α sma  (Proteintech)

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: SLC25A33 deficiency induces pericyte differentiation via mitochondrial ROS accumulation, which were regulated by the TGF‐β/Smad2 signaling pathway. (A) Flow cytometry analysis using MitoSOX Red shows increased mtROS production in SLC25A33‐suppressed HBVPs compared to control and SLC25A33‐overexpressing groups. (B) Quantification of mtROS fluorescence intensity confirms this increase. (C, D) qPCR analysis reveals upregulated α‐Sma and downregulated E‐cadherin mRNA levels in Slc25a33 ‐knockdown HBVPs. (E) Representative Western blot images of α‐SMA and E‐cadherin protein expression. (F, G) Densitometric quantification shows increased α‐SMA and decreased E‐cadherin protein levels. (H) qPCR analysis shows upregulated Tgfb1 mRNA in SLC25A33‐deficient HBVPs. (I–K) No significant changes are observed in Smad2 , Smad3 , or Smad4 mRNA levels among groups. (L) Representative western blots of TGF‐β, p‐Smad2, Smad2, p‐Smad3, Smad3, Smad4, and GAPDH. (M) TGF‐β protein levels are significantly elevated in the knockdown group. (N) Phosphorylated Smad2 (p‐Smad2) is markedly increased, indicating pathway activation. (O–R) No significant differences are detected in p‐Smad3, total Smad2, Smad3, or Smad4 protein expression. Control: HBVPs without treatment; SLC25A33 (−): HBVPs treated with LV‐ Slc25a33 ‐shRNA; SLC25A33 (+): HBVPs treated with LV‐ Slc25a33 ‐OE. Data are mean ± SEM; n = 3.

Article Snippet: Membranes were blocked in 5% non‐fat milk for 1 h and incubated overnight at 4°C with primary antibodies against: E‐cadherin (Bioworld, BS80070, 1:500), α‐SMA (Bioss, bs‐10196R, 1:2000), TGFβ1 (Bioworld, BS80030, 1:500), Smad2 (Bioworld, BS1425, 1:500), Smad3 (Bioworld, BS1426, 1:500), Smad4 (Bioworld, BS45204, 1:1000), p‐Smad2 (Bioworld, MB11895, 1:500), p‐Smad3 (Bioworld, BZ16381, 1:500), and GAPDH (Cell Signaling Technology, 1:5000).

Techniques: Flow Cytometry, Analysis, Control, Fluorescence, Knockdown, Western Blot, Expressing, Activation Assay, shRNA

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: SLC25A33 modulates α‐SMA and E‐cadherin expression through the TGF‐β/Smad2 pathway in vivo. (A) Immunofluorescence staining shows α‐SMA (red) and E‐cadherin (green) expression in the stria vascularis. Nuclei are counterstained with DAPI (blue). (B, C) Quantification reveals increased α‐SMA and decreased E‐cadherin in the Slc25a33 ‐knockdown group ( n = 5, five typical sections from five individual mice were analyzed per group). (D, E) qPCR confirms upregulation of α‐Sma and downregulation of E‐cadherin mRNA levels in the Slc25a33 ‐knockdown group ( n = 3 mice). (F, G) TGF‐β and (H, I) p‐Smad2 immunoreactivity are enhanced in the knockdown group and reduced in the overexpression group ( n = 5, five typical sections from five individual mice were analyzed per group). (J) qPCR confirms upregulation of Tgf‐β mRNA levels in the knockdown group and downregulation levels in the overexpression group ( n = 3 mice). Control: wild‐type C57BL/6J mice without treatment; SLC25A33 (−): mice treated with AAV‐ie‐sh Slc25a33 ; SLC25A33 (+): mice treated with AAV‐ie‐ Slc25a33 ‐OE. Data are mean ± SEM.

Article Snippet: Membranes were blocked in 5% non‐fat milk for 1 h and incubated overnight at 4°C with primary antibodies against: E‐cadherin (Bioworld, BS80070, 1:500), α‐SMA (Bioss, bs‐10196R, 1:2000), TGFβ1 (Bioworld, BS80030, 1:500), Smad2 (Bioworld, BS1425, 1:500), Smad3 (Bioworld, BS1426, 1:500), Smad4 (Bioworld, BS45204, 1:1000), p‐Smad2 (Bioworld, MB11895, 1:500), p‐Smad3 (Bioworld, BZ16381, 1:500), and GAPDH (Cell Signaling Technology, 1:5000).

Techniques: Expressing, In Vivo, Immunofluorescence, Staining, Knockdown, Over Expression, Control

