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

Journal: The Journal of Cell Biology

Article Title: The glycoprotein GP130 governs the surface presentation of the G protein–coupled receptor APLNR

doi: 10.1083/jcb.202004114

Figure Lengend Snippet: GP130 contributes to APLNR availability at the plasma membrane. (A) Flow cytometry analysis of APLNR and GP130 in patient-derived GSCs (mesenchymal GSC#1, mesenchymal GSC#4, and classical GSC#9) transfected with nonsilencing (sic, blue) and GP130 targeting siRNA duplexes (si GP130 , green). Ig control staining plots are shown (red). Histograms present the mean fluorescence intensity (MFI) normalized to respective sic conditions for GP130 and APLNR staining as indicated. Data are presented as the mean ± SEM of three independent experiments. (B) Flow cytometry analysis of APLNR and GP130 in GSC#1 transfected with nonsilencing (sic, blue) and APLNR targeting siRNA duplexes (si APLNR , green). Ig control staining plots are shown (Ig, red). Data are representative of three independent experiments. (C) Flow cytometry analysis of GP130, APLNR and JAMC in WT (blue) and GP130 KO (#2, green, and #7, orange) GSC#1. Ig control staining plots are shown (Ig, red). Data are representative of three independent experiments. (D) Flow cytometry analysis of GP130 and APLNR in GSC#1 WT (parental, blue), GP130 KO (#2, green, and #7, orange), and GP130 KO reconstituted with GP130 cDNA (#2+GP130, light blue, and #7+GP#130, pink). Ig control staining plots are shown (Ig, red). Data are representative of three independent experiments. Histograms present the MFI normalized to respective sic conditions for GP130 and APLNR staining as indicated. Data are presented as the mean ± SEM on three independent experiments. (E) Confocal analysis of GP130 (green), APLNR (red) and nuclei (DAPI, blue) in WT and GP130 KO (#2 and #7) permeabilized GSC#1. Scale bars, 10 µm. Fluorescence mean intensity for APLNR signal (arbitrary unit) was quantified by high-content microscopy in WT and GP130 KO (#2 and #7) GSC#1 and represented as violin diagram. Lines delineate the mean. Data are representative of three independent experiments, with n > 950 cells. (F) WT and GP130 KO (#2 and #7) GSC#1 were fixed and analyzed by confocal microscopy for GPI-enriched domains (FLAER, green) and APLNR (red). Nuclei are shown in blue (DAPI). Scale bars, 10 µm. Data are representative of at least three independent experiments. (G) Schematic diagram for the anti-APLNR uptake to analyze internalization (iAPLNR) and further recycling (rAPLNR). (H) Anti-APLNR antibody uptake (iAPLNR, green) was assessed in WT and GP130 KO#2 cells after 15 min at 37°C. Following acid wash and fixation, cells were stained for RAB5 (red) or RAB7 (red) and analyzed by confocal microscopy. Nuclei are shown in blue (DAPI). Merge images are shown. Scale bars, 5 µm. Data are representative of three independent experiments. (I) Similarly, anti-APLNR antibody uptake was assessed by flow cytometry after incubation at 37°C at the indicated times (0, 2.5, 5, 10, 15, and 30 min) in WT (blue) and GP130 KO#2 (green) GSC#1. Ig control staining plots are shown (red). Data are representative of three independent experiments. Boxplots depict the APLNR uptake in WT (blue) and GP130 KO#2 (green) GSC#1, calculated from normalized MFI at the indicated time points (minutes) following 37°C incubation. Line delineates the mean, and boxes show upper and lower quartiles. (J) Anti-APLNR antibody recycling (rAPLNR, red) was evaluated in WT and GP130 KO#2 cells after a 60-min chase at 37°C (following a 15-min pulse at 37°C and acid washes, as depicted in G). Cells were fixed and not permeabilized to analyze recycling APLNR (red) by confocal microscopy. Nuclei are shown in blue (DAPI). Merge images are shown. Scale bars, 10 µm. (K) Similarly, anti-APLNR antibody recycling was assessed by flow cytometry after incubation at 37°C at the indicated times (0, 5, 15, 30, and 60 min) in WT (blue) and GP130 KO#2 (green) GSC#1. Ig control staining plots are shown (red). Data are representative of three independent experiments. Boxplots depict the APLNR recycling in WT (blue) and GP130 KO#2 (green) GSC#1, calculated from normalized MFI at the indicated time points (minutes) following 37°C chasing incubation. Line delineates the mean, and boxes show upper and lower quartiles. All data are representative of at least three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 using ANOVA tests.

Article Snippet: The following primary antibodies were used (dilution 1:1,000 for Western blot, 1:200 for immunofluorescence and flow cytometry): GAPDH (mouse SC-25778; Santa Cruz Biotechnology), GP130 (rabbit SC-656 for Western blot, mouse SC-376280 for immunofluorescence; Santa Cruz Biotechnology; and mouse ab34324 for flow cytometry; Abcam), APLNR (mouse FAB8561R and MAB856; R&D Systems), JAMC (mouse FAB11891A; R&D Systems), STAT3 (rabbit 9132; Cell Signaling), pS727-STAT3 (rabbit 9134; Cell Signaling), pY705-STAT3 (rabbit 9145; Cell Signaling), pS235/S236-S6 (rabbit 2211 for Western blot and rabbit 5316 for flow cytometry; Cell Signaling), pS9-GSK3β (rabbit 9336; Cell Signaling), ELMOD1 (rabbit ab127541; Abcam), RAB5 (rabbit ab218624; Abcam) and RAB7 (rabbit 9367; Cell Signaling).

Techniques: Clinical Proteomics, Membrane, Flow Cytometry, Derivative Assay, Transfection, Control, Staining, Fluorescence, Microscopy, Confocal Microscopy, Incubation