Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 serves as a promoter of glycolysis in esophageal squamous cell carcinoma. A) KEGG metabolism analysis of the differential genes of shPDIA3P1 versus shNC by RNA‐seq. B,C) 2‐NBDG uptake was measured by flow cytometry to monitor glucose uptake in cells with transfection PDIA3P1‐siRNA (B) or PDIA3P1 expression plasmid (C) (MFI: Mean fluorescence intensity). D,E) Lactate production was measured in silenced PDIA3P1 (D) or overexpressed PDIA3P1 cells (E). F,G) Detection of glucose uptake in knocking down PDIA3P1 cells (F) or overexpressing PDIA3P1 cells (G). H,I) Seahorse metabolic analysis of ECAR, glycoPER (glycolytic proton efflux rate), basal glycolysis and compensatory glycolysis in PDIA3P1 knockdown cells (H) or PDIA3P1 overexpression cells (I) (Rot/AA: Rotenone/Antimycin A; 2‐DG: 2‐Deoxy‐D‐glucose). J,K) Relative level of acetyl‐CoA was measured in silenced PDIA3P1 cells (J) or overexpressed PDIA3P1 cells (K). L,M) Detection of relative α‐ketoglutarate levels in knocking down PDIA3P1 cells (L) or overexpressing PDIA3P1 cells (M). These data represent the mean ± S.D. of triplicates. ns: no significance; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: RNA Sequencing, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Fluorescence, Knockdown, Over Expression

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: Glycolysis mediates PDIA3P1 regulation of esophageal squamous cell carcinoma progression. A) Schematic diagram of glycolysis and inhibition methods target. B–G) Stable silencing of PDIA3P1 in TE‐1 and Eca‐109 cells was treated with exogenous lactate (15m m ) for 24h. B) The proliferative abilities were investigated via CCK‐8 assays. C) ESCC cell growth was analyzed using colony formation assay. The upper panel is for statistical analysis. D) EdU assays were performed to assess the proliferative ability of ESCC cells. The upper panel is for statistical analysis. Scale bar: 100 µm. E) Annexin V‐APC/7‐AAD staining for detecting apoptosis in TE‐1 and Eca‐109 cells by flow cytometry. The right panel is for statistical analysis. F) Transwell assays were used to detect changes in the cell migration and invasion. The upper panel is for statistical analysis. Transwell Scale bar: 10 µm. G) Western blot shows expression levels of E‐Cadherin, N‐Cadherin, Vimentin, and Snail. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Inhibition, CCK-8 Assay, Colony Assay, Staining, Flow Cytometry, Migration, Western Blot, Expressing

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 upregulates GLUT1 and HK2 to promote the glycolysis in ESCC cells. A,B) Western blot analysis of GLUT1, HK2, PFKFB3, PKM2, and LDHA after PDIA3P1 stable silencing in TE‐1 and Eca‐109 cells (A) or PDIA3P1 stable overexpressing in KYSE‐30 and KYSE‐150 cells (B). C,E,G,I) In Eca‐109 cells with stable knockdown of PDIA3P1, overexpression plasmids of GLUT1 and HK2 were transfected separately. D,F,H,J) In KYSE‐150 cells stably overexpressing PDIA3P1, GLUT1 siRNA and HK2 siRNA were transfected separately. C,D) Relative 2‐NBDG uptake detected by flow cytometry (MFI: Mean fluorescence intensity). E,F) Relative lactate production was measured using lactate assay kit. G,H) Detection of relative glucose uptake in ESCC. I,J) Cells were subjected to a Seahorse metabolic analyzer to determine the glycolytic rate including ECAR, glycoPER, basal glycolytic rate, and compensatory glycolytic rate. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Western Blot, Knockdown, Over Expression, Transfection, Stable Transfection, Flow Cytometry, Fluorescence, Lactate Assay

