Journal: Advanced Science

Article Title: Endowing Flexibility of Alveolar Type II Cells to Recapitulate Development From Lung Bud to Alveoli

doi: 10.1002/advs.77773

Figure Lengend Snippet: Establishment and characterization of alveolar progenitor‐like organoids (APLOs), bud tip‐like organoids (BTLOs), and alveolar‐like organoids (ALOs). (A) Schematic diagram illustrating the differentiation protocol from APLCs to APLOs, BTLOs, and ALOs. Created in BioRender. Ma, H. (2026) https://BioRender.com/91lb473 (B) Representative immunofluorescence (IF) images showing expression of the bud tip marker SOX9 in P5‐APLOs and P15‐BTLOs. Scale bars, 50 µm. (C) Quantitative PCR (qPCR) analysis of AT2 and AT1 cell marker expression in APLC‐P5‐2D, P5‐APLOs, P5‐ALOs, and human lung tissue. Data are presented as mean ± SD from three independent experimental replicates using samples derived from a single donor (n = 3). Significance was evaluated by one‐way ANOVA with Tukey's multiple comparisons test; ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also Supplementary Figure . (D) Representative transmission electron microscopy (TEM) images of APLOs and ALOs with lamellar body structures. Scale bars, 2 µm (left), 500 nm (upper right), 1 µm (lower right). (E) Immunofluorescence staining of the pan‐epithelial marker E‐cadherin and AT2 markers (proSFTPC, SFTPC, and HT2‐280) in APLOs, BTLOs, and ALOs. Scale bars, 50 µm. (F) Western blot analysis of bud tip markers (SOX9, SOX2), AT2 markers (proSFTPC, SFTPB dimer), and the lung lineage marker NKX2‐1 in APLOs, BTLOs, and ALOs.

Article Snippet: Primary antibodies were as follows: Pro‐surfactant protein C (abcam, ab90716, 1:800), E‐cadherin (Proteintech, 20874‐1‐AP, 1:500), SFTPC (ABclonal, A1835, 1:100), HT2‐280 (terracebiotech, TB‐27AHT2‐280, 1:250), NKX2‐1(Beyotime, AG3409, 1:200), Influenza A virus Nucleoprotein (GeneTex, GTX636247, 1:100).

Techniques: Organoids, Immunofluorescence, Expressing, Marker, Real-time Polymerase Chain Reaction, Analysis, Tissue, Derivative Assay, Transmission Assay, Electron Microscopy, Staining, Western Blot

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: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

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: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

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: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

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