Journal: bioRxiv

Article Title: Human iPSC-derived CNS and retinal microvasculature-on-a-chip models recapitulate hallmarks of diabetic microvascular pathology

doi: 10.64898/2026.01.15.699708

Figure Lengend Snippet: Gene expression changes in CNS-mv-on-a-chip induced by high glucose level. (A) The principal component analysis (PCA) plot shows clustering of samples cultured under physiological (5.5 mM glucose) and hyperglycemic (75 mM glucose) conditions. (B) Volcano plot illustrating differentially expressed genes between physiological and hyperglycemic CNS-mv-on-a-chip conditions. Orange and blue dots represent significantly upregulated and downregulated genes, respectively (FDR < 0.05, |FC| >2). (C) Heatmap showing upregulated genes associated with inflammation. Values are shown as fold changes relative to control group mean. (D) Over-representation analysis (ORA) plot of genes upregulated under hyperglycemia conditions. (E) Quantitative PCR analysis of IL1B, JUN and NFKBIA expression in ECs and PCs isolated from CNS-mv-on-a-chip cultured under physiological 5.5 mM glucose concentration (5.5G), hyperglycemic conditions 25 mM (25G) and 75 mM glucose (75G), or corresponding osmotic controls with mannitol (19.5M and 69.5M). Gene expression was normalized to GAPDH and HMBS as housekeeping genes and calculated using the ΔΔCt method. Data are presented as mean ± SD, n = 3-4.

Article Snippet: First, a pre-amplification step with TaqMan primers (Thermo Fisher Scientific) was carried out for GAPDH (Hs99999905_m1), HMBS (Hs00609297_m1), IL1B (Hs01555410_m1), JUN (Hs01103582_s1), and NFKBIA (Hs00355671_g1).

Techniques: Gene Expression, Cell Culture, Control, Real-time Polymerase Chain Reaction, Expressing, Isolation, Concentration Assay

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Tet2 deficiency induces downregulation of Nfkbia expression in germinal center B‐cells . (A) Normalized gene expression level of Nfkbia by RNAseq of Tet2 ‐WT and Tet2 ‐KO GC B‐cells, data from. ²⁹ (B) Gene expression levels by RT‐PCR of Nfkbia in Tet2 ‐WT and Tet2 ‐KO GC B‐cells at D8 (IgG1+) and D4 (IgM+ and IgG1+) ( n = 4). (C) Quantification of iPC (GFP+ CD19+ CD138+), iGCB (GFP+ CD19+ CD138− Fas+ GL7+), IgG1+ iGCB (GFP+ CD19+ CD138− Fas+ GL7+ IgG1+), and MFI of IgG1 in IgG1+ iGCB at D11 of the iGCB transduction model, after transduction of Tet2 ‐WT B‐cells by a MSCV containing sh Renilla , a MSCV containing shRNA Nfkbia 1224, or a MSCV containing shRNA Nfkbia 1485 ( n = 4). For A and B, P values were calculated using an unpaired two‐tailed t ‐test. For (C) P values were calculated using a paired RM one‐way ANOVA test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant in all experiments. ANOVA, analysis of variance; iGCB, induced germinal center B‐cells; iPC, induced plasma cells; MFI, mean fluorescence intensity; MSCV, murine stem cell virus.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Transduction, shRNA, Two Tailed Test, Clinical Proteomics, Fluorescence, Virus

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: The effect of Vitamin C on plasma cell differentiation of Tet2 ‐WT and Tet2 ‐KO B‐cells . (A) Schematic illustration of Vitamin C treatment in the in vitro iGCB culture system. (B) Number of live Tet2 ‐WT and Tet2‐KO cells cultured on Day 4 (D4) and Day 8 (D8), in the presence and absence of vitamin C ( n = 5). (C) Graph shows iPC (CD19+ CD138+) percentages at D8 upon treatment of Tet2 ‐WT ( n = 6) and Tet2 ‐KO ( n = 6) B‐cells with Vitamin C (250 µM) or vehicle. (D) Quantification of the MFI of IgG1 staining on the cell surface of D8 iGCB (CD19+ CD95+ GL7+) from Tet2 ‐WT and Tet2 ‐KO mice, in the presence and absence of vitamin C ( n = 9). (E) Gene expression levels by RT‐PCR of Nfkbia in Tet2 ‐WT and Tet2 ‐KO iGCB cells at D8 in the presence and absence of vitamin C ( n = 3). (F) Snapshot from UCSC genome browser showing 5hmC mark distribution at the Nfkbia locus (32,380 bp, mm10, chr12: 55.474.813−55.507.192) in D4 iGCB cells, in the presence and absence of vitamin C. All P values were calculated using an unpaired two‐tailed t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant, in all experiments. 5hmC, 5‐hydroxymethylcytosine; D, Day; iGCB, induced germinal center B‐cells; iPC, induced plasma cells; MFI, mean fluorescence intensity.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Clinical Proteomics, Cell Differentiation, In Vitro, Cell Culture, Staining, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Fluorescence

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Diagram depicting the effects of Tet2 inactivation on IgG1 + GC B cells during primary and secondary immunizations, alongside a proposed model of the underlying molecular mechanisms . At the cellular level, Tet2 ‐deficient mice exhibit germinal center (GC) hyperplasia and impaired plasma cell differentiation following T‐dependent immunization. During primary immunization, Tet2 inactivation reduces the generation of IgG1+ GC B‐cells by impairing isotype switching to IgG1 and hindering their differentiation into plasma cells and memory B‐cells. This deficiency enhances the proliferative response of IgG1+ GC B‐cells to external cues, leading to their accumulation during subsequent immunizations and a persistent differentiation blockade. At the molecular level, Tet2 deficiency directly impacts Prdm1 (encoding Blimp1) expression through hypermethylation of its regulatory elements and induces sustained DNA methylation at the Nfkbia locus. This results in the downregulation of Nfkbia , leading to increased c‐Rel activity, which further represses Blimp1 expression. Elevated IgG1 BCR expression likely sustains c‐Rel activation. Consequently, there is an increased proliferation capacity of IgG1+ GC B‐cells, coupled with a blockade in memory and plasma cell differentiation.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Clinical Proteomics, Cell Differentiation, Expressing, DNA Methylation Assay, Activity Assay, Activation Assay