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 acted as a sponge of miR‐152‐3p to regulate GLUT1 expression. A,B) qRT‐PCR analyses of GLUT1 mRNA levels in stably PDIA3P1‐KD cells (A) or stably PDIA3P1‐OE cells (B). C) RIP assay for AGO2 was conducted to detect the levels of endogenous PDIA3P1 in the IP pellet of AGO2. D) The miRNAs that sponged by PDIA3P1 was predicted by miRNet and ENCORI and miRNAs that targeting GLUT1 by miRNet, TargetScan, miRDB, and ENCORI, the intersection predicted five miRNAs. E,F) AGO2‐RIP assays showed the enrichment of the predicted five miRNAs in stably PDIA3P1‐KD cells (E) or stably PDIA3P1‐OE cells (F). G) Putative binding sequence between PDIA3P1 and miR‐152‐3p. H) TE‐1 and Eca‐109 cells co‐transfected with wild‐type or mutant lncRNA PDIA3P1 and miR‐152‐3p mimics or control were detected by dual luciferase reporter assay. I) Luciferase reporters containing WT or MUT PDIA3P1 transcript were co‐transfected with miR‐152‐3p inhibitor or miR‐control in KYSE‐30 and KYSE‐150 cells. J) FISH results showing the colocalization of PDIA3P1 and miR‐152‐3p in cytoplasm of Eca‐109 cells. K) Enrichment of PDIA3P1 pulled down by biotin‐miR‐152‐3p or biotin‐miR‐control. L) Putative binding sequence of miR‐152‐3p in the 3′‐UTR of GLUT1. M) ESCC cells co‐transfected with wild‐type or mutant 3′‐UTR of GLUT1 and miR‐135b‐3p mimics or control were detected by dual luciferase reporter assay. N) Luciferase reporters containing WT or MUT 3′‐UTR of GLUT1 were co‐transfected with miR‐152‐3p inhibitor or miR‐control in ESCC cells. O) Transfection of miR‐152‐3p inhibitor into stably PDIA3P1‐KD cells, the expression levels of GLUT1 were detected by qRT‐PCR. P) Transfection of miR‐152‐3p mimics into stably PDIA3P1‐OE cells, the expression levels of GLUT1 were detected by qRT‐PCR. Q, R) Transfection of miR‐152‐3p inhibitor into stably PDIA3P1‐KD cells (Q) or miR‐152‐3p mimics into stably PDIA3P1‐OE cells (R), the expression levels of GLUT1 were detected by Western blot. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, Binding Assay, Sequencing, Transfection, Mutagenesis, Control, Luciferase, Reporter Assay, Western Blot

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 increases H4K8la level by promoted glycolysis in ESCC cells. A) Western blot shows the levels of Pan Kla in normal cell line HEEC and five ESCC cell lines (KYSE‐30, KYSE‐150, KYSE‐520, TE‐1, and Eca‐109). B,C) Western blot analysis of Pan‐ Kla after PDIA3P1 stable silencing in TE‐1 and Eca‐109 cells (B) or PDIA3P1 stable overexpressing in KYSE‐30 and KYSE‐150 cells (C). D,E) Representative images of IF staining revealing the effect of PDIA3P1 knockdown in Eca‐109 (D) or PDIA3P1 overexpressed in KYSE‐150 (E) on the expression of Pan‐ Kla. F, G) Western blot analysis of site‐specific histone lactylation in PDIA3P1‐KD cells (F) or PDIA3P1‐OE cells (G). H, I) Representative images of IF staining revealing the effect of PDIA3P1 knockdown in Eca‐109 (H) or PDIA3P1 overexpressed in KYSE‐150 (I) on the expression of H4K8la. J) PDIA3P1‐KD cells were treated with lactate for 24 h, Western blot measured the level of H4K8 lactylation. K) H4K18la levels were detected in PDIA3P1‐OE cells cultured in 2‐DG or oxamate for 24 h by western blot.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Western Blot, Staining, Knockdown, Expressing, Cell Culture

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: BMP7 is a target of H4K8 lactylation in ESCC cells. A) The binding density of H4K8la was visualized by deepTools: the heatmap illustrates the CUT&Tag tag counts on the various H4K8la enrichment peaks in shNC and shPDIA3P1 cells. B) Distribution of H4K8la sites relative to translation start site (TSS). C Genome‐wide distribution of downregulated H4K8la‐binding peaks in shPDIA3P cells. D) KEGG analysis of the decreased H4K8la binding peaks at candidate target genes. E) Transcriptome sequencing was performed in control and shPDIA3P1 cells. F) Venn Diagram of CUT&Tag, RNA‐seq, GEO, and pubmed database to identify the potential downstream targets of H4K8la. G) Integrative Genomics Viewer tracks of CUT&Tag showing enriched H4K8la in the promotors of BMP7. The red rectangles indicate the peak regions of H4K8la on target‐gene promoters. H‐K) BMP7 mRNA and protein levels were measured in PDIA3P1‐KD cells (H, J) or PDIA3P1‐OE cells (I, K). L) Western blotting analysis of BMP7 expression in PDIA3P1‐KD cells cultured in lactate for 24 h. M) Western blot analysis of BMP7 expression in PDIA3P1‐OE cells cultured in 2‐DG or oxamate for 24 h. N, O) Using antibodies against H4K8la, CUT&Tag‐qPCR analysis for binding status at the BMP7 promotor of PDIA3P1‐KD cells (N) or PDIA3P1‐OE cells (O). These data represent the mean ± S.D. of triplicates. ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Binding Assay, Genome Wide, Sequencing, Control, RNA Sequencing, Western Blot, Expressing, Cell Culture

