hsp 47  (Proteintech)

Journal: Biomolecules and Biomedicine

Article Title: Glioblastoma induces CAF-like astrocyte activation via the AKT/mTOR–SERPINH1/COL5A1 axis

doi: 10.17305/bb.2025.11898

Figure Lengend Snippet: Construction of CAF prognostic model, and gene screening and validation. (A and B) Survival curves for TCGA (A) and GEO (B) data obtained using the four CAF-scoring algorithms; (C and D) Weighted correlation network analysis (WGCNA) of TCGA (C) and GEO data (D); Horizontal coordinates are the scored items, and vertical coordinates are the module names. Red color represents positive correlation, and blue represents negative correlation. The correlation coefficient is shown at the top of the module, and the P value used for correlation assessment is shown at the bottom. P < 0.05 is correlated with CAFs expression levels; (E) Intersection genes in TCGA and GEO datasets; (F) Univariate Cox regression analysis. Red indicates high risk; (G and H) Lasso Cox regression analysis (lasso Lambda and lasso Cvfit); (I) Kaplan–Meier curves for the survival analysis of the high- and low-risk groups in TCGA and GEO datasets, respectively; (J and K) Prognostic analyses of SERPINH1 (J) and COL5A1 (K) using mRNA data from the Chinese Glioma Genome Atlas (CGGA); (L) Correlation between CAF scores and patient risk scores. Correlation coefficient is shown at the top right. Scatter plot of correlations is shown in the lower left quadrant of the figure. The diagonal sequence of squares represents the type of scoring algorithm. The last column represents the correlation between the CAF scores and patient scores; (M) Heat map of two identified CAF-related genes and CAF genes reported in the literature; (N) Correlation analysis of CAF genes reported in the literature, the two identified CAF-related genes, and risk scores. CAF: Cancer-associated fibroblast; TCGA: The Cancer Genome Atlas; GEO: Gene Expression Omnibus.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (ST023, Beyotime, China) in TBST for 2 h and then incubated overnight at 4 ∘ C with the following primary antibodies: SERPINH1 (sc-5293, Santa Cruz, 1:1000), COL5A1 (sc-133162, Santa Cruz, 1:1000), GAPDH (GB15002, Servicebio, 1:2000), AKT (#4685, CST, 1:1000), P-AKT (#4060, CST, 1:1000), mTOR (ET1608-5, HUABIO, 1:1000), P-mTOR ( HA600094 , HUABIO, 1:1000), FAP (AF5344, Affinity, 1:800), and S100A4 (CY5799, Abways, 1:1000).The membrane was then incubated for 3 h at 4 ∘ C with the following secondary antibodies: HRP-conjugated Goat Anti-Mouse IgG(H+L) (A0216, Beyotime, 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H+L) (A0208, Beyotime, 1:1000).

Techniques: Biomarker Discovery, Expressing, Sequencing, Gene Expression

Journal: Biomolecules and Biomedicine

Article Title: Glioblastoma induces CAF-like astrocyte activation via the AKT/mTOR–SERPINH1/COL5A1 axis

