Journal: bioRxiv

Article Title: Coordinated inhibition of SOX9 and cell cycle progression by microRNA-200 restricts sebaceous gland fate specification

doi: 10.64898/2026.01.09.698672

Figure Lengend Snippet: a. Conserved miR-200 targeting sequence on the 3’UTR sequences of Csnk1a1 and Btrc . b & c. Luciferase reporter assay for Csnk1a1 (b) & Btrc (c) showing the functional relevance of miR-200 binding sites to their respective 3’UTR. A Student’s t-test was performed for statistical analysis. d. Spatial transcriptomics clusters of control and induced skin at P3. e. Schematics of scHolography analysis from ST and scRNA-seq together. f. Expression of selected marker genes along the epidermal-to-matrix axis. The solid line connects the median gene expression level at each computed distance bin along the epidermal-to-matrix axis. g-i. Expression of Wnt10a (g), Wnt10b (h) , and Sox9 (i) along the epidermal-to-matrix axis shows upregulation of Wnt10a and Wnt10b , as well as downregulation of Sox9 in the upper HF region. Solid lines connect the mean expression level for individual genes for each sample across the spatial bins. Wilcoxon test was performed for statistical analysis. j. Representative contour plot for HF-keratinocytes marked by EpCAM-APC + /SOX9-eGFP + from 2 control and 3 induced skin samples harvested at P1. k. Modal distribution of GFP intensity of the EpCAM + /SOX9-eGFP + of both control (red) and induced (blue) cells. l. SOX9 IF shows uHF-specific downregulation in induced HF compared to control. Scale bar: 20 μm. m. NFATc1 and PPARγ downregulation in induced HF reveals compromised SOX9 function. Scale bar: 20 μm. n. In SOX9 cKO and miR-200 induced samples, LEF1+ cells were detected in the uHF region marked by a white bracket whereas these LEF1+ cells were absent in the control uHF. White brackets annotate the uHF region. Scale bar: 20 μm.

Article Snippet: Primary antibodies and dilutions used in this study are: PPARγ (1:400; Cell Signaling Technology #2443), SCD1 (1:400; Santa Cruz Biotechnology #sc-14719), LEF1 (1:400; Cell Signaling Technology #2230), FOXC1 (1:100; Cell Signaling Technology #8758), KRT5 (1:2000; Covance #SIG-3475), β4 integrin (1:400; clone 346-11A; BD Pharmingen #553745), SOX9 (1:200; Milipore Sigma #HPA001758), Alexa Fluor 488 conjugated with SOX9 (1:200; Millipore Sigma #AB5535-AF488), NFATc1 (1:10; Developmental Studies Hybridoma Bank #7A6).

Techniques: Sequencing, Luciferase, Reporter Assay, Functional Assay, Binding Assay, Control, Expressing, Marker, Gene Expression

Journal: Frontiers in Immunology

Article Title: Mitochondrial calcium shapes B cell signaling and mitochondrial function

doi: 10.3389/fimmu.2025.1710128

Figure Lengend Snippet: The loss of MCU alters cell signaling and oxygen consumption. (A–G) Mcu fl/fl x mb1 CreERT2 (MCU -/- ) and control (Ctrl) mice were injected with tamoxifen for three consecutive days to induce B cell-specific Mcu deletion. (A) Representative immunoblot analysis of MCU protein levels in MCU -/- and Ctrl mouse B cells stimulated with anti-IgM for 2 days. Representative of 3 independent experiments. (B) Mitochondrial Ca 2+ levels in MCU -/- and Ctrl mouse B cells stimulated with anti-mouse IgM (10µg/ml) for 1 (left) and 2 days (right) were determined by Rhod-2 AM staining. Pooled data from 6 and 5 independent experiments, respectively. (d1: n=9 for MCU -/- and 10 for Ctrl, d2: n=8 for MCU -/- and Ctrl mice). (C) Measurement of mouse B cell proliferation by eFluor 670 dilution. MCU -/- and Ctrl mouse B cells were stimulated with anti- IgM and tracked for 3 days. Representative of 3 independent experiments. (n=6 MCU -/- and 5 Ctrl mice). (D) Cell survival analysis of mouse B cells shown in (C) . Forward scatter (FSC) and side scatter (SSC) properties were used to determine the percentage of living cells. Pooled data from 3 experiments. (n=6 MCU -/- and 5 Ctrl mice). (E) Cytosolic Ca 2+ was assessed in MCU -/- and Ctrl B cells using indo-1 AM. Shown are basal and peak Ca 2+ levels after anti-IgM stimulation as well as the difference between peak and basal levels (delta). Pooled data from 5 independent experiments. (n=7 for MCU -/- and for Ctrl). (F) Representative immunoblot analysis of NFAT2 protein levels in MCU -/- mouse B cells compared to Ctrl B cells. Cells were stimulated with anti-mouse IgM (10µg/ml) for 48 h. Blots were probed for NFAT2 and actin. Representative of 3 independent experiments. (G) Quantification of (F) was performed by normalization of NFAT2 levels to actin levels. Then, fold change in NFAT2 levels of MCU -/- mouse B cells relative to Ctrl B cells was calculated. Pooled data from 3 independent experiments. (n=5 Ctrl and 7 MCU -/- mice). (H–N) Mcu fl/fl and Ctrl B cells were treated with TAT-CRE and stimulated with anti-IgM. (H) Representative immunoblot analysis of MCU protein levels in MCU -/- and Ctrl mouse B cells stimulated with anti-IgM for 48h. Blots were probed with MCU and actin antibodies. Representative of 3 independent experiments. (I) Mitochondrial Ca 2+ levels in MCU -/- and Ctrl mouse B cells stimulated as in (H) was determined with Rhod-2 AM staining. Pooled data from 5 independent experiments. (n=6 MCU -/- and 7 Ctrl mice). (J) MCU -/- and Ctrl B cells were stimulated with anti-IgM. Proliferation was assessed by eFluor 670 dilution. Representative of 3 independent experiments. (K) Representative immunoblot analysis of NFAT2 protein expression in MCU -/- and Ctrl mouse B cells shown in (H) . Blots were probed for NFAT2 and actin. Representative of 3 independent experiments. (L) Quantification of (K) was performed by normalization of NFAT2 levels to actin levels. Then, fold change of NFAT2 levels of MCU-deficient mouse B cells relative to Ctrl B cells was calculated. Pooled data from 3 independent repeats. (n=4 for MCU -/- and Ctrl mice). (M) Representative oxygen consumption measurement of MCU -/- and Ctrl mouse B cells as in (H) . Oxygen consumption rates (OCR) were measured by Seahorse flux technology after sequential injection of oligomycin (1µM), FCCP (1µM) and rotenone + antimycin A (1µM). Representative of 3 independent experiments. (N) Summary graphs showing calculated basal and maximal respiration in anti-IgM stimulated MCU-deficient and Ctrl mouse B cells shown in (M) . Pooled data from 3 independent experiments. (n=3 for MCU -/- and Ctrl). Data are presented as mean. Paired (B, I, N) and unpaired (G, L) Student’s t tests were used to compare groups. *p < 0.05; ns, not significant.

Article Snippet: Membranes were probed with antibodies against MCU, MCUR1, NFAT2, total and cleaved caspase 3, pPLCγ2(Y1217), pBtk(Y223), pS6(S235/236), pAkt(S473), PLCγ2, Btk, Akt (Cell Signaling Technology), and β-actin (Proteintech) overnight at 4°C.

Techniques: Control, Injection, Western Blot, Staining, Expressing