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 promotes tumorigenesis of esophageal squamous cell carcinoma through BMP7 both in vitro and in vivo. A) Western blot detection of BMP7 expression to reflect the effect of transfection of BMP7 by lentivirus. B‐G) Using lentivirus to construct BMP7 stable overexpression cell lines in TE‐1 and Eca‐109 cells with stably PDIA3P1‐KD. B) Proliferation of cells was analyzed using CCK8 assay. C) Tumor growth of TE‐1 and Eca‐109 cells was evaluated by colony formation assay. D) The proliferative abilities of ESCC cells were investigated using EdU assays. EdU scale bar: 100 µm. E) The flow cytometry showing apoptosis of cells by Annexin V‐APC and 7‐AAD staining. F) The migration and invasion ability was assessed by transwell assays. Transwell scale bar: 10 µm. G) Western blot shows expression levels of E‐Cadherin, N‐Cadherin, Vimentin, Snail, and BMP7. H‐K) Eca‐109 cells transfected with control, shPDIA3P1 or shPDIA3P1 and BMP7 were injected subcutaneously to establish a tumorigenesis model in nude mice ( n = 6). H) Photograph and comparison of tumor sizes in the indicated groups. I) The parameters of subcutaneous tumors were measured and recorded every 5 days, and calculated the volume of the tumor according to the formula below: tumor volume = 0.5 × length × width × width (mm3). J) Tumor weights in the indicated groups. K) Ki‐67 and BMP7 expression of representative IHC of nude mice tumor tissues. Scale bar, 20 µm. IF staining of TUNEL in subcutaneous tumor tissue sections. Scale bar: 50 µm. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Transfection, Construct, Over Expression, Stable Transfection, CCK-8 Assay, Colony Assay, Flow Cytometry, Staining, Migration, Control, Injection, Comparison, TUNEL Assay

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: Increased stability of PDIA3P1 is via IGF2BP1 recognition of WTAP‐mediated m6A modification. A) The enriched and specific m6A peak distribution of PDIA3P1 predicted by SRAMP. B) MeRIP‐qPCR analysis of m6A modification level of PDIA3P1 in normal cell line HEEC and five ESCC cell lines (KYSE‐30, KYSE‐150, KYSE‐520, TE‐1, and Eca‐109). C) Correlation analysis showing a positive correlation between WTAP and PDIA3P1 expression. D) qRT‐PCR analysis of PDIA3P1 expression in TE‐1 and Eca‐109 cells with WTAP knockdown. E) Relative luciferase activity in TE‐1 and Eca‐109 cells co‐transfected with luciferase reporter pmirGLO‐PDIA3P1 and WTAP siRNA. F) WTAP silencing cells treated with actinomycin D (10 µg mL −1 ) for the various time points; the level of PDIA3P1 was examined by qRT‐PCR. G) The m6A modification level of PDIA3P1 was examined in WTAP‐silencing cells by MeRIP‐qPCR. H) PDIA3P1 pull‐down followed by Western blot validated its interaction with WTAP. I) RIP assay was performed using the WTAP antibody in KYSE‐150 and Eca‐109 cells. J) Relative luciferase activity of the wild‐type and its mutants pmirGLO‐PDIA3P1 reporter vectors when transfected with WTAP siRNA in TE‐1 and Eca‐109 cells (WT: wild‐type; A–G mut: adenine residues substituted by guanine; A–T mut: adenine residues substituted by thymine; A–Del mut: adenine residues deleted). K) Relative luciferase activity of the wild‐type and its mutants pmirGLO‐PDIA3P1 reporter vectors during IGF2BP1 silencing in ESCC cells. L) PDIA3P1 stability in control and IGF2BP1‐silenced cells, the level of PDIA3P1 was examined by qRT‐PCR. M) PDIA3P1 pull‐down followed by Western blot validated its interaction with IGF2BP1. N) RIP assay was performed using the IGF2BP1 antibody. O) FISH and IF double staining showing the co‐localization of PDIA3P1 and IGF2BP1 in the cytoplasm. P) qRT‐PCR analysis of PDIA3P1 expression in KYSE‐30 and KYSE‐150 cells co‐transfected with WTAP overexpression vector and the IGF2BP1 siRNA. Q) Relative luciferase activity in KYSE‐30 and KYSE‐150 cells co‐transfected with WTAP overexpression vector and the IGF2BP1 siRNA. R) PDIA3P1 stability in ESCC cells co‐transfected with WTAP overexpression vector and the IGF2BP2 siRNA, the level of PDIA3P1 was examined by qRT‐PCR. S) Transfection or co‐transfection of OCT4 cDNA or WTAP cDNA separately, and expression of PDIA3P1 was detected by qRT‐PCR. These data represent the mean ± S.D. of triplicates. ns: no significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Modification, Expressing, Quantitative RT-PCR, Knockdown, Luciferase, Activity Assay, Transfection, Western Blot, Control, Double Staining, Over Expression, Plasmid Preparation, Cotransfection