doi: 10.17305/bb.2025.11898

Figure Lengend Snippet: Detection, localization, and expression of SERPINH1 and COL5A1 . (A) Expression of SERPINH1/COL5A1 in central nervous system (CNS) tumors and fibroblasts from the Cancer Cell Line Encyclopedia (CCLE) database; (B and C) Immunolabeling of SERPINH1 (B) and COL5A1 (C) in the GBM (left) and normal tissue (right) from the HPA database; (D) Tumor volumes were analyzed using bioluminescence imaging; (E–H) Multicolor IHC labeling of SERPINH1 (E), COL5A1 (F), FAP (G), and S100A4 (H) expression in the GBM mouse model. Green indicates G422-GFP expression. Red indicates the four CAF-associated proteins (SERPINH1, COL5A1, FAP, S100A4). Nuclei were stained using DAPI. Merge panel shows the combined image. White arrow indicates peritumor tissues with high expression of the indicated protein; (I–J) Western blotting analysis of the four CAF proteins in peritumor and normal tissues (FAP, 1.632 ± 0.279, n ═ 3; S100A4, 1.269 ± 0.165, n ═ 3; SERPINH1, 3.014 ±0.855, n ═ 3; COL5A1, 1.188 ±0.104, n ═ 3); (K) HE staining in the brain tissues of GBM mice. The experiment was repeated three times, and the results are representative of three independent experiments. The statistical results are shown in Figure S7 . GBM: Glioblastoma multiforme; CAF: Cancer-associated fibroblast; HPA: Human Protein Atlas; HE: Hematoxylin and eosin.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (ST023, Beyotime, China) in TBST for 2 h and then incubated overnight at 4 ∘ C with the following primary antibodies: SERPINH1 (sc-5293, Santa Cruz, 1:1000), COL5A1 (sc-133162, Santa Cruz, 1:1000), GAPDH (GB15002, Servicebio, 1:2000), AKT (#4685, CST, 1:1000), P-AKT (#4060, CST, 1:1000), mTOR (ET1608-5, HUABIO, 1:1000), P-mTOR ( HA600094 , HUABIO, 1:1000), FAP (AF5344, Affinity, 1:800), and S100A4 (CY5799, Abways, 1:1000).The membrane was then incubated for 3 h at 4 ∘ C with the following secondary antibodies: HRP-conjugated Goat Anti-Mouse IgG(H+L) (A0216, Beyotime, 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H+L) (A0208, Beyotime, 1:1000).

Techniques: Expressing, Immunolabeling, Imaging, Labeling, Staining, Western Blot

Journal: Biomolecules and Biomedicine

Article Title: Glioblastoma induces CAF-like astrocyte activation via the AKT/mTOR–SERPINH1/COL5A1 axis

doi: 10.17305/bb.2025.11898

Figure Lengend Snippet: Localization of SERPINH1 and COL5A1. (A–C) Multicolor IHC staining of astrocytes (A, gfap), microglia (B, Iba1), and oligodendrocytes (C, MBP) in the GBM mouse model. Green indicates G422-GFP and Red indicates glial cells. Nuclei were stained using DAPI. The Merge panel shows the combined image. Red arrows indicate the localized recruitment of glial cells. The statistical results are shown in Figure S7 . (D–G) Co-localization labeling of SERPINH1 (D, 10×; E, 63×) and COL5A1 (F, 10×; G, 63×) with astrocytes in the GBM mouse model. Green indicates G422-GFP, and Red indicates astrocytes. White indicates SERPINH1/COL5A1 expression. Nuclei were stained using DAPI. The Merge panel shows the combined image. White arrows indicate areas with high levels of co-localization and the cells that co-localized. (H–K) Co-localization labeling of FAP (H, 10×; I, 63×) and S100A4 (J, 10×; K, 63×) with astrocytes in the GBM mouse model. Green indicates G422-GFP, and Red indicates astrocytes. White indicates FAP/S100A4 expression. Nuclei were stained using DAPI. The Merge panel shows the combined image. White arrows indicate areas with high levels of co-localization and the cells that co-localized. Red arrows indicate FAP/S100A4-positive and GFAP-negative cells. (L) Statistical chart of tissue immunofluorescence localization (E, G, I, and K) results SERPINH1,0.557 ± 0.011, n ═ 3; COL5A1, 0.590 ± 0.006, n ═ 3; FAP, 0.505 ± 0.013, n ═ 3; S100A4, 0.481 ± 0.003, n ═ 3. The experiment was repeated three times, and the results are representative of three independent experiments. In the statistical figures, P values are indicated with asterisks, where *represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. GBM: Glioblastoma multiforme.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (ST023, Beyotime, China) in TBST for 2 h and then incubated overnight at 4 ∘ C with the following primary antibodies: SERPINH1 (sc-5293, Santa Cruz, 1:1000), COL5A1 (sc-133162, Santa Cruz, 1:1000), GAPDH (GB15002, Servicebio, 1:2000), AKT (#4685, CST, 1:1000), P-AKT (#4060, CST, 1:1000), mTOR (ET1608-5, HUABIO, 1:1000), P-mTOR ( HA600094 , HUABIO, 1:1000), FAP (AF5344, Affinity, 1:800), and S100A4 (CY5799, Abways, 1:1000).The membrane was then incubated for 3 h at 4 ∘ C with the following secondary antibodies: HRP-conjugated Goat Anti-Mouse IgG(H+L) (A0216, Beyotime, 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H+L) (A0208, Beyotime, 1:1000).

