Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: OGPs identification in LFH via Integrated Transcriptomic, Proteomic, and Bioinformatics Analyses. ( A ) LFH microarray dataset GSE113212 boxplot pre-standardization. ( B ) Standardized LFH microarray dataset GSE113212 boxplot. ( C ) Volcano plot showing 735 DEGs in GSE113212 from transcriptomic analysis. ( D ) Volcano plot displaying 125 DEPs identified in proteomic analysis. ( E ) Venn diagrams showing 29 OGPs. Blue section: 735 DEGs dataset, yellow section: 125 DEPs dataset. ( F ) Heatmap of the 29 OGPs in Non-LFH and LFH samples. ( G ) KEGG analysis of the overlapping genes. ( H ) GO analysis of the overlapping genes. ( I ) STRING protein-protein interaction network, highlighting key interactions involving CILP, TGF-β1, COL1A2, and α-SMA in a red square

Article Snippet: To induce fibrosis in LF cells in vitro , the cells were treated with 10 ng/mL of recombinant TGF-β1 protein (HY-P7118, MCE, NJ, USA) for 24 hours.

Techniques: Microarray

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: Fibrosis phenotype and the expression of CILP/TGF-β1 were markedly elevated. ( A ) Dashed lines delineate the middle layer of the L4/5 disc, while solid lines indicate the LF thickness. Blue coloration signifies normal LF, whereas red denotes LFH. ( B ) Histological sections from Non-LFH and LFH groups (n = 6 per group) groups were assessed using hematoxylin & eosin (H&E), Masson’s trichrome, and Verhoeff–Van Gieson (EVG) staining, scale bar: 50 μm. ( C ) Fibrosis scores in Non-LFH and LFH groups (n = 6 per group). ( D ) Fibrosis severity showed a direct linear relationship with LF thickening (R² = 0.6730, P < 0.0001). ( E ) IHC staining for CILP, TGF-β1, Collagen Ⅰ, and α-SMA in Non-LFH and LFH groups (n = 6 per group), with cell quantification. Scale bar: 50 μm. ( F ) qRT-PCR showed elevated CILP, TGF-β1, ACTA2, COL1A2 mRNA in Non-LFH and LFH groups (n = 6 per group). ( G ) Western blotting for CILP, TGF-β1, α-SMA and Collagen Ⅰ in Non-LFH and LFH groups (n = 6 per group), with GAPDH as loading control. ( H ) Quantified relative protein expression from WB. Significance levels are denoted as follows: ** P < 0.01;*** P < 0.001;**** P < 0.0001

Article Snippet: To induce fibrosis in LF cells in vitro , the cells were treated with 10 ng/mL of recombinant TGF-β1 protein (HY-P7118, MCE, NJ, USA) for 24 hours.

Techniques: Expressing, Staining, Immunohistochemistry, Quantitative RT-PCR, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: TGF-β1 induces fibrosis and enhances CILP expression in LF cells. ( A ) Double-labeling immunofluorescence was employed to stain Collagen I and vimentin. Nuclei were highlighted with DAPI (blue), Collagen Ⅰ appeared green, and vimentin was marked red. Scale bar: 20 µm. ( B ) Western blotting was conducted to evaluate the protein levels of CILP, α-SMA, and Collagen Ⅰ in LF cells stimulated by TGF-β1 at 0, 8, 16, and 24 h time points, and relative protein expression were quantified. ( C ) qRT-PCR analysis was performed to measure mRNA concentration of CILP, ACTA2 (α-SMA), and COL1A2 in LF cells treated with TGF-β1 at 0, 8, 16, and 24 h time points. ( D ) IF showed TGF-β1 and CILP after TGF-β1 treatment at 0, 8, 16, and 24 h time points. Nuclei stained blue with DAPI, CILP in green, and TGF-β1 in red. Scale bar: 20 µm. ( E ) ELISA assays determined CILP and Collagen Ⅰ protein expression levels in culture supernatant post-TGF-β1 induction at specified time points (0, 8, 16, and 24 h). Significance levels are denoted as follows: ns, not significant;* P < 0.05;** P < 0.01

Article Snippet: To induce fibrosis in LF cells in vitro , the cells were treated with 10 ng/mL of recombinant TGF-β1 protein (HY-P7118, MCE, NJ, USA) for 24 hours.

Techniques: Expressing, Labeling, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: CILP inhibits TGF-β1-induced SMAD3 signaling. ( A ) After 24 hours of different treatments, p-SMAD3 and fibrosis-related protein levels in LF cells were evaluated. ( B ) Examination of p-SMAD3 and fibrotic protein expression in LF cells treated with TGF-β1 and varying concentrations of CILP for 24 hours. ( C , D ) qRT-PCR analysis determined COL1A2 and ACTA2 (α-SMA) gene expression levels in LF cells under varied treatments. ( E ) Double-labeling IF staining visualized Collagen I and Vimentin in LF cells post 24-hour treatments. Nuclei stained blue with DAPI, Collagen Ⅰ in green, and Vimentin in red. Scale bar: 20 µm. ( F ) Confocal microscopy demonstrated that CILP attenuates TGF-β1-induced enhancement of p-SMAD3 nuclear localization in LF cells. Cells treated with TGF-β1 for 24 hours and CILP for 0, 8, 16, and 24 hours. Nuclei stained blue with DAPI, p-SMAD3 in green, and Vimentin in red. Scale bar: 10 µm. Significance levels are denoted as follows: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.01

Article Snippet: To induce fibrosis in LF cells in vitro , the cells were treated with 10 ng/mL of recombinant TGF-β1 protein (HY-P7118, MCE, NJ, USA) for 24 hours.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Labeling, Staining, Confocal Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: CILP attenuates LF fibrosis via the TGF-β1/SMAD3/SERPINE2 axis. ( A ) A violin plot depicted differential SERPINE2 expression between Non-LFH and LFH groups (n = 6 per group), showing significant differences. ( B ) IHC of SERPINE2 in LF samples from Non-LFH and LFH groups (n = 6 per group). Scale bar: 50 µm. ( C , D ) Western blotting revealed elevated SERPINE2 protein levels between Non-LFH and LFH groups (n = 6 per group), with relative protein expression quantified. And mRNA concentration showed consistent results. ( E , F ) Western blotting demonstrated SERPINE2 expression in LF cells treated with TGF-β1 at 0, 8, 16, and 24 h time points, with relative protein expression quantified. And mRNA concentration showed consistent results. ( G ) Western blotting assessed protein expression levels of CILP, Collagen I, p-SMAD3, and α-SMA in LF cells subjected to various SERPINE2 treatments. ( H ) qRT-PCR assessed transcriptional profiles of CILP, ACTA2 (α-SMA), and COL1A2 across experimental conditions in LF cells. ( I ) WB analysis evaluated p-SMAD3 and fibrotic protein levels 48 hours post-transfection with CILP-overexpressing plasmids under different conditions in LF cells. ( J ) WB analysis examined p-SMAD3 and fibrotic protein expression 48 hours post-transfection with CILP-silencing plasmids under various regimens. Significance levels are denoted as follows: ns, not significant;* P < 0.05;** P < 0.01;*** P < 0.01

Article Snippet: To induce fibrosis in LF cells in vitro , the cells were treated with 10 ng/mL of recombinant TGF-β1 protein (HY-P7118, MCE, NJ, USA) for 24 hours.

Techniques: Expressing, Western Blot, Concentration Assay, Quantitative RT-PCR, Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: CILP attenuates TGF-β1-mediated LFH in vivo. ( A - D ) IHC analysis of SERPINE2, TGF-β1, Collagen I, and p-SMAD3 in mouse tissues, with quantification of positive cell percentages. Scale bar: 50 µm. Significance levels are denoted as follows: ns, not significant;* P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: To induce fibrosis in LF cells in vitro , the cells were treated with 10 ng/mL of recombinant TGF-β1 protein (HY-P7118, MCE, NJ, USA) for 24 hours.

Techniques: In Vivo

Journal: Science Advances

Article Title: TDRD3, a Tudor domain-containing protein, regulates Klf2 -dependent T reg differentiation and function to modulate immune tolerance

doi: 10.1126/sciadv.aea3960

Figure Lengend Snippet: ( A and B ) Immunoblot analysis of TDRD3 in CD4 + YFP + T regs (A) or CD4 + cells (B) isolated from the spleen of indicated mice. Right: qPCR analysis of Tdrd3 mRNA ( n ≥ 3). ( C ) Representative flow cytometric analysis (left two panels), percentage (third panel) and number (right panel) of T reg (Foxp3 + ) cells in thymocytes from indicated mice ( n ≥ 6). ( D and E ) Representative flow cytometric analysis (left two panels) and percentage (third panel) of Foxp3 + iT regs among CD4 + T cells from Foxp3 YFP-Cre or Tdrd3 fl/fl /Foxp3 YFP-Cre mice (D) or from Tdrd3 fl/fl and Tdrd3 fl/fl /CD4 Cre mice (E), polarized in the presence of TGF-β (5 ng/ml) for 48 hours ( n ≥ 6 per treatment cohort). Foxp3 mRNA levels in differentiated iT regs were detected by qPCR (right). ( F ) Representative flow cytometric analysis (two left panels) and percentage (right panel) of live cells among Foxp3 + iT regs differentiated from naïve CD4 + T cells of indicated mice ( n ≥ 6). ( G ) Representative flow cytometric analysis (two left panels) and percentage (right panel) of the proliferating Foxp3 YFP-Cre or Tdrd3 fl/fl /Foxp3 YFP-Cre CD4 + T cells in indicated peaks shown on left, labeled with CellTrace Violet (CTV) and polarized under T reg conditions for 48 hours ( n ≥ 6). Boxed area: cell population of interest. Data are from three independent experiments [(A), (B), (C), (E), (F), and (G), right panels; presented as means ±SEM] or are from one representative of three independent experiments [(A), (B), (C), (E), (F), and (G), left panels]. ** P < 0.01, *** P < 0.001, and **** P < 0.0005; ns, not significant (two-tailed Student’s t test).

Article Snippet: Recombinant TGF-β (#130-095-067) was from Miltenyi Biotec.

Techniques: Western Blot, Isolation, Labeling, Two Tailed Test