Journal: Redox Biology

Article Title: Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes

doi: 10.1016/j.redox.2019.101142

Figure Lengend Snippet: Validation of ELISA assay for specific detection of PDI . A. PDI concentration curves (0.375–3 ng/mL) were generated using purified PDI quantified by ELISA. Measurements were analyzed on 4 independent assays. Data represent mean ± SEM. B-C . Human cell lysates (HUVEC, HCT116 and HKE3) were (B) quantified by ELISA ( n = 4; mean ± SEM) and (C) submitted to protein separation by reducing SDS-PAGE and immunoblotted with anti-PDI ELISA's capture antibody or anti-β actin (loading control) ( n = 3). Uncropped western blots are shown in Supplementary . D. Cross-reactivity with other PDI family members was analyzed using recombinant PDI, ERp57 and ERp5 (1.5–3 ng/mL) quantified by ELISA. Measurements were analyzed on 4 independent assays. Data represent mean ± SEM. E. Plasma PDI immunoprecipitation. Platelet-poor plasma was diluted in lysis buffer and immunoprecipitated using rabbit anti-PDI antibody. Immunoblotting was performed using mouse anti-PDI (RL90) ( n = 3 from independent experiments). F. Detection of plasma PDI reductase activity (di-eosin-GSSG assay) by formation of the reduced fluorogenic product eosin-5-isothiocyanate-coupled reduced glutathione (EGSH). It was measured in platelet-poor plasma exposed or not to 60 μM of rutin. Bar graphs represent the percentage cleavage of di-eosin-GSSG as compared to total reductase activity – Control (100%) in 10 min. The main result in this case is the rutin-inhibitable fraction of di-eosin-GSSG reductase activity. Data represent mean ± SEM from 5 independent experiments. *p < 0.05; ***p < 0.001 vs. control (One-way ANOVA followed by Tukey's post test).

Article Snippet: Platelet-poor plasma (no dilution), recombinant PDI solutions (reduced, oxidized, S-nitrosylated), human cell lysates, conditioned medium, and recombinant Erp57 (OriGene-TP305940) and ERp5 (OriGene-TP301710) were analyzed.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Concentration Assay, Generated, Purification, SDS Page, Control, Western Blot, Recombinant, Clinical Proteomics, Immunoprecipitation, Lysis, Activity Assay, GSSG Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Ganoderic acid A ameliorates non-alcoholic streatohepatitis (NASH) induced by high-fat high-cholesterol diet in mice

doi: 10.3892/etm.2022.11237

Figure Lengend Snippet: GAA hepatoprotection is associated with hepatic oxidative stress and the ER stress response. (A) MDA and (B) SOD levels were detected in murine livers. (C and G) Western blotting was performed to determine the relative protein levels of (D) GRp78, (E) p-eIF-2α, (F) p-JNK, (H) ERp57, (I) p-MAPK and (J) p-AKT. Mice were fed an ND or HFHC diet with or without indicated doses of GAA treatment (n=6-8). Assays were repeated three times. Data are expressed as the mean ± SEM. * P<0.05, ** P<0.01 and *** P<0.001 vs. the HFHC group. eIF-2α, eukaryotic initiation factor-2α GAA, ganoderic acid A; GAAH, GAA 50 mg/kg/day; GAAL, GAA 25 mg/kg/day; GRp78, glucose-regulated protein 78; HFHC, high-fat high-cholesterol; MDA, malondialdehyde; ND, normal diet; ns, not significant; p, phosphorylated; SOD, superoxide dismutase.