Techniques: Immunohistochemistry, Staining, Labeling, Expressing, Immunofluorescence

Journal: Biomolecules and Biomedicine

Article Title: Glioblastoma induces CAF-like astrocyte activation via the AKT/mTOR–SERPINH1/COL5A1 axis

doi: 10.17305/bb.2025.11898

Figure Lengend Snippet: The expression of SERPINH1 /COL5A1 and cell function assays in a GBM-astrocytes co-culture model. (A and B) Western blotting (A) (SERPINH1: SVGP12, 1 ± 0, n ═ 3; U87, 0.664 ± 0.054, n ═ 3; T98G, 0.490 ± 0.146, n ═ 3; LN229, 0.547 ± 0.023, n ═ 3; U343, 0.487 ± 0.067, n ═ 3. COL5A1: SVGP12, 1 ± 0, n ═ 3; U87, 0.819 ± 0.169, n ═ 3; T98G, 0.809 ± 0.089, n ═ 3; LN229, 1.280 ± 0.130, n ═ 3; U343, 0.789 ± 0.026, n ═ 3) and RT-qPCR (B) (SERPINH1: SVGP12, 1 ± 0, n ═ 3; U87, 0.646 ± 0.041, n ═ 3; T98G, 0.077 ± 0.012, n ═ 3; LN229, 0.147 ± 0.042, n ═ 3; U343, 0.250 ± 0.029, n ═ 3. COL5A1: SVGP12, 1 ± 0, n ═ 3; U87, 0.509 ± 0.054, n ═ 3; T98G, 0.268 ± 0.089, n ═ 3; LN229, 0.065 ± 0.023, n ═ 3; U343, 0.007 ± 0.002, n ═ 3) in SVGP12 and GBM cell lines. (C and D) Western blotting (C) (SERPINH1: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 1.871 ± 0.077, n ═ 3; SVGP12-T98G, 1.509 ± 0.117, n ═ 3; SVGP12-LN229, 1.628 ± 0.166, n ═ 3; SVGP12-U343, 1.635 ± 0.106, n ═ 3. COL5A1: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 1.514 ± 0.088, n ═ 3; SVGP12-T98G, 1.659 ± 0.108, n ═ 3; SVGP12-LN229, 1.836 ± 0.410, n ═ 3; SVGP12-U343, 1.990 ± 0.228, n ═ 3) and RT-qPCR (D) (SERPINH1: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 2.367 ± 0.217, n ═ 3; SVGP12-T98G, 2.211 ± 0.125, n ═ 3; SVGP12-LN229, 1.308 ± 0.128, n ═ 3; SVGP12-U343, 1.324 ± 0.348, n ═ 3. COL5A1: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 2.759 ± 0.425, n ═ 3; SVGP12-T98G, 7.595 ± 1.252, n ═ 3; SVGP12-LN229, 2.027 ± 0.629, n ═ 3; SVGP12-U343, 1.561 ± 0.246, n ═ 3) in a co-culture model. (E) Schematic diagram of GBM-astrocytes in co-culture. (F) Wound healing assay in co-cultured SVGP12 (24 h: SVGP12, 0.692 ± 0.016, n ═ 3; U87, 0.264 ± 0.051, n ═ 3; T98G, 0 ± 0, n ═ 3; LN229, 0.174 ± 0.151, n ═ 3; U343, 0.137 ± 0.017, n ═ 3). (G) Migration assay in co-cultured SVGP12. (SVGP12, 1 ± 1, n ═ 3; U87, 603 ± 49.96, n ═ 3; T98G, 314 ± 55.05, n ═ 3; LN229, 856.7 ± 100.3, n ═ 3; U343,124.0 ± 26.21, n ═ 3). The statistical results are shown in Figure S7 . The experiment was repeated three times, and the results are representative of three independent experiments. In the statistical figures, P values are indicated with asterisks, where *represents P < 0.05, **represents P < 0.01, ***represents P < 0.001, and ****represents P < 0.0001. GBM: Glioblastoma multiforme.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (ST023, Beyotime, China) in TBST for 2 h and then incubated overnight at 4 ∘ C with the following primary antibodies: SERPINH1 (sc-5293, Santa Cruz, 1:1000), COL5A1 (sc-133162, Santa Cruz, 1:1000), GAPDH (GB15002, Servicebio, 1:2000), AKT (#4685, CST, 1:1000), P-AKT (#4060, CST, 1:1000), mTOR (ET1608-5, HUABIO, 1:1000), P-mTOR ( HA600094 , HUABIO, 1:1000), FAP (AF5344, Affinity, 1:800), and S100A4 (CY5799, Abways, 1:1000).The membrane was then incubated for 3 h at 4 ∘ C with the following secondary antibodies: HRP-conjugated Goat Anti-Mouse IgG(H+L) (A0216, Beyotime, 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H+L) (A0208, Beyotime, 1:1000).

