Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Single‐cell RNA sequencing showed cellular heterogeneity in the injured spinal cord and identified Hcar2 as a highly expressed gene in activated microglia following SCI. (A) Schematic of the bioinformatic workflow, which integrates public scRNA‐seq data (GEO: GSE162610 ) and employs nonnegative matrix factorization (NMF) and pseudobulk differential gene expression (DEG) analysis. (B) UMAP visualization of 12 distinct cell types identified from 59 040 cells in the mouse spinal cord. The cell clusters are color‐coded by annotation. (C) NMF consensus matrix heatmap identifying 10 stable gene meta‐programs (MPs). MP3, the immune activation module, is highlighted. (D) Gene Ontology (GO) enrichment heatmap for genes within the top MP. MP3 is highly enriched in neuroinflammatory and immune response pathways. (E) Volcano plot of DEGs (SCI vs sham). The upregulated genes ( n = 373; adjusted p < .001, log2FC > 1) are red; the downregulated genes ( n = 297; adjusted p < .001, log2FC < ‐1) are blue. (F) Venn diagram illustrating the intersection of significantly upregulated DEGs and the immune meta‐programme (MP3) signature. (G) Key genes, including Hcar2 , identified at the intersection of MP3 and upregulated DEGs, contributing to the ‘immune score’.

Article Snippet: To establish Hcar2 ‐knockdown microglial cell lines, lentiviral vectors expressing short hairpin RNA (shRNA) targeting mouse Hcar2 (sh Hcar2 ) and a scrambled negative control (NC) were constructed and packaged by Obio Technology.

Techniques: Single Cell, RNA Sequencing, Gene Expression, Activation Assay

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Hcar2 was specifically upregulated in an activated microglial subtype (AM3) following SCI. (A) UMAP plot showing Hcar2 expression, primarily localized to immune cell clusters. (B) Kernel density plot confirming high Hcar2 expression density within microglia and neutrophil populations. (C) Dotplot illustrating Hcar2 expression levels and percentage of expression across all identified cell types at 1‐, 3‐, and 7‐day post‐injury (dpi) compared to sham controls. (D) UMAP plot of the reclustered microglial population, identifying 7 distinct subtypes: homeostatic microglia (HM), differentiating microglia (DM), activated microglia (AM1‐3), and interferon‐related microglia (IRM). (E and F) Ridge plot (E) and density plot overlay (F) showing Hcar2 expression is highly and specifically concentrated within the AM3 microglial subtype. (G) Heatmap displaying scaled expression of selected marker genes defining each microglial subtype. (H and I) Pseudotime trajectory analysis using Monocle3 (H) and Slingshot (I), both of which identify a differentiation path from HM toward the AM3 subtype. (J) Hcar2 expression plotted along the pseudotime trajectory, demonstrating progressive upregulation during differentiation. (K) Gene ontology (GO) enrichment analysis for marker genes of each microglial subtype.

Article Snippet: To establish Hcar2 ‐knockdown microglial cell lines, lentiviral vectors expressing short hairpin RNA (shRNA) targeting mouse Hcar2 (sh Hcar2 ) and a scrambled negative control (NC) were constructed and packaged by Obio Technology.

Techniques: Expressing, Marker

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Hcar2 protein expression was upregulated in perilesional microglia following SCI. (A) Schematic timeline of the in vivo experimental design, indicating spinal cord tissue collection points (days post‐injury, dpi) and the definition of the IA and SA regions. (B) Representative Western blot analysis of Hcar2 protein levels in spinal cord lysates at 1, 3, 7 and 14 dpi. β‐Tubulin served as the loading control ( n = 4). (C) Densitometric quantification of Hcar2 protein levels relative to those of β‐Tubulin ( n = 4). (D) Representative confocal images of spinal cord sections at 7 dpi stained for Iba‐1 (microglia, grey) and Hcar2 (green). Nuclei were counterstained with DAPI (blue). Scale bars: 500 µm (overview), 100 µm (inset). (E) Fluorescence intensity line scan profile across a representative Iba‐1 + Hcar2 + cell in the IA region, demonstrating signal colocalization ( n = 5). (F) Quantification of the Hcar2 + area as a percentage of the total Iba‐1 + microglial area ( n = 5). (G) Representative immunofluorescence images of BV2 cells treated with vehicle or LPS (100 ng/mL) for 24 h and stained for Iba‐1 (red) and Hcar2 (green). Scale bar: 100 µm. (H) Quantification of Hcar2 immunofluorescence intensity in BV2 cells ( n = 3 independent experiments). (I) Representative Western blot analysis of Hcar2 protein levels in BV2 cell lysates. (J) Densitometric quantification of Hcar2 protein levels relative to those of β‐tubulin ( n = 3 independent experiments). The data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA with Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. ** p < .01, *** p < .001 vs. Sham. ### p < .001 vs. SCI‐IA; ns, not significant.