Article Snippet: The primary antibodies (all dilutions were 1:500) including glucose-regulated protein 78 (GRp78; cat. no. 3177), phosphorylated (p)-eukaryotic initiation factor-2α (eIF-2α; cat. no. 3398), eIF-2α (cat. no. 5324), p-JNK (cat. no. 4668), JNK (cat. no. 9252), ERp57 (cat. no. 2887S), p-AKT (cat. no. 4060), AKT (cat. no. 9272) p-MAPK (cat. no. 4370), MAPK (cat. no. 9102) and GAPDH (cat. no. 8884) were obtained from Cell Signaling Technology, Inc. Immunoreactive signals were detected with Chemi-Lumi One Ultra (Tanon Science and Technology Co., Ltd.).

Techniques: Western Blot

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: ERp57 KO mice display severe structural ECM defects in knee joint cartilage High-magnification transmission electron microscopic (TEM) analysis of articular cartilage isolated from 18-week-old WT and ERp57 KO mouse knees. KO samples exhibit a significantly lower ECM density around chondrocytes with holes in the territorial/interterritorial matrix (marked with arrows) (A). In microphotographs of WT samples, an average of 96% of the total area was covered with dense matrix, compared to 79% in the KO (B). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. N (number of animals per genotype) ≥ 4; n (number of analyzed images per genotype) = 8; scale bars = 1 μm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Transmission Assay, Isolation

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Primary ERp57 KO chondrocytes produce less fibrillar matrix than WT cells Transmission electron microscopic (TEM) analysis of micromass cultures of primary WT and ERp57 KO chondrocytes isolated from knee joints of newborn mice revealed fewer and shorter cartilage fibrils in KO samples compared to WT controls (A), although the cell number is comparable (B). In microphotographs of WT samples, an average of 37% of the total area was covered with fibrils, compared to 25% in the KO (C). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. ns indicates non-significant p -values. N (number of animals per genotype) = 4; n (number of micromasses per genotype) ≥ 10; scale bars = 500 nm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Transmission Assay, Isolation

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Cultured C28/I2 ERp57 KO cells exhibit a reduced extracellular network of fibronectin 1 but unchanged collagen II fibrils Immunofluorescence analyses of the extracellular matrix (ECM) produced by C28/I2 WT and C28/I2 ERp57 KO chondrocytes, examined after fixation (Cells + Matrix) or after decellularization and fixation (Matrix) to visualize the ECM fibrils without cell-derived signals. The figure shows the projections of z-stacks. Punctate Col II signals in non-decellularized samples (Cells + Matrix) reveal Col II-containing vesicles near/above the nuclei of chondrocytes. Fibronectin (FN1) and collagen II (Col II) fibrils were detected in WT samples, including cells and matrix, and also in decellularized samples containing only matrix. The FN1 network was significantly reduced in KO samples (A). In contrast, the Col II network was comparably well developed in ERp57 KO and WT cells (B). Quantitative analysis of the decellularized samples revealed a reduction in the mean staining intensity of the FN1 matrix by more than 60% in the KO samples compared to WT controls and no statistically significant difference in Col II staining in samples of both genotypes. (C) Statistical evaluation was performed with the Student’s t test. Data are mean ± SD. ∗∗∗∗ represents a p -value of <0.0001, ns indicates non-significant p -values. N ≥ 8 (number of experiments), n ≥ 30 (technical replicates). Scale bars = 20 μm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Cell Culture, Immunofluorescence, Produced, Derivative Assay, Staining

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Extracellular ERp57 colocalizes with fibronectin 1 fibrils Co-Immunofluorescence analysis of FN1/ERp57 (A, top panel) and Col II/ERp57 (B, bottom panel) on decellularized matrices. In C28/I2 WT samples, ERp57 was detected on FN1 fibrils in different quantities (← ERp57 high, FN1 high, < ERp57 high, FN1 low, ∗ ERp57 low, FN1 high). The Col II network showed no direct colocalization with ERp57, however ERp57 was detectable in close vicinity to Col II structures (◄). N = 3. Scale bars = 20 μm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Immunofluorescence