Techniques: Expressing, Cell Function Assay, Co-Culture Assay, Western Blot, Quantitative RT-PCR, Wound Healing Assay, Cell Culture, Migration

Journal: Biomolecules and Biomedicine

Article Title: Glioblastoma induces CAF-like astrocyte activation via the AKT/mTOR–SERPINH1/COL5A1 axis

doi: 10.17305/bb.2025.11898

Figure Lengend Snippet: The expression of SERPINH1/ COL5A1 and AKT/m-TOR pathway in a GBM and astrocytes co-culture model. (A) KEGG pathway enrichment. (B) Western blot shows the expression of the AKT/m-TOR pathway in co-cultured SVGP12 cells (p-mTOR: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 4.776 ± 0.467, n ═ 3; SVGP12-T98G, 3.234 ± 0.316, n ═ 3; SVGP12-LN229, 1.497 ± 0.349, n ═ 3; SVGP12-U343, 5.107 ± 0.456, n ═ 3. mTOR: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 1.367 ± 0.054, n ═ 3; SVGP12-T98G, 1.349 ± 0.043, n ═ 3; SVGP12-LN229, 1.350 ± 0.330, n ═ 3; SVGP12-U343, 1.261 ± 0.438, n ═ 3. p-AKT: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 1.847 ± 0.168, n ═ 3; SVGP12-T98G, 1.880 ± 0.152, n ═ 3; SVGP12-LN229, 1.877 ± 0.053, n ═ 3; SVGP12-U343, 2.127 ± 0.213, n ═ 3. AKT: SVGP12, 1 ± 0, n ═ 3; SVGP12-U87, 1.089 ± 0.287, n ═ 3; SVGP12-T98G, 1.075 ± 0.044, n ═ 3; SVGP12-LN229, 1.183 ± 0.269, n ═ 3; SVGP12-U343, 1.237 ± 0.077, n ═ 3). (C) Western blot shows AKT/m-TOR pathway expression in peritumor and normal tissues of GBM mice (p-mTOR: Normal, 1 ± 0, n ═ 3; Peritumor, 2.578 ± 0.981, n ═ 3. mTOR: Normal, 1 ± 0, n ═ 3; Peritumor,0.944 ± 0.413, n ═ 3. p-AKT: Normal, 1 ± 0, n ═ 3; Peritumor, 1.554 ± 0.349, n ═ 3. AKT: Normal, 1 ± 0, n ═ 3; Peritumor, 1.011 ± 0.218, n ═ 3). (D) Western blot shows SERPINH1 and COL5A1 expression in SVGP12 cells treated with the AKT agonist sc79 (SERPINH1: SVGP12, 1 ± 0, n ═ 3; SVGP12+SC79, 5.263 ± 0.584, n ═ 3. COL5A1: SVGP12, 1 ± 0, n ═ 3; SVGP12+SC79, 1.160 ± 0.033, n ═ 3). (E) Migration of SVGP12 cells treated with the AKT agonist sc79 (right) and of untreated SVGP12 cells (left). (F and G) Western blot shows the expression of the AKT-mTOR pathway (F) (p-mTOR: SVGP12, 1 ± 0, n ═ 3; SVGP12+Perifosine, 0.782 ± 0.007, n ═ 3; SVGP12-U87+Perifosine, 0.812 ± 0.018, n ═ 3; SVGP12-T98G+Perifosine, 0.588 ± 0.026, n ═ 3; SVGP12-LN229+Perifosine, 0.451 ± 0.035, n ═ 3; SVGP12-U343+Perifosine, 0.574 ± 0.023, n ═ 3. p-AKT: SVGP12, 1 ± 0, n ═ 3; SVGP12+Perifosine, 0.555 ± 0.036, n ═ 3; SVGP12-U87+Perifosine, 0.690 ± 0.006, n ═ 3; SVGP12-T98G+Perifosine, 0.714 ± 0.030, n ═ 3; SVGP12-LN229+Perifosine, 0.665 ± 0.023, n ═ 3; SVGP12-U343+Perifosine, 0.677 ± 0.021, n ═ 3) and that of SERPINH1 and COL5A1 (G) in co-cultured SVGP12 cells, and in co-cultured SVGP12 cells treated with the AKT inhibitor perifosine (SERPINH1: SVGP12, 1 ± 0, n ═ 3; SVGP12+Perifosine, 0.876 ± 0.048, n ═ 3; SVGP12-U87+Perifosine, 0.959 ± 0.023, n ═ 3; SVGP12-T98G+Perifosine, 0.817 ± 0.050, n ═ 3; SVGP12-LN229+Perifosine, 0.748 ± 0.070, n ═ 3; SVGP12-U343+Perifosine, 0.807 ± 0.046, n ═ 3.COL5A1:SVGP12, 1 ± 0, n ═ 3; SVGP12+Perifosine, 0.868 ± 0.072, n ═ 3; SVGP12-U87+Perifosine, 0.923 ± 0.034, n ═ 3; SVGP12-T98G+Perifosine, 0.859 ± 0.074, n ═ 3; SVGP12-LN229+Perifosine, 0.920 ± 0.011, n ═ 3; SVGP12-U343+Perifosine, 0.830 ± 0.028, n ═ 3). (H) Statistical graph of changes in SERPINH1 and COL5A1 protein expression with the addition of perifosine. The experiment was repeated three times, and the results are representative of three independent experiments. The statistical results are shown in Figure S7 . In the statistical figures, P values are indicated with asterisks, where * represents P < 0.05, ** represents P < 0.01, ***represents P < 0.001, and **** represents P < 0.0001. GBM: Glioblastoma multiforme; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (ST023, Beyotime, China) in TBST for 2 h and then incubated overnight at 4 ∘ C with the following primary antibodies: SERPINH1 (sc-5293, Santa Cruz, 1:1000), COL5A1 (sc-133162, Santa Cruz, 1:1000), GAPDH (GB15002, Servicebio, 1:2000), AKT (#4685, CST, 1:1000), P-AKT (#4060, CST, 1:1000), mTOR (ET1608-5, HUABIO, 1:1000), P-mTOR ( HA600094 , HUABIO, 1:1000), FAP (AF5344, Affinity, 1:800), and S100A4 (CY5799, Abways, 1:1000).The membrane was then incubated for 3 h at 4 ∘ C with the following secondary antibodies: HRP-conjugated Goat Anti-Mouse IgG(H+L) (A0216, Beyotime, 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H+L) (A0208, Beyotime, 1:1000).

Techniques: Expressing, Co-Culture Assay, Western Blot, Cell Culture, Migration

Journal: Wellcome Open Research

Article Title: Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

doi: 10.12688/wellcomeopenres.23776.1

Figure Lengend Snippet: A ) Dendra2::Col1a2 NIH3T3 cells were imaged using Airyscan microscopy after 18 h of culture, 1000x magnification, and 2.5 × digital zoom; a maximum intensity projection is shown. Vesicles containing Dendra2 signals were observed at the sites of fibril assembly. The Dendra2 signal was observed at the periphery of the vesicles; the scale bar represents 1 µm. The area outlined by the box is enlarged in the right-hand panel. Small panels show looped structures containing Dendra2-positive collagen. B ) Individual frames taken from Extended data 14 demonstrate that a single fibripositor (highlighted in the red box) is in contact numerous times by both the endoplasmic reticulum and electron-dense vesicles. The Golgi apparatus did not contact fibripositor (Extended data 15). C ) Immunofluorescence imaging of type I collagen in mouse embryonic fibroblasts using the ER marker PDI. Scale bar represents 20 µm. D ) Fractionation of NIH3T3 cells using a sucrose density gradient, type I collagen is co-resident with the ER protein calreticulin (Calr), but also with the lysosomal protein Lamp1 and the collagen chaperone Hp47. E ) Proteomic analysis of lysosomal fractions 6 and 7 revealed significant enrichment of proteins identified by proteomic analysis of fractions 6 and 7 based on GO Cellular component terms. F ) Col1a1 (Red and Magenta) and Col1a2 (Blue and Green)-derived procollagen peptides were present in fractions 6 and 7, suggesting that the newly synthesized collagen transitioned to these compartments. G ) Dendra2::Col1a2 NIH3T3 cells were imaged by Airyscan microscopy 48 h after transfection with LAMP1-YFP. Images were recorded at 1000x magnification using a 2.5 × digital zoom. The maximum intensity projection of the 31 images is shown. LAMP1-YFP vesicles (green) containing photoswitched Dendra2 signals were observed. Three LAMP1-positive areas were also observed. H ) Live super-resolution microscopy of NIH3T3 cells stably transduced with Hsp47-BFP-RDEL lentivirus and transfected with LAMP1-YFP. Scale bar, 20 µm.

Article Snippet: Antibodies used in this study were collagen (Gentaur, OARA02579, dilution 1:2000 (WB), 1:500 (IF)), Dendra2 (Origene, TA180094, dilution 1:500), GAPDH (Sigma, G8795, dilution 1:10,000), Calreticulin (Stressgen; SPA-601, dilution 1:1000), Lamp1 (Santa Cruz, sc-20011, 1:500), vinculin (Chemicon; CBL233, 1:2000), Hp47 (Santa Cruz, sc-398579, 1:1000 (WB), 1:500 (IF)), and syntaxin 5 (Santa Cruz, sc-365124, 1:500(WB)) PDI (Abcam, ab180993, dilution 1:500 (IF)).

Techniques: Microscopy, Immunofluorescence, Imaging, Marker, Fractionation, Derivative Assay, Synthesized, Transfection, Super-Resolution Microscopy, Stable Transfection, Transduction