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: To analyze PDI, extracellular ERp57 or Col II-containing fibrils, a rabbit polyclonal PDI antibody (sc-20132, Santa Cruz Biotechnology, USA, 1:50), a rabbit polyclonal ERp57 antibody (NBP1-84796, Novus, Centennial, USA,1:200) or monoclonal mouse Collagen II antibodies (MAB8887, Merck Darmstadt, Germany, 1:200, or DSHB II6B3, 1:200) were applied.

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: To analyze PDI, extracellular ERp57 or Col II-containing fibrils, a rabbit polyclonal PDI antibody (sc-20132, Santa Cruz Biotechnology, USA, 1:50), a rabbit polyclonal ERp57 antibody (NBP1-84796, Novus, Centennial, USA,1:200) or monoclonal mouse Collagen II antibodies (MAB8887, Merck Darmstadt, Germany, 1:200, or DSHB II6B3, 1:200) were applied.

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: To analyze PDI, extracellular ERp57 or Col II-containing fibrils, a rabbit polyclonal PDI antibody (sc-20132, Santa Cruz Biotechnology, USA, 1:50), a rabbit polyclonal ERp57 antibody (NBP1-84796, Novus, Centennial, USA,1:200) or monoclonal mouse Collagen II antibodies (MAB8887, Merck Darmstadt, Germany, 1:200, or DSHB II6B3, 1:200) were applied.

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: To analyze PDI, extracellular ERp57 or Col II-containing fibrils, a rabbit polyclonal PDI antibody (sc-20132, Santa Cruz Biotechnology, USA, 1:50), a rabbit polyclonal ERp57 antibody (NBP1-84796, Novus, Centennial, USA,1:200) or monoclonal mouse Collagen II antibodies (MAB8887, Merck Darmstadt, Germany, 1:200, or DSHB II6B3, 1:200) were applied.

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: To analyze PDI, extracellular ERp57 or Col II-containing fibrils, a rabbit polyclonal PDI antibody (sc-20132, Santa Cruz Biotechnology, USA, 1:50), a rabbit polyclonal ERp57 antibody (NBP1-84796, Novus, Centennial, USA,1:200) or monoclonal mouse Collagen II antibodies (MAB8887, Merck Darmstadt, Germany, 1:200, or DSHB II6B3, 1:200) were applied.

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: To analyze PDI, extracellular ERp57 or Col II-containing fibrils, a rabbit polyclonal PDI antibody (sc-20132, Santa Cruz Biotechnology, USA, 1:50), a rabbit polyclonal ERp57 antibody (NBP1-84796, Novus, Centennial, USA,1:200) or monoclonal mouse Collagen II antibodies (MAB8887, Merck Darmstadt, Germany, 1:200, or DSHB II6B3, 1:200) were applied.

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: To assess cellular uptake of ERp57, ERp57 KO cells were cultured in conditioned media from ERp57 WT cells or treated with 0.1 μM recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA).

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: To assess cellular uptake of ERp57, ERp57 KO cells were cultured in conditioned media from ERp57 WT cells or treated with 0.1 μM recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA).

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: To assess cellular uptake of ERp57, ERp57 KO cells were cultured in conditioned media from ERp57 WT cells or treated with 0.1 μM recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA).

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: To assess cellular uptake of ERp57, ERp57 KO cells were cultured in conditioned media from ERp57 WT cells or treated with 0.1 μM recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA).

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: To assess cellular uptake of ERp57, ERp57 KO cells were cultured in conditioned media from ERp57 WT cells or treated with 0.1 μM recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA).

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: To assess cellular uptake of ERp57, ERp57 KO cells were cultured in conditioned media from ERp57 WT cells or treated with 0.1 μM recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA).

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: To analyze extracellular disulfide bridge formation or the role of extracellular ERp57, cultures were supplemented during the entire culture period with 60 and 300 μM Monobrom (trimethylammonio) bimanbromid (QBBR) (Merck, Darmstadt, Germany), 1.5 and 15 μM p-Cholromercuriphenylsulphonate (pCMPS) (LGC Standards, Wesel, Germany) or 0.1 μM active recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA), respectively.

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: To analyze extracellular disulfide bridge formation or the role of extracellular ERp57, cultures were supplemented during the entire culture period with 60 and 300 μM Monobrom (trimethylammonio) bimanbromid (QBBR) (Merck, Darmstadt, Germany), 1.5 and 15 μM p-Cholromercuriphenylsulphonate (pCMPS) (LGC Standards, Wesel, Germany) or 0.1 μM active recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA), respectively.

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: To analyze extracellular disulfide bridge formation or the role of extracellular ERp57, cultures were supplemented during the entire culture period with 60 and 300 μM Monobrom (trimethylammonio) bimanbromid (QBBR) (Merck, Darmstadt, Germany), 1.5 and 15 μM p-Cholromercuriphenylsulphonate (pCMPS) (LGC Standards, Wesel, Germany) or 0.1 μM active recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA), respectively.

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: To analyze extracellular disulfide bridge formation or the role of extracellular ERp57, cultures were supplemented during the entire culture period with 60 and 300 μM Monobrom (trimethylammonio) bimanbromid (QBBR) (Merck, Darmstadt, Germany), 1.5 and 15 μM p-Cholromercuriphenylsulphonate (pCMPS) (LGC Standards, Wesel, Germany) or 0.1 μM active recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA), respectively.

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: To analyze extracellular disulfide bridge formation or the role of extracellular ERp57, cultures were supplemented during the entire culture period with 60 and 300 μM Monobrom (trimethylammonio) bimanbromid (QBBR) (Merck, Darmstadt, Germany), 1.5 and 15 μM p-Cholromercuriphenylsulphonate (pCMPS) (LGC Standards, Wesel, Germany) or 0.1 μM active recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA), respectively.