Article Snippet: To establish Hcar2 ‐knockdown microglial cell lines, lentiviral vectors expressing short hairpin RNA (shRNA) targeting mouse Hcar2 (sh Hcar2 ) and a scrambled negative control (NC) were constructed and packaged by Obio Technology.

Techniques: Expressing, In Vivo, Western Blot, Control, Staining, Fluorescence, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Hcar2 deficiency induced a metabolic shift toward oxidative phosphorylation and exacerbated neuroinflammation. (A) Schematic illustration of the Hcar2 knockout (KO) strategy. (B) qRT‒PCR confirmation of Hcar2 mRNA expression in the spinal cords of Hcar2 +/+ and Hcar2 −/− mice ( n = 6). (C) Schematic workflow of the in vivo study, including SCI modelling, tissue harvesting at 7 dpi, RNA sequencing, and qRT‒PCR validation. (D and E) KEGG (D) and Reactome (E) pathway enrichment analyses of differentially expressed genes (DEGs) between Hcar2 +/+ and Hcar2 −/− mice, highlighting the downregulation of metabolic pathways ( n = 3). (F–H) qRT‒PCR analysis of the expression of the microglial markers Arg1 (F), Cd206 (G), and Cd86 (H) in the spinal cord at 7 dpi ( n = 6). (I) Representative images of immunofluorescence staining for Iba‐1 (green) and Arg‐1 (red) in the lesion core from Hcar2 +/+ and Hcar2 −/− Hcar2 +/+ mice at 7 dpi. Scale bars: 500 µm (overview), 100 µm (inset) ( n = 4). (J, K) Quantification of Iba‐1 + Arg‐1 + cell density (J) and the percentage of Arg‐1 + cells within the Iba‐1 + population (K) ( n = 4). (L‐N) Multiplex flow cytometric analysis of IL‐6 (L), IL‐1β (M) and TNF‐α (N) in spinal cord lysates ( n = 4). (O) Representative Western blots of M2‐like markers (Arg‐1, CD206 and TGF‐β) ( n = 4). (P–R) Densitometric quantification of Arg‐1 (P), CD206 (Q) and TGF‐β (R) protein levels normalized to those of β‐actin ( n = 4). The data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA with Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. * p < .05, ** p < .01, *** p < .001 vs. Hcar2 +/+ +Sham. # p < .05, ### p < .001 vs. Hcar2 +/+ +SCI; ns, not significant.

Article Snippet: To establish Hcar2 ‐knockdown microglial cell lines, lentiviral vectors expressing short hairpin RNA (shRNA) targeting mouse Hcar2 (sh Hcar2 ) and a scrambled negative control (NC) were constructed and packaged by Obio Technology.