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Extracellular ERp57 interacts directly with fibronectin 1 fibrils Proximity ligation assays (PLA) showed FN1/ERp57 interactions, visible as red dots on fibrillar structures of the extracellular matrix (ECM) (A). The corresponding statistical analysis (B) revealed a mean staining intensity of 0.284 ± 0.1065, which differed significantly from the mean staining intensities in the matrix of ERp57 KO cells and in the negative control (WT matrix without primary antibodies). In contrast, no interactions between Col II and ERp57 were detectable using PLA. The mean staining intensity in the WT-produced ECM did not exceed the background staining of the fibrils produced by ERp57 KO cells or the negative control (WT matrix without both primary antibodies). Short-term incubation with the reducing agent dithiothreitol (DTT) reduced PLA signals (C and D) significantly. Omission of ERp57 or FN1 antibodies reduced PLA signals to background levels (D). Statistical evaluation was performed with one-way ANOVA with Tukey’s post-hoc-test. Data are mean ± SD. ∗ represents a p -value of <0.05. N = 3 (number of experiments), n = 12 (technical replicates). Scale bars = 20 μm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Ligation, Staining, Negative Control, Produced, Incubation

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Active recombinant ERp57 protein added to the culture medium increases the fibronectin 1 fibrillogenesis around ERp57 KO cells in vitro Immunofluorecence analysis of FN1 and Col II on decellularized matrices of C28/I2 WT and C28/I2 ERp57 KO cells. Some of the KO cells were cultured for the entire culture period of 72 h in the presence of 0.1 μM active recombinant ERp57 protein or in the presence of 0.1 μM active recombinant ERp57 protein with the addition of 5 μM p -Chloromercuriphenylsulfonate (pCMPS) or 300 μM Monobromo (trimethylammonio) bimanbromide (QBBR) (A). KO cells showed in the quantitative analysis a strongly reduced staining intensity of FN1 and an unchanged staining intensity of Col II (B). The addition of active recombinant ERp57 protein to the cell culture medium of KO cells led to an increase in the mean staining intensity of FN1 (partial rescue), which was reduced again by the simultaneous addition of pCMPS and QBBR. The staining intensity of Col II was not significantly affected by the addition of active recombinant ERp57 protein in the presence or absence of pCMPS or QBBR. Statistical evaluation was performed with two-way ANOVA with Tukey’s post-hoc-test. Data are mean ± SD. ∗∗∗∗ represents a p -value of p < 0.0001, ∗∗ represents a p -value of p < 0.01, ns indicates non-significant p -values. N ≥ 5 (number of experiments), n ≥ 16 (technical replicates). Scale bars = 20 μm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Recombinant, In Vitro, Cell Culture, Staining

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: ERp57 KO mice display severe structural ECM defects in knee joint cartilage High-magnification transmission electron microscopic (TEM) analysis of articular cartilage isolated from 18-week-old WT and ERp57 KO mouse knees. KO samples exhibit a significantly lower ECM density around chondrocytes with holes in the territorial/interterritorial matrix (marked with arrows) (A). In microphotographs of WT samples, an average of 96% of the total area was covered with dense matrix, compared to 79% in the KO (B). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. N (number of animals per genotype) ≥ 4; n (number of analyzed images per genotype) = 8; scale bars = 1 μm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Transmission Assay, Isolation

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Primary ERp57 KO chondrocytes produce less fibrillar matrix than WT cells Transmission electron microscopic (TEM) analysis of micromass cultures of primary WT and ERp57 KO chondrocytes isolated from knee joints of newborn mice revealed fewer and shorter cartilage fibrils in KO samples compared to WT controls (A), although the cell number is comparable (B). In microphotographs of WT samples, an average of 37% of the total area was covered with fibrils, compared to 25% in the KO (C). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. ns indicates non-significant p -values. N (number of animals per genotype) = 4; n (number of micromasses per genotype) ≥ 10; scale bars = 500 nm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Transmission Assay, Isolation