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: To analyze extracellular disulfide bridge formation or the role of extracellular ERp57, cultures were supplemented during the entire culture period with 60 and 300 μM Monobrom (trimethylammonio) bimanbromid (QBBR) (Merck, Darmstadt, Germany), 1.5 and 15 μM p-Cholromercuriphenylsulphonate (pCMPS) (LGC Standards, Wesel, Germany) or 0.1 μM active recombinant human ERp57 protein (NBP2-52140, Novus, Centennial, CO, USA), respectively.

Techniques: Recombinant, In Vitro, Cell Culture, Staining

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Mean diameter of ensemble particles in solution ( Z -average) of PDIA1, PDIA3, PDIA4, PDIA6, PDIA10 and PDIA15 under various Ca 2+ concentrations. The values are the mean ± s.d. of three independent experiments. b , Liquid droplets observed by DIC microscopy when 50 μM PDIA6 and 4 mM CaCl 2 were mixed at pH 7.2. This experiment was replicated three times independently. c , PDIA6 phase diagram obtained by DIC microscopy when 5–100 μM PDIA6 and 0.5–10 mM CaCl 2 were mixed at pH 7.2. Dominant PDIA6 states at varying protein and Ca²⁺ concentrations are indicated by symbols: black circles, dispersed state; black triangles, condensed state. The dashed black line represents the critical droplet concentration. Three independent experiments were performed. d , Confocal fluorescence images of PDIA6 droplets before and after photobleaching. The white arrowhead indicates the laser irradiation area. Rapid recovery of mCherry–PDIA6 fluorescence after photobleaching (left). Increases in the normalized fluorescence intensity of mCherry–PDIA6 after photobleaching (five replicates; right). The fluorescence recovery t 1/2 was calculated from the normalized fluorescence intensity of the five replicates. e , Liquid droplets observed by DIC microscopy when 50 μM PDIA6 and 4 mM CaCl 2 were mixed with (right) or without (left) NaCl. This experiment was replicated three times independently. f , Liquid droplets observed by DIC microscopy when 50 μM PDIA6 and 4 mM CaCl 2 were mixed in solutions with different pH values. This experiment was replicated three times independently. g , Time course of representative two-dimensional (2D) RI distribution (top), and bright-field (middle) and fluorescence images (bottom) of FUS and PDIA6 droplets monitored by 3D holographic imaging (green, ThT fluorescence; three independent experiments). [Ca 2+ ], Ca 2+ concentration; BF, bright field; FI, fluorescence image; [PDIA6], PDIA6 concentration.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Microscopy, Concentration Assay, Fluorescence, Irradiation, Imaging

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Condensation of the green fluorescent protein (GFP)-tagged PDI family members in PDIA6 droplets. This experiment was performed three times independently. Statistical significance was examined using a one-way ANOVA with Tukey’s honest significant difference post-hoc test; the test was two-sided. **** P < 0.0001. b , Changes in the RI inside PDIA6 droplets with increasing concentrations of different PDI family members. Representative 2D RI distributions are indicated. Data were analysed for 198 particles for PDIA6 only, 217 particles for 1 µM PDIA1, 210 particles for 5 µM PDIA1, 212 particles for 10 µM PDIA1, 215 particles for 1 µM PDIA3, 220 particles for 5 µM PDIA3, 212 particles for 10 µM PDIA3, 215 particles for 1 µM PDIA4, 219 particles for 5 µM PDIA4, 212 particles for 10 µM PDIA4, 214 particles for 1 µM PDIA10, 212 particles for 5 µM PDIA10, 217 particles for 10 µM PDIA10, 217 particles for 1 µM PDIA15, 218 particles for 5 µM PDIA15 and 226 particles for 10 µM PDIA15 pooled from three independent replicates. a , b , Data are presented as the mean ± s.d. c , Representative images of the 2D RI distribution of PDIA6 droplets with untagged PDI family members, monitored by 3D holographic imaging from the same dataset analysed in Fig. 5b. d , Co-localization of endogenous PDIA3 and PDIA6 in U2OS cells (top). The fluorescence intensity (bottom) was analysed against the yellow line (top). The merged figure shows the fluorescence intensities of mCherry–PDIA6 and PDIA3 as a solid line, which means that their respective fluorescence intensities overlap (bottom). This experiment was performed three times independently.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Imaging, Fluorescence

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Condensation of GFP-tagged PDI family members into PDIA6 droplets. Representative images of bright-field (BF) and fluorescence microscopy of PDIA6 droplets with GFP-tagged PDI family members in the uptake assay. Three independent experiments were performed. b , Signal-enhanced images of Extended Data Fig. 8a. c , Condensation of PDI family members into PDIA6 droplets. The average refractive index (RI) inside the droplet and droplet radius were calculated by enclosing the RI image in a circle. Data were analysed for 198 particles for PDIA6 only, 217 particles for 1 µM PDIA1, 210 particles for 5 µM PDIA1, 212 particles for 10 µM PDIA1, 215 particles for 1 µM PDIA3, 220 particles for 5 µM PDIA3, 212 particles for 10 µM PDIA3, 215 particles for 1 µM PDIA4, 219 particles for 5 µM PDIA4, 212 particles for 10 µM PDIA4, 214 particles for 1 µM PDIA10, 212 particles for 5 µM PDIA10, 217 particles for 10 µM PDIA10, 217 particles for 1 µM PDIA15, 218 particles for 5 µM PDIA15 and 226 particles for 10 µM PDIA15, pooled from three independent replicates.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Fluorescence, Microscopy, Refractive Index