Techniques: Phospho-proteomics, Knock-Out, Expressing, In Vivo, RNA Sequencing, Biomarker Discovery, Immunofluorescence, Staining, Multiplex Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Niacin promoted an anti‐inflammatory phenotype in vitro through reprogramming microglial immunometabolism. (A) Schematic of the in vitro experimental design involving BV2 microglia challenged with LPS (100 ng/mL) and treated with niacin (NA). (B) Representative images of Iba‐1 (green) and Arg‐1 (red) immunofluorescence staining in BV2 cells treated with increasing concentrations of NA (0.1, 0.3 or 1 mM) for 24 h. Scale bar: 100 µm. (C) Quantification of OCR in BV2 cells, showing the NA‐mediated rescue of mitochondrial respiration. (D) Quantification of the red/green fluorescence intensity ratio from JC‐1 staining, which represents the relative mitochondrial membrane potential (ΔΨm). (E) Representative images of JC‐1 staining showing J‐aggregates (red, high potential) and J‐monomers (green, low potential). Scale bar: 100 µm. (F) Quantification of intracellular ATP levels. (G and H) qRT‒PCR analysis of the mRNA expression levels of the proinflammatory cytokines IL‐6 (G) and IL‐1β (H) normalized to that of β‐actin. (I) Schematic of the in vitro experimental design involving shRNA‐mediated Hcar2 knockdown in BV2 cells followed by LPS (100 ng/mL) and NA (0.3 mM) treatment. (J) qRT‒PCR confirmation of Hcar2 mRNA expression indicating knockdown efficiency. (K) Quantification of OCR in Hcar2 ‐knockdown BV2 cells. (L) Quantification of the red/green fluorescence intensity ratio from JC‐1 staining, representing the relative ΔΨm in Hcar2 ‐knockdown cells. (M) Representative images of JC‐1 staining in Hcar2 ‐knockdown BV2 cells showing J‐aggregates (red) and J‐monomers (green). Scale bar: 100 µm. (N) Quantification of intracellular ATP levels in Hcar2 ‐knockdown cells. (O and P) qRT‒PCR analysis of the mRNA expression levels of the proinflammatory cytokines IL‐6 (O) and IL‐1β (P) in Hcar2 ‐knockdown cells, normalized to that of β‐actin. Data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA with Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. In A‐H, * p < .05, *** p < .001 vs. Control. # p < .05, ## p < .01, ### p < .001 vs. LPS; In I‐P, * p < .05, ** p < .01, *** p < .001 vs. NC+LPS. ### p < .001 vs. NC+LPS+NA; ns, not significant; n = 3 independent experiments.

Article Snippet: To establish Hcar2 ‐knockdown microglial cell lines, lentiviral vectors expressing short hairpin RNA (shRNA) targeting mouse Hcar2 (sh Hcar2 ) and a scrambled negative control (NC) were constructed and packaged by Obio Technology.

Techniques: In Vitro, Immunofluorescence, Staining, Fluorescence, Membrane, Expressing, shRNA, Knockdown, Control

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Niacin promoted neuroprotection and locomotor recovery after SCI in a Hcar2‐dependent manner. (A) Experimental timeline for niacin (NA) administration and behavioural assessments. (B) Representative double immunofluorescence images of the perilesional spinal cord at 7 dpi showing Iba‐1 (microglia, red) and HK2 (glycolytic enzyme, green) expression. Scale bar: 100 µm. (C) Representative immunofluorescence images of the perilesional spinal cord at 28 dpi showing GFAP (astrocytes, green) and ChAT (motor neurons, red) expression. The high‐magnification inset displayed a representative region rostral to the lesion core, as indicated by the white box. Scale bars: 500 µm (overview), 100 µm (inset). (D) Quantification of the HK2 + area within the Iba‐1 + microglial population at 7 dpi ( n = 4). (E) Quantification of surviving ChAT + motor neurons in the ventral horn (n = 4). (F, G) CatWalk gait analysis at 28 dpi, displaying representative footprint patterns (F) and 3D intensity maps (G). (H–J) Quantification of key gait parameters: average run speed (H), mean intensity (I), and maximum contact mean intensity (J) of the hindlimbs ( n = 9). (K) Time course of locomotor recovery assessed by Basso Mouse Scale (BMS) scores over 28 days ( n = 9). The data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA (D‐E, H–J) or two‐way repeated‐measures ANOVA (K) followed by Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. *** p < .001 vs. Sham. # p < .05, ## p < .01, ### p < .001 vs. SCI+NA; ns, not significant.

Article Snippet: To establish Hcar2 ‐knockdown microglial cell lines, lentiviral vectors expressing short hairpin RNA (shRNA) targeting mouse Hcar2 (sh Hcar2 ) and a scrambled negative control (NC) were constructed and packaged by Obio Technology.

Techniques: Immunofluorescence, Expressing

Journal: Cell Death & Disease

Article Title: HOXA9 orchestrates EMT and metastasis in oral cancer via transcriptional activation of vimentin and β-catenin signaling

