Journal: bioRxiv

Article Title: HDAC5 -encoded Microprotein NISM Mediates Nucleolar Formation and Ribosomal RNA Synthesis

doi: 10.64898/2026.02.21.707204

Figure Lengend Snippet: (A-B) BedGraph tracks of HEK293T Ribo-seq coverage across the entire HDAC5 transcript (A) and zoomed in on the NISM smORF within the 5′-UTR (B). In (A), the NISM smORF is highlighted in yellow. HDAC5 is on the negative strand and thus the 5′ to 3′ orientation runs right to left. The top tracks show read coverage for cells pre-treated with harringtonine (Harr) to capture translation initiation sites and the bottom tracks show read coverage for untreated cells lysed in the presence of cycloheximide to capture elongating ribosomes. PhyloCSF scores for each reading frame are shown below the RefSeq transcript tracks. Possible start codons in the NISM smORF are denoted by ‘M’ in (B). (C) Sequence alignment showing amino acid level conservation of NISM across distant mammals. (D) Immunoblot analysis of short and long NISM-ALFA expression in HEK293T cells. (E) ESMFold predicted structure of short NISM. Low confidence predictions are colored yellow and very low confidence predictions are colored orange. (F) Representative immunofluorescence images of NPM1 (red), FBL (cyan), and NISM-ALFA (green) in HEK293T cells transfected with empty vector (EV), short NISM-ALFA, or long NISM-ALFA for 48 h. Nuclei were counter-stained with DAPI (blue). Scale bar, 5 µm. All data are representative of at least two biological replicates.

Article Snippet: Expression of HDAC5 at the protein level was also validated by SDS-PAGE and immunoblot using the HDAC5 rabbit monoclonal antibody (Cell Signaling, 20458) following IP of HDAC5 from 500 μg of total protein from WT or NISM KO U2OS cells.

Techniques: Sequencing, Western Blot, Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Staining

Journal: Biomedicines

Article Title: Secosteroid–2-Pyrazoline Hybrids: Design, Synthesis, Biological Evaluation and Development of Therapeutic Combinations Against ERα-Positive Breast Cancer Cells

doi: 10.3390/biomedicines13123057

Figure Lengend Snippet: Effect of lead secosteroid–2-pyrazoline hybrids 3f , 3j , and 3k on signaling pathways in MCF-7 breast cancer cells. Antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were used for immunoblotting.

Article Snippet: Primary antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were employed (Cell Signaling Technology, Danvers, MA, USA). α-Tubulin served as the internal loading control.

Techniques: Protein-Protein interactions, Western Blot

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A, VS-6063 reduced levels of phosphorylated HDAC4 and HDAC5. n=4 biological replicates in each group. B, Silencing FAK also reduced the phosphorylation of HDAC4 and HDAC5 in HASMCs grown in common medium or osteogenic medium. n=4 biological replicates in each group. C, FAK inhibition using VS6063 decreased cytosolic localization and increased nuclear localization of HDAC4 and HDAC5 in osteogenic media as shown by immunofluorescence images (60x). 30 cells in each group were used for analysis.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Phospho-proteomics, Immunofluorescence