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Cultured C28/I2 ERp57 KO cells exhibit a reduced extracellular network of fibronectin 1 but unchanged collagen II fibrils Immunofluorescence analyses of the extracellular matrix (ECM) produced by C28/I2 WT and C28/I2 ERp57 KO chondrocytes, examined after fixation (Cells + Matrix) or after decellularization and fixation (Matrix) to visualize the ECM fibrils without cell-derived signals. The figure shows the projections of z-stacks. Punctate Col II signals in non-decellularized samples (Cells + Matrix) reveal Col II-containing vesicles near/above the nuclei of chondrocytes. Fibronectin (FN1) and collagen II (Col II) fibrils were detected in WT samples, including cells and matrix, and also in decellularized samples containing only matrix. The FN1 network was significantly reduced in KO samples (A). In contrast, the Col II network was comparably well developed in ERp57 KO and WT cells (B). Quantitative analysis of the decellularized samples revealed a reduction in the mean staining intensity of the FN1 matrix by more than 60% in the KO samples compared to WT controls and no statistically significant difference in Col II staining in samples of both genotypes. (C) Statistical evaluation was performed with the Student’s t test. Data are mean ± SD. ∗∗∗∗ represents a p -value of <0.0001, ns indicates non-significant p -values. N ≥ 8 (number of experiments), n ≥ 30 (technical replicates). Scale bars = 20 μm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Cell Culture, Immunofluorescence, Produced, Derivative Assay, Staining

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Extracellular ERp57 colocalizes with fibronectin 1 fibrils Co-Immunofluorescence analysis of FN1/ERp57 (A, top panel) and Col II/ERp57 (B, bottom panel) on decellularized matrices. In C28/I2 WT samples, ERp57 was detected on FN1 fibrils in different quantities (← ERp57 high, FN1 high, < ERp57 high, FN1 low, ∗ ERp57 low, FN1 high). The Col II network showed no direct colocalization with ERp57, however ERp57 was detectable in close vicinity to Col II structures (◄). N = 3. Scale bars = 20 μm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Immunofluorescence

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Extracellular ERp57 interacts directly with fibronectin 1 fibrils Proximity ligation assays (PLA) showed FN1/ERp57 interactions, visible as red dots on fibrillar structures of the extracellular matrix (ECM) (A). The corresponding statistical analysis (B) revealed a mean staining intensity of 0.284 ± 0.1065, which differed significantly from the mean staining intensities in the matrix of ERp57 KO cells and in the negative control (WT matrix without primary antibodies). In contrast, no interactions between Col II and ERp57 were detectable using PLA. The mean staining intensity in the WT-produced ECM did not exceed the background staining of the fibrils produced by ERp57 KO cells or the negative control (WT matrix without both primary antibodies). Short-term incubation with the reducing agent dithiothreitol (DTT) reduced PLA signals (C and D) significantly. Omission of ERp57 or FN1 antibodies reduced PLA signals to background levels (D). Statistical evaluation was performed with one-way ANOVA with Tukey’s post-hoc-test. Data are mean ± SD. ∗ represents a p -value of <0.05. N = 3 (number of experiments), n = 12 (technical replicates). Scale bars = 20 μm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Ligation, Staining, Negative Control, Produced, Incubation

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Active recombinant ERp57 protein added to the culture medium increases the fibronectin 1 fibrillogenesis around ERp57 KO cells in vitro Immunofluorecence analysis of FN1 and Col II on decellularized matrices of C28/I2 WT and C28/I2 ERp57 KO cells. Some of the KO cells were cultured for the entire culture period of 72 h in the presence of 0.1 μM active recombinant ERp57 protein or in the presence of 0.1 μM active recombinant ERp57 protein with the addition of 5 μM p -Chloromercuriphenylsulfonate (pCMPS) or 300 μM Monobromo (trimethylammonio) bimanbromide (QBBR) (A). KO cells showed in the quantitative analysis a strongly reduced staining intensity of FN1 and an unchanged staining intensity of Col II (B). The addition of active recombinant ERp57 protein to the cell culture medium of KO cells led to an increase in the mean staining intensity of FN1 (partial rescue), which was reduced again by the simultaneous addition of pCMPS and QBBR. The staining intensity of Col II was not significantly affected by the addition of active recombinant ERp57 protein in the presence or absence of pCMPS or QBBR. Statistical evaluation was performed with two-way ANOVA with Tukey’s post-hoc-test. Data are mean ± SD. ∗∗∗∗ represents a p -value of p < 0.0001, ∗∗ represents a p -value of p < 0.01, ns indicates non-significant p -values. N ≥ 5 (number of experiments), n ≥ 16 (technical replicates). Scale bars = 20 μm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Recombinant, In Vitro, Cell Culture, Staining