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: Exogenous IGF‐1 rescues blood‐labyrinth barrier integrity and improves hearing function in aged mice. (A) TEM images reveal ultrastructural changes in the stria vascularis. In the IGF‐1‐inhibited group, pericytes exhibit abnormal morphology, loose association with endothelial cells, and widened tight junctions (yellow arrows). These defects are ameliorated by exogenous IGF‐1 administration. (B) Click‐evoked ABR thresholds are significantly elevated in IGF‐1‐suppressed mice but partially rescued by IGF‐1 supplementation. (C–E) At 12 kHz, ABR thresholds are significantly lower in the IGF‐1‐treated group compared to control and suppressed groups, indicating improved auditory sensitivity. (F) Proposed model of IGF‐1/SLC25A33‐mediated regulation of pericyte phenotype and BLB integrity in age‐related hearing loss. Under physiological conditions, pericytes maintain a quiescent state characterized by low α‐SMA and high E‐cadherin expression, contributing to BLB stability. With aging, declining IGF‐1 levels lead to reduced SLC25A33 expression, resulting in mtROS accumulation and activation of the TGF‐β/Smad2 signaling pathway. This drives pericyte transition toward a contractile, pro‐fibrotic phenotype (high α‐SMA, low E‐cadherin), disrupting BLB integrity and promoting cochlear dysfunction. Targeting this axis may offer therapeutic potential for ARHL. Control: wild‐type C57BL/6J mice without treatment; IGF‐1 (−): mice treated with NVP‐AEW541; IGF‐1 (+): mice treated with recombinant IGF‐1. Data are mean ± SEM; n = 6 mice.

Article Snippet: Membranes were blocked in 5% non‐fat milk for 1 h and incubated overnight at 4°C with primary antibodies against: E‐cadherin (Bioworld, BS80070, 1:500), α‐SMA (Bioss, bs‐10196R, 1:2000), TGFβ1 (Bioworld, BS80030, 1:500), Smad2 (Bioworld, BS1425, 1:500), Smad3 (Bioworld, BS1426, 1:500), Smad4 (Bioworld, BS45204, 1:1000), p‐Smad2 (Bioworld, MB11895, 1:500), p‐Smad3 (Bioworld, BZ16381, 1:500), and GAPDH (Cell Signaling Technology, 1:5000).

Techniques: Morphology, Control, Sensitivity, Expressing, Stability, Activation Assay, Recombinant

Journal: Signal Transduction and Targeted Therapy

Article Title: Breast cancer-associated fibroblast-derived exosomal miR-1290 promotes triple-negative breast cancer cell proliferation

doi: 10.1038/s41392-026-02922-y

Figure Lengend Snippet: miR-1290 is upregulated in breast cancer and BCAF/MB-231-derived exosomes. a Nanoparticle tracking analysis of exosomes isolated from BCAFs and MB-231 cells showing their size distribution. The mean size and standard deviation (SD) are indicated for each sample. b TEM images of exosomes isolated from BCAFs (upper) and MB-231 cells (lower) showing the characteristic morphology of the exosomes. Scale bar, 100 nm. c Western blot analysis of exosome markers (CD63, CD81, and flotillin-1), CAF markers (α-SMA and FAP) and cellular markers (GM130 and H3) in BCAF cells, MB-231 cells and their derived exosomes. d BCAF-derived exosome uptake by TNBC cells. PKH67-labeled BCAF-derived exosomes (green) were incubated with BT-549 and Hs578T cells for 1 hour. The cell nuclei were stained with Hoechst 33342 (blue). Scale bar, 20 μm. e Heatmap showing miRNA expression profiles in exosomes derived from BCAFs and MB-231 cells. Red indicates high expression, and blue indicates low expression. miR-1290 (highlighted in red) is highly expressed in both exosome types. f Volcano plots showing differentially expressed miRNAs between breast cancer or TNBC samples and normal controls in five independent GEO datasets ( GSE37963 , GSE45666 , GSE45498 , GSE38167 , and GSE61438 ). Red and blue dots represent upregulated and downregulated miRNAs, respectively, selected using fold-change and nominal p value cutoffs (|log2(fold change)| > 0.58 and nominal p < 0.05, two-tailed Welch’s t test). g Venn diagram analysis of upregulated miRNAs across the five GEO datasets revealed four commonly upregulated miRNAs. h Violin plot showing miR-1290 expression levels in serum samples from normal individuals ( N , n = 2686) and breast cancer patients ( P , n = 1280) from the GSE73002 dataset. The expression of miR-1290 was significantly upregulated in the breast cancer patient samples (*** p < 0.001, Mann‒Whitney U test). i Analysis of serum exosomal miR-1290 levels in normal ( N , n = 20) and TNBC patient ( P , n = 39) samples collected at Keimyung University Dongsan Hospital. The results support the potential of miR-1290 as a candidate diagnostic marker in an independent cohort (*** p < 0.001, Mann‒Whitney U test). j Kaplan‒Meier plot of overall survival for TNBC patients based on serum exosomal miR-1290 expression levels from Keimyung University Dongsan Hospital ( p = 0.006, log-rank test)

Article Snippet: Membranes were incubated with the following primary antibodies: CD63 (1:200; ABclonal, A5271), CD81 (1:200; ABclonal, A5270), flotillin-1 (1:1000; ABclonal, A6220), α-SMA (1:1000; ABclonal, A17910), FAP (1:1000; ABclonal, A23789 ), GM130 (1:1000; Abcam, ab52649), H3 (1:1000; Cell Signaling Technology, 4620S), ACTB (1:20,000; ABclonal, AC026), TBC1D4 (1:200; Cell Signaling Technology, 2670S), P70S6K (1:500; Cell Signaling Technology, 9202S), phospho-P70S6K (1:500; ABclonal, AP0564), and Ki67 (1:1000; Abcam, ab16667).

Techniques: Derivative Assay, Exosomes, Analysis, Isolation, Standard Deviation, Morphology, Western Blot, Labeling, Incubation, Staining, Expressing, Two Tailed Test, Blood/Plasma/Serum Samples, Diagnostic Assay, Marker