doi: 10.1038/s41419-026-08664-7

Figure Lengend Snippet: A Bar plot showing the results of qRT-PCR showing the efficient knockdown of HOXA9 in HSC-3 and CAL-27 cells, at the mRNA level. B Representative western blot images confirming the knockdown of HOXA9 in HSC-3 and CAL-27 cells with β-actin as an endogenous control. Bar plot showing the results of quantitative densitometric analysis confirming the HOXA9-knockdown in HSC-3 and CAL-27 cells at protein level. C Bright field images showing the changes in the morphology of HSC-3 and CAL-27 cells upon successful HOXA9-knockdown (enlarged 10X magnification). D Line plots showing the gradual reduction in the proliferation rate upon HOXA9-knockdown with increase in the cell-doubling time of HSC-3 cells (32.68 h) and E in CAL-27 cells (46.90 h), compared to the corresponding scrambled cells (23.7 hours and 32.27 h), respectively. F Representative images of the anchorage-dependent colony formation assay (upper panel) and its quantitative analysis confirming the reduction in the number and size of the colonies upon HOXA9-knockdown in HSC-3 and CAL-27 cells, when compared to the scrambled cells (lower panel). G Cell cycle analysis of scrambled and HOXA9-knockdown cells using flow cytometry. Silencing of HOXA9 in HSC-3 cells showed a significant increase in the number of cells undergoing apoptosis, and G0/G1 cell cycle arrest (Scr v/s Sh-HOXA9: apoptotic phase 5.461% ± 0.80 v/s 8.58% ± 0.57; G0/G1 arrest 22.39% ± 3.80 v/s 36.60% ± 2.56). H In CAL-27 cells, the silencing of HOXA9 has significantly induced apoptosis (Scr v/s Sh-HOXA9: apoptotic phase 4.84% ± 0.12 v/s 13.70% ± 1.86), compared to the scrambled cells. I Confocal images of actin-phalloidin staining showing substantial changes in actin cytoskeletal rearrangements and the cell morphology upon HOXA9-knockdown in OC cells. The arrows indicate the presence of filopodia (63X magnification). J Box plots confirming the significant reduction in the number and length of filopodia upon HOXA9-knockdown in HSC-3 and CAL-27, respectively. Quantitatively, the number of filopodia in HSC-3 cells decreased from 22 ± 3 (mean ± SD) in scrambled (Scr) to 13 ± 2 in knockdown cells (Sh-HOXA9), and the length of the filopodia decreased from 0.26 µm±0.006 to 0.11 µm ± 0.009. In CAL-27 cells, the number of filopodia decreased from 82 ± 6 (mean ± SD) to 22 ± 2.64, and the length of the filopodia decreased from 0.37 µm ± 0.07 to 0.16 µm ± 0.003 following HOXA9 knockdown. The data represents mean ± SD of the experiments performed in duplicates, repeated twice and P < 0.05 was considered statistically significant.

Article Snippet: To generate a stable knockdown cell line, HOXA9 shRNA retroviral plasmids (pGFP-V-RS) were purchased from Origene Technologies (TG307647) and transfected into Phoenix packaging cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Control, Colony Assay, Cell Cycle Assay, Flow Cytometry, Staining

Journal: Cell Death & Disease

Article Title: HOXA9 orchestrates EMT and metastasis in oral cancer via transcriptional activation of vimentin and β-catenin signaling

doi: 10.1038/s41419-026-08664-7

Figure Lengend Snippet: A Representative images of tumors extracted from nude mice ( n = 4). The mice that received scrambled HSC-3 cells showed progressively growing tumors. B The line graph represents the difference in the tumor volume of nude mice of scrambled and knockdown group as measured at regular time intervals. C Representative images of H&E staining and IHC staining for the tumor tissues stained with Pan-cytokeratin (CK-Pan) antibody. The tissues of scrambled group showing higher positive staining than that of knockdown group. D Bar graph showing the significant reduction in the IHC optical density score of the tissues of knockdown group mice when compared to scrambled group, stained with CK-Pan antibody. E Representative images of metastasized lung tissues in the presence and absence of HOXA9 expression. The arrows represent the metastatic nodules in mice lungs. F Bar graph showing the quantitative analysis of number of metastatic nodules in nude mice receiving scrambled and HOXA9-knockdown HSC-3 cells. G Representative images of H&E staining performed for mice lung tissues under 10X magnification. Mice that received scrambled cells exhibited extensive lung metastasis. P < 0.05 was considered statistically significant.

Article Snippet: To generate a stable knockdown cell line, HOXA9 shRNA retroviral plasmids (pGFP-V-RS) were purchased from Origene Technologies (TG307647) and transfected into Phoenix packaging cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA).

Techniques: Knockdown, Staining, Immunohistochemistry, Expressing