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: Leptomycin B (LEP) inhibited nuclear export of HDAC4 and HDAC5 in A , normal media and B , in osteogenic media as shown by immunofluorescence. 12–46 cells in each group were used for analysis. C, Leptomycin B reduced phosphorylated HDAC4 and HDAC5 and inhibited the expression of RUNX2 and ALPL in osteogenic media. n=4 biological replicates in each group.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Immunofluorescence, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A , Increased expression of HDAC4/5 was achieved by adenovirus transduction (Ad.HDAC4 or Ad.HDAC5). B, Ad.HDAC4 or Ad.HDAC5 resulted in enhanced calcification that was inhibited with the treatment of leptomycin B (5nM) n=3 biological replicates in each group (with 2 representative replicates shown). Quantitative calcium assay in cells treated with C, Ad.HDAC4 and D , Ad.HDAC5 in the presence or absence of leptomycin demonstrated reduced calcification with leptomycin treatment. n=3 biological replicates in each group. E and F, Cytosolic and nuclear localization of HDAC4 and HDAC5 in AdHDAC4 and AdHDAC5 treated cells in the presence and absence of leptomycin (a nuclear export inhibitor, 10 nM for 3 hours) in osteogenic media. 12–13 cells in each group were used for analysis.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Over Expression, Expressing, Transduction, Calcium Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A, Treatment of HASMCs with siHDAC4 resulted in >70% and >65% knockdown of HDAC4 mRNA level in common or osteogenic medium, respectively. n=6 biological replicates in each group. B, Protein levels of RUNX2 and ALPL were increased with osteogenic medium. However, siHDAC4 decreased the levels of RUNX2 and ALPL induced by osteogenic medium. n=4 biological replicates in each group. C, Treatment of HASMCs with siHDAC5 resulted in >55% and >50% knockdown of HDAC5 mRNA level in normal or osteogenic medium, respectively, and decreased HDAC5 protein levels in siHDAC5-treated cells. n=6 biological replicates in each mRNA group. D, siHDAC5 decreased the protein levels of RUNX2 and ALPL induced by osteogenic medium. n=4 biological replicates in each group. E, Treatment with siHDAC4, siHDAC5, or the combination inhibited calcification of HASMCs grown in osteogenic medium for 14 days, as evidenced by Alizarin Red staining. n=3 biological replicates in each group (with 2 representative replicates shown). F, Treatment of HASMCs with LMK-235 (a pharmacologic inhibitor of HDAC4 and HDAC5) inhibited calcification induced by osteogenic medium in a dose-dependent manner. n=2 biological replicates in each group G, LMK-235 reduced the migration of VSMCs induced by osteogenic medium. The experiments in figure 1H and 7G were performed at the same time. n=6 biological replicates in each group.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Knockdown, Staining, Migration

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A and B, Increased HDAC4 or HDAC5 expression by adenovirus resulted in augmented calcification of HASMCs that was inhibited by treatment with the FAK inhibitor VS-6063 (1μM). n=3 biological replicates in each group (with 2 representative replicates shown). C and D, Reduction of FAK expression with siFAK significantly attenuated the calcification of HASMCs induced by Ad.HDAC4 or Ad.HDAC5 in osteogenic medium or induced by osteogenic medium alone. n=3 biological replicates in each group (with 2 representative replicates shown). E and F, Osteogenic medium induced calcification of mouse aortas and human carotid arteries after culturing for 21 days. However, the calcification induced by osteogenic medium was inhibited by treatment with VS-6063 (2μM or 4μM). n=4 biological replicates in each group.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Over Expression, Cell Culture, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: Localization of PTK2 , HDAC5 , and HDAC4 gene expression in modulated SMC subtypes using an integrated human atherosclerosis reference. Uniform Manifold Approximation and Projection (UMAP) embeddings from an integrated human atherosclerosis single-cell RNA-seq reference dataset (see Methods ), highlighting ( A ) PTK2 , ( B ) HDAC5 , and ( C ) HDAC4 normalized gene expression. Individual sequencing libraries across four studies were harmonized after QC and batch correction with reciprocal PCA (rPCA). A broad SMC cluster was annotated using transfer learning with cell labels from the Tabula Sapiens vasculature subset. SMC subtypes were further annotated by extracting gene modules from a scRNA meta-analysis of murine SMCs (contractile SMC, transitional SMC, fibromyocyte, and fibrochondrocyte) and calculating their enrichment in cells within the main SMC cluster. PTK2 and HDAC5 expression were enriched in transitional SMCs and fibromyocytes.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Gene Expression, RNA Sequencing, Sequencing, Expressing

