Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C inhibits TAZ activation in RPE. (A-E) Western blot showing the expression of JAM-C, TAZ, p-TAZ and YAP after JAM-C knockdown in RPE cells. n = 3. (F-H) Western blot analysis for TAZ after JAM-C knockdown and treatment with 25 µg/ml Cycloheximide (CHX) in RPE cells. n = 3. (I) Co-immunoprecipitation (co-IP) using anti-JAM-C antibody or IgG followed by Western blot showing the interaction of JAM-C, TAZ and YAP in RPE cells. n = 3. (J) Schematic representation of full-length JAM-C and its ΔCyto truncate. (K) Results of co-immunoprecipitation (co-IP) followed by Western blot showing the interaction between Flag-tagged JAM-C FL but not ΔCyto and TAZ. n = 3. (L-N) Western blot showing the subcellular portion of TAZ in RPE cells after JAM-C knockdown. GAPDH and HISTONE 3 were used as cytoplasmic or nuclear controls respectively. n = 3. (O-P) Immunostaining of JAM-C (red) and TAZ (green) in RPE cells after JAM-C knockdown. Nuclei are stained by DAPI. Scale bar: 20 μm. Quantification of the percentage of TAZ + cells in the nucleus are shown in P, n = 4. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA in G, H, M−N and Student’s t -test in B-E, and P. ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The cells were then blocked using 5 % bovine serum albumin (BSA, B2064, Sigma-Aldrich) at room temperature for 1 h, and incubated overnight at 4 °C with the indicated primary antibodies, including anti-JAM-C (AF1189, R&D Systems) and anti-fibronectin (F3648, Sigma-Aldrich) and anti-TAZ (HPA007415 Sigma-Aldrich).

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunostaining, Staining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: Genetic deletion of Jam-c exacerbates ocular fibrosis. (A) ELISA results showing lower levels of JAM-C in the vitreous humor of PVR patients. n = 9 for CTRL and n = 13 for PVR. (B-F) RPE cells were treated with TGF-β2 (2 and 4 ng/ml) for 48 h and subjected to Western blot for indicated proteins. n = 3. (G) Diagram showing the generation of the PVR mouse model in Jam-c RPE cko and CTRL mice. (H-J) Western blot showing the protein levels of JAM-C and RPE marker (RPE65) in the RPE-choroid complex of CTRL and Jam-c RPE cko mice. n = 3. (K-M) H&E staining showing epiretinal (black arrows) and subretinal (yellow arrows) membrane formation in Jam-c RPE cko and CTRL mice in a PVR model (K). RPE: retinal pigment epithelium, INL: inner nuclear layer, ONL: outer nuclear layer. Scale bar: 50 μm. The area of the membranes (K) are shown in L and M. n = 9 for CTRL and n = 8 for Jam-c RPE cko. (N-Q) DIC (differential interference contrast) and immunostaining of fibronectin in Jam-c RPE cko and CTRL mice in a PVR model (N). Green: fibronectin; Blue: DAPI. Red asterisks: misplaced melanosome; Orange arrows: subretinal membrane (SRM); Red arrows: epiretinal membrane (ERM); Yellow lines: RPE-choroid complex. Scale bar: 50 μm. The thickness of RPE-choroid complex and the fibronectin-positive membrane area (N) are shown in O-Q. n = 7 for CTRL and n = 6 for Jam-c RPE cko. Data are shown as mean ± SD. Statistical significance was assessed by one-way ANOVA in C-F, and Student’s t -test in A, I-J, L-M, and O-Q. * p < 0.05, ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Marker, Staining, Membrane, Immunostaining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C knockdown results in RPE EMT. (A) qRT-PCR results showing the knockdown efficiency of JAM-C by two different siRNA oligos in RPE cells. n = 4. (B-F) Western blot showing the protein levels of JAM-C, mesenchymal markers (fibronectin, N-cadherin) and the epithelial marker E-cadherin in ARPE-19 cells with JAM-C knockdown. n = 3. (G-L) Western blot showing the protein levels of JAM-C, mesenchymal markers (fibronectin, vimentin, N-cadherin) and the epithelial marker E-cadherin in primary human RPE cells with JAM-C knockdown. n = 3. (M−N) Immunostaining of fibronectin in RPE cells with JAM-C knockdown (M). Nuclei are stained by DAPI. Scale bar: 10 μm. Quantification of fibronectin staining intensity is shown in N. n = 6. (O) CCK8 assay results showing the cell proliferation status after JAM-C knockdown in RPE cells at 72 h. n = 6. (P-Q) Wound healing assay results showing the migration potential of RPE cells after JAM-C knockdown (P). Scale bar: 200 μm. Quantification of wound closure areas are shown in Q. n = 5. (R-S) Gel contraction assay results showing the cell contraction ability after JAM-C knockdown (R). Quantification of the relative remaining areas of the gels are shown in S. n = 4. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA in A, C-F, and Student’s t -test in H-L, N, O, Q and S. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Marker, Immunostaining, Staining, CCK-8 Assay, Wound Healing Assay, Migration, Collagen Gel Contraction Assay

