Fig 1: miR-221–3p is necessary for JAM-A 3′ UTR to promote VCAN expression. (A) Representative immunofluorescence imaging of AP expression in miR-221–3p transfected hDPCs. (B) CCK-8 assays were conducted to determine the cell proliferation of hDPCs with miR-221–3p transfection. The right panel shows the cell viability analysis of miR-221–3p transfected hDPCs. (C) Representative immunofluorescence imaging of AP expression in miR-221–3p inhibitor transfected hDPCs. (D) CCK-8 assays and FCM were used to observe the effects of miR-221–3p expression on the proliferation and cell cycle of hDPCs. (E and F) Quantitative analysis of JAM-A and VCAN expression in miR-221–3p mimics and inhibitor transfected hDPCs. (G) The immunofluorescence imaging of JAM-A and VCAN expression in miR-221–3p overexpressed or inhibited hDPCs. (H and I) Quantitative analysis of JAM-A and VCAN expression in miR-221–3p overexpression (H) or inhibition (I) treated hDPCs in different groups. The agglutinative properties of treated hDPCs were shown using bright field microscopy in the left panel. All quantifications were done with three independent repeats, and the expression of GAPDH was used as PCR internal references. Data are represented as means ± SD. *P < 0.05, **P < 0.01. Scale bars show 50 μm.
Fig 2: JAM-A 3′ UTR expression promotes hair regeneration in AA mice. (A) C3H/HeJ mice with induced AA were used to examine the decreased numbers of anagen phase hair follicle microstructures in AA skin tissue. The morphology and histology analysis and imaging were performed. (B) Quantifications of anagen HFs in the AA area were calculated and compared. (C) Representative imaging showing the integration of injected lentivirus in the HFs using anti-GFP immunofluorescence on days 3 and 14 after the injection. The results showed that injected lentivirus successfully integrated into the HFs after 14 days. (D) Distinct hair regeneration was observed in mice injected with JAM-A 3′ UTR Lentivirus at 14 days after engraftment (left). The histology showing the status of hair follicles in different treatment groups were shown in the right panel. (E) The number of anagen phase hair follicles in the JAM-A 3′ UTR injection group was significantly higher than among other groups (right). (F) Distinct hair regeneration was observed in mice injected with miR-221–3p mimics or inhibitor at 14 days after engraftment (left). The histology showing the status of hair follicles in different treatment groups were shown in the right panel. (G) Quantifications of anagen HFs in the AA area were calculated and compared. (H) Illustration summarizes the mechanism of how JAM-A protects VCAN expression in AA by sponging up miR-221–3p.
Fig 3: JAM-A promotes agglutinative growth and VCAN expression of hDPCs through the 3′ UTR region. (A) Quantitative analysis of JAM-A and VCAN expression in JAM-A knockdown hDPCs, the phenotypic agglutinative colonies were lost in the siJAM-A group. NC, transfected with a scrambled siRNA negative control. (B) Western blot analysis revealed the expression of JAM-A and VCAN were decreased in the siJAM-A group compared to NC. (C) CCK-8 assays were conducted to determine the cell proliferation. (D) Cell viability was assessed by FCM in hDPCs with and without JAM-A knockdown. (E) Quantitative analysis of JAM-A and VCAN expression in JAM-A overexpressed hDPCs, the phenotypic agglutinative colonies were shown in the upper panel. The JAM-A CDS represents overexpression of only JAM-A coding region, while JAM-A 3′ UTR represents overexpression of JAM-A 3′ UTR region only. (F) Western blot analysis showing the protein expression of JAM-A and VCAN in JAM-A overexpressed hDPCs. (G) CCK-8 assays were conducted to determine the cell proliferation of hDPCs with JAM-A CDS or 3′ UTR overexpression. (H) Cell viability was assessed by FCM. (I) Quantitative analysis of JAM-A and VCAN expression in hDPCs of different treatments, the phenotypic agglutinative colonies were shown in the left panel. The JAM-A CDS represents overexpression of only the JAM-A coding region, while JAM-A 3′ UTR represents overexpression of only the JAM-A 3′ UTR region. (J) CCK-8 assays were conducted to determine the cell proliferation of hDPCs with of different treatments. (K) Cell viability analysis of hDPCs with different treatments by FCM. All quantifications were done with three independent repeats, and the expression of GAPDH was used as PCR internal references. CTL means control. Data are represented as means ± SD. *P < 0.05, **P < 0.01. Scale bars show 50 μm.
Fig 4: Expression of the JAM-A 3′ UTR promotes hair growth in nude mice. BALB/c-nu/nu mice were divided into six groups: saline, hDPCsscr, hDPCssiJAM-A, hDPCssiJAM-A+vec, hDPCssiJAM-A+UTR, and hDPCssiJAM-A+UTR mut. (A) Gross observation. (B) Histological observation. (C) Immunohistofluorescence observation. (D) Quantification of regenerated hair diameter in six groups. (E) Quantification of VCAN expression in hair follicles in different groups. All quantifications were done with three independent repeats. Data are represented as means ± SD. *P < 0.05, **P < 0.01. Scale bars show 50 μm.
Fig 5: JAM-A and VCAN are down regulated in AA. (A) Heat map of differentially expressed adhesion-related genes with green indicating down-regulation of genes and red indicating up-regulation of genes (top bar: blue samples are scalp skin from normal tissue; red samples are scalp skin from patients with AA). Data Sources GSE68801. (B) Compared with healthy people, the relative expression levels of JAM-A and VCAN in the skin and scalp samples of patients with AA were lower. (C) The results of hematoxylin and eosin staining showed that in the scalp tissue of the control group, the hair follicles penetrated deep into the fat cells of the subcutaneous tissue, while in the scalp of patients with AA the hair follicle was shorter in length and confined to the dermis tissue. The results of immunohistochemistry showed that VCAN and JAM-A were enriched in the papilla of hair follicles, which was brown. (D) Relative expression levels of JAM-A and VCAN in AA patient's tissue were lower compared to normal samples. (E) Western blot analysis showed that the relative expression level of JAM-A and VCAN in the scalp of patients with AA was lower than that of normal people. (F) We downregulated JAM-A expression in AA model mice (left) (subcutaneous injection JAM-A shRNA expressing lentivirus). After 14 days, we observed the hair regrowth of AA was delayed in siJAM-A group mice. (G) Quantification of germinal area. In the JAM-A interference group, the number and speed of hair growth were lower than those in the SCR group. (H) Western blot analysis showed that the relative expression level of JAM-A, AP, and VCAN in JAM-Akd group was lower than that of SCR group. Three repetitions, data are represented as means ± SD. *P < 0.05, **P < 0.01.
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