Journal: Scientific Reports

Article Title: Somatostatin regulates the clock sensitivity to evening light

doi: 10.1038/s41598-025-26904-2

Figure Lengend Snippet: SST m/m mice show higher light sensitivity at dusk in DD condition. ( A ), 200-lx light pulse at CT12 induced larger phase delays in SST m/m mice. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower) illuminated with a light pulse (200 lx, 30 min) at CT12. Yellow boxes indicate the time of light illumination. Right , Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 9 ( SST m/m ); ** P < 0.01, unpaired t -test). ( B ), Effect of dim light pulse (2 lx, 30 min) at CT12. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower ). Yellow boxes indicate the time of light illumination. Middle, Magnified presentations of the activities shown in left panels. Right, Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 11 ( SST m/m ); *** P < 0.001, unpaired t -test). ( C) , Dim light pulse (2 lx, 30 min) at CT12 induced higher cFos mRNA expression and ERK phosphorylation in the SCN of SST m/m mice. Expression of cFos mRNA was examined by in situ hybridization using digoxigenin-labelled probe. Phosphorylated ERK was immunohistochemically stained with pERK antibody. Representative micrographs of cFos mRNA expression ( Left panels ) and phosphorylated ERK ( Right panels ). Arrows indicate the cFos mRNA positive cells and phosphorylated ERK-positive cells. Scale bars, 100 μm. ( D ), Confocal microscopy revealed abundant cells (DAPI, blue) in the VL SCN surrounded by both RHT (green) and SST (red) terminals in SST +/+ mice. Note the dense distribution of SST-terminals proximal to the RHT-terminals in the VL SCN of SST +/+ mice, but none in SST m/m mice. Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( E ), Exposure to a dim light pulse (2 lx, 30 min) at CT12 induced cFOS protein expression in SSTR1 -positive cells in the VL SCN of SST m/m mice. Left , Expression of SSTR1 mRNA (blue) and cFOS protein (brown) was examined. The lower panels show a magnified image of the area indicated by the red rectangle displayed in the upper panels. Arrowheads indicate the cells expressing both SSTR1 mRNA and cFOS protein. Right , Ratio of cFOS-positive cells in SSTR1 -positive cells (mean ± s.e.m; n = 4 (both genotypes); ** P < 0.01, unpaired t -test). Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( F ), Electron microscopy revealed that SST neurons innervate cells in the VL SCN. Left , asymmetrical axo-dendritic input (red arrow) from SST-negative axon to SST-positive dendrite. N, nucleus. Right , symmetrical axo-somatic output (innervation: blue arrows) from SST-positive axon to SST-negative soma. Scale bars, 500 nm. ( G ), Increased SSTR1 mRNA expression in the SCN of SST m/m mice. Quantitative RT-PCR analysis of SSTR1 mRNA was performed using SCN tissue samples precisely collected from SST m/m and SST +/+ mice using a laser-microdissection technique. ( H ), CH-275, an SSTR1 agonist, ameliorated the abnormal delay in locomotor activity in SST m/m mice under long photoperiod. Representative double-plotted actograms of vehicle- ( upper ) or CH-275-treated SST m/m mice ( lower ) entrained for more than three weeks in LD18h:6 h and released to DD at ZT12. CH-275 was administered into the SCN of SST m/m mice using an osmotic mini pump. The time of the surgery is indicated by the stars. Amounts of phase advance (mean ± s.e.m; n = 6 (vehicle) and 7 (CH-275); * P < 0.05, unpaired t -test). The phase was determined by fitting a regression line across several days in DD and extrapolating it back to the first day.

Article Snippet: We used a rabbit polyclonal antibody specific for the phosphorylated forms of ERK 1 and 2 (Cell Signaling; catalog code 9101) and a rabbit antibody for cFOS protein identification (Abcam; catalog code 7963).

Techniques: Expressing, Phospho-proteomics, In Situ Hybridization, Staining, Confocal Microscopy, Electron Microscopy, Quantitative RT-PCR, Laser Capture Microdissection, Activity Assay