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C inhibits TAZ activation in RPE. (A-E) Western blot showing the expression of JAM-C, TAZ, p-TAZ and YAP after JAM-C knockdown in RPE cells. n = 3. (F-H) Western blot analysis for TAZ after JAM-C knockdown and treatment with 25 µg/ml Cycloheximide (CHX) in RPE cells. n = 3. (I) Co-immunoprecipitation (co-IP) using anti-JAM-C antibody or IgG followed by Western blot showing the interaction of JAM-C, TAZ and YAP in RPE cells. n = 3. (J) Schematic representation of full-length JAM-C and its ΔCyto truncate. (K) Results of co-immunoprecipitation (co-IP) followed by Western blot showing the interaction between Flag-tagged JAM-C FL but not ΔCyto and TAZ. n = 3. (L-N) Western blot showing the subcellular portion of TAZ in RPE cells after JAM-C knockdown. GAPDH and HISTONE 3 were used as cytoplasmic or nuclear controls respectively. n = 3. (O-P) Immunostaining of JAM-C (red) and TAZ (green) in RPE cells after JAM-C knockdown. Nuclei are stained by DAPI. Scale bar: 20 μm. Quantification of the percentage of TAZ + cells in the nucleus are shown in P, n = 4. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA in G, H, M−N and Student’s t -test in B-E, and P. ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunostaining, Staining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: TAZ increases KLF6′s expression and pro-EMT function. (A) Volcano plot showing the up- and down-regulated genes in TAZ overexpressing RPE cells. (B) qRT-PCR showing KLF6 level in TAZ overexpressing RPE cells. n = 3. (C-E) Western blot showing the protein levels of TAZ and KLF6 after TAZ knockdown in primary human RPE cells. n = 3. (F) Dual luciferase reporter gene assay showing the luciferase activity of FN1 promoter after the overexpression of TAZ and/or KLF6 in RPE cells. n = 3. (G) ChIP-qPCR assay detected the enrichment of KLF6 at FN1 promoter region in RPE cells. n = 3. (H) qRT-PCR results showing the expression level of FN1 in RPE cells overexpressing KLF6 and/or TAZ. n = 3. (I) qRT-PCR result showing KLF6 mRNA level after knockdown of JAM-C and/or TAZ in RPE cells. n = 4. (J-N) Western blot showing the protein levels of JAM-C, TAZ, KLF6 and fibronectin after the knockdown of JAM-C and/or TAZ in RPE cells. n = 3. Data are presented as mean ± SD. Statistical significance was determined using Student’s t-test in B and one-way ANOVA in D-I, K-N. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Luciferase, Reporter Gene Assay, Activity Assay, Over Expression, ChIP-qPCR

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: KLF6 mediates JAM-C depletion-induced RPE EMT and ocular fibrosis. (A-E) Western blot showing the protein levels of KLF6 and mesenchymal markers (fibronectin, N-cadherin, vimentin) after KLF6 knockdown in RPE cells. n = 3. (F) CCK8 assay showing the cell proliferation after KLF6 knockdown in RPE cells at 72 h. n = 4. (G-I) Western blot showing the protein levels of JAM-C and KLF6 after knockdown of JAM-C and/or KLF6 in RPE cells. n = 3. (J) CCK8 assay showing cell proliferation after knockdown of JAM-C and/or KLF6 in RPE cells. n = 4. (K-L) Fibronectin immunostaining in RPE cells with JAM-C and/or KLF6 knockdown (K). Nuclei are stained by DAPI. Scale bar: 10 μm. Quantification of fibronectin staining intensity is shown in L. n = 6. Data are presented as mean ± SD. Statistical significance was determined using Student’s t -test in B-F and one-way ANOVA in H, I, J and L. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Western Blot, Knockdown, CCK-8 Assay, Immunostaining, Staining

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C overexpression alleviates ocular fibrosis. (A) Diagram showing the generation of the PVR model in WT mice with RPE-specific overexpression of JAM-C or CTRL. AAV, adeno-associated virus. Ad, adenovirus. (B-D) Western blot showing the protein levels of JAM-C and the RPE marker RPE65 in the RPE-choroid complex from mice with JAM-C OE in PVR models. n = 3. (E-H) DIC and immunostaining of fibronectin in the RPE-choroid complex from mice with JAM-C OE in PVR model (E). Green: fibronectin, Blue: DAPI. Red asterisks: displaced melanosome, orange arrows: subretinal membrane (SRM), red arrows: epiretinal membrane (ERM), yellow lines: thickness of RPE-choroid complex. Scale bar: 50 μm. Quantification of the thickness of RPE-choroid complex and the fibronectin-positive membrane areas in E are shown in F-H. n = 5 for each group. (I-K) Immunostaining of KLF6 in the retina of mice with JAM-C OE in a PVR model (I). Green: KLF6, Blue: DAPI, Red arrows: epiretinal membrane (ERM) Orange arrows: subretinal membrane (SRM). Scale bar: 50 μm. Quantification of the KLF6 positive areas in I are shown in J and K. n = 5 for each group. (L-N) Western blot showing the protein levels of KLF6 and the EMT marker fibronectin in the RPE-choroid complex of mice with JAM-C OE in PVR models. n = 3. Data are presented as mean ± SD. Statistical significance was determined using Student’s t -test in C-D, F-H, J-K, M−N. * p < 0.05, ** p < 0.01, *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Over Expression, Virus, Western Blot, Marker, Immunostaining, Membrane

Journal: Journal of Advanced Research

Article Title: JAM-C prevents ocular fibrosis by suppressing the TAZ/KLF6 pathway

doi: 10.1016/j.jare.2025.05.037

Figure Lengend Snippet: JAM-C prevents RPE EMT and ocular fibrosis by suppressing the TAZ/KLF6 axis. In wild-type mice, JAM-C reduces TAZ expression, stability and nuclear transportation to inhibit its EMT-promoting function to prevent EMT and fibrosis, thus maintaining normal RPE and retina. In Jam-c knockout mice, in the absence of JAM-C, TAZ expression, stability and nuclear translocation increases, leading to the upregulation of KLF6 and KLF6-induced expression of many EMT causing genes, resulting in RPE EMT and fibrosis.

Article Snippet: Cell lysates were incubated with JAM-C antibody (AF1189, R&D Systems, MN, USA), or IgG (AB-108-C, R&D Systems) at 4°C overnight, followed by Protein A/G-Sepharose (sc-2003, Santa Cruz, USA) incubation for 1.5 h at 4 °C.

Techniques: Expressing, Knock-Out, Translocation Assay