Fig 2: Tcf4 protein expression in excitatory and inhibitory neurons in adult brain.Representative immunofluorescence images showing Tcf4 protein expression in the nucleus of excitatory and inhibitory neurons in A cortex, B hippocampus (CA1 region), C striatum (caudate putamen) and D cerebellum; solid arrow: TCF4+ PC, empty arrow: TCF4− PC; PC Purkinje cells, ML molecular layer, GL granule cell layer (shown in dash-lined box), representative of 3–5 adult mice. Scale bar = 10 µm.

Fig 3: ERLR expression is associated with clinical PVR.A PhRPE cells were cultured with 25% PVR vitreous (N = 8 samples) or normal vitreous samples (N = 4 samples) for 48 h. ERLR expression was detected by RT-PCR. Data are presented as means ± SEM. **P < 0.01 by two-tailed Students’s t test. B PVR subretinal membranes were obtained from surgery. ERLR expression was detected by FISH. Pan-CK was detected by IF. White arrow marks the co-localization of ERLR and pan-CK. C A diagram depicting the mechanism of ERLR in EMT of RPE cells (modified with permission from our previous published paper in Discovery Medicine [3]). TGF-β1 upregulates TCF4 expression, which activates the transcription of ERLR. ERLR binds to MYH9 and