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: In further experiments, cells were exposed to varying concentrations of CILP protein (10, 25, 50 ng/ml, RPC382Hu01, Cloud-Clone, TX, USA) and/or SERPINE2 protein (25 μg/ml, HY- P71085 , MCE) for 24 h. The plasmids and siRNA were obtained from Hanbio Biotechnology Co., Ltd, located in Shanghai, China.

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: In further experiments, cells were exposed to varying concentrations of CILP protein (10, 25, 50 ng/ml, RPC382Hu01, Cloud-Clone, TX, USA) and/or SERPINE2 protein (25 μg/ml, HY- P71085 , MCE) for 24 h. The plasmids and siRNA were obtained from Hanbio Biotechnology Co., Ltd, located in Shanghai, China.

Techniques: In Vivo

Journal: Communications Biology

Article Title: Integrated methylome analysis identifies an epigenetically silenced tumor suppressor RASA4 in small cell lung cancer

doi: 10.1038/s42003-025-09440-7

Figure Lengend Snippet: A Volcano plot of differentially expressed genes in SBC-2 cells with stable RASA4 knockdown, relative to those transfected with scramble shRNA. B qRT-PCR shows that SERPINE2 mRNA levels were significantly lower in DMS114 and SHP77 cells with stable over-expression of RASA4, compared to cells stably transfected with an empty vector. SERPINE2 mRNA levels were significantly higher in H446 and SBC-2 cells with stable knockdown of RASA4, compared to cells stably transfected with scramble shRNA. Data are shown as mean ± SD from three independent biological replicates ( n = 3, unpaired Student’s t -test). C ELISA analysis shows that H446 and SBC-2 cells with RASA4 knockdown secreted more SERPINE2 protein to cell culture medium, compared to cells stably transfected with scramble shRNA. Data are shown as mean ± SD from three independent biological replicates ( n = 3, unpaired Student’s t -test). D Immunofluorescence staining of SERPINE2 protein in SBC-2 and H446 cells with stable transfections of scramble shRNA and RASA4 shRNA. (scale bar = 10 μm for SBC-2 cells, scale bar = 20 μm for H446 cells). E Co-IP analysis of protein interaction between RASA4 and SERPINE2 in DMS114 cells with RASA4 stable over-expression. Cell lysates from DMS114 cells with stable transfection of FLAG-tagged RASA4 were immunoprecipitated with anti-FLAG antibody, followed by western blot with anti-SERPINE2 antibody. IgG was used as a negative control. F Western blot shows that the protein expression of SERPINE2 and EMT markers (N-cadherin, E-cadherin, Vinmintin, and snail) following RASA4 stable knockdown and overexpression in SBC-2 and DMS114 cells. G qRT-PCR analysis of stemness markers NANOG , OCT4 , SOX2, and MYC in SBC-2 and DMS114 cells with RASA4 stable knockdown and overexpression. Data are shown as mean ± SD from three independent biological replicates ( n = 3, unpaired Student’s t -test). H Sphere formation assay in SBC-2 and H446 cells with RASA4 stable knockdown, and in DMS114 and SHP77 cells with RASA4 stable overexpression. (scale bar = 50 μm).

Article Snippet: SERPINE2 concentration in cell culture medium was quantified using the Human Glia-Derived Nexin (SERPINE2) ELISA Kit (#CSB-EL021082HU, Cusabio, Wuhan, China) following the manufacturer’s protocol.

Techniques: Knockdown, Transfection, shRNA, Quantitative RT-PCR, Over Expression, Stable Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Negative Control, Expressing, Tube Formation Assay

Journal: Communications Biology

Article Title: Integrated methylome analysis identifies an epigenetically silenced tumor suppressor RASA4 in small cell lung cancer

doi: 10.1038/s42003-025-09440-7

Figure Lengend Snippet: A Representative images of H&E staining in tumor and adjacent normal tissues from FFPE specimens of three surgical SCLC patients. B IHC staining of RASA4 in tumor and adjacent normal tissues from FFPE specimens of the same three SCLC patients. The results show significantly reduced RASA4 expression in the tumor tissues compared to the adjacent normal tissues. C IHC staining of SERPINE2 in tumor and adjacent normal tissues from FFPE specimens of the same three SCLC patient samples. The results show significantly increased SERPINE2 expression in the tumor tissues compared to the adjacent normal tissues. (scale bar = 20 μm for 40×, scale bar = 100 μm for 8×) ( D ) IHC staining shows that SERPINE2 expression is elevated in the invasive front of the SCLC tumor. (scale bar = 40 μm for 20×, scale bar = 200 μm for 5×) ( E ) IHC scores of RASA4 and SERPINE2 in tumor and adjacent normal tissues from 29 SCLC patients who underwent surgery ( n = 29, Mann–Whitney test). F Statistical analysis indicates the negative association between RASA4 and SERPINE2 expression in SCLC tumor and adjacent normal tissues ( n = 29, Chi-square test for R × C contingency tables). G Kaplan–Meier survival analysis of 29 SCLC patients who underwent surgery, stratified by RASA4 IHC scores in tumor tissues. The results show that SCLC patients with moderate scores of RASA4 expression have significantly better survival compared to those with weak scores. The statistical significance of the difference in survival was determined by the log-rank test.

Article Snippet: SERPINE2 concentration in cell culture medium was quantified using the Human Glia-Derived Nexin (SERPINE2) ELISA Kit (#CSB-EL021082HU, Cusabio, Wuhan, China) following the manufacturer’s protocol.

Techniques: Staining, Immunohistochemistry, Expressing, MANN-WHITNEY