expression quantified by flow cytometry in the indicated human and murine cell lines following genetic NMD inhibition via siRNA-mediated SMG1-KD and UPF1-KD ( H ) or doxycycline-inducible SMG1-KD ( I ). J) In vivo assessment of MHC-I (H2-Kb) surface expression in control vs NMD-inhibited murine RP1380 TetO-shSMG1 tumors (following doxycycline diet, DOXY) grown subcutaneously in C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in C57BL/6 mice significantly correlating tumor-specific H2-Kb surface expression with levels of immune cell infiltration (CD45+ infiltration). L) In vivo levels of tumor-specific MHC-I (H2-Kb) surface expression in control vs NMD-inhibited RP1380 TetO-shSMG1 tumors (DOXY) grown subcutaneously in RAG1-KO C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in RAG1-KO C57BL/6 mice correlating tumor-specific H2-Kb surface expression with levels of immune cell (CD45+) infiltration. L) Representative flow cytometry contour plot showing T cells (CD45+/CD3+) and B cells (CD45+/CD19+) in the blood from WT C57BL/6 and RAG1-KO C57BL/6 mice.

Article Snippet: Human primary antibodies used were B2M (Molecular probes #A15770), MHC-ABC (Invitrogen #11998342), CD45 (Miltenyi #130110636), CD3 (Miltenyi #130113142), CD4 (Biolegend #357410), CD8 (Miltenyi #130110678), CD56 (Miltenyi #130113310), CD11b (Miltenyi #130110556).

Techniques: Sequencing, Amplification, CRISPR, Clone Assay, Mutagenesis, Inhibition, Activation Assay, Derivative Assay, Control, Flow Cytometry, Staining, Co-Culture Assay, In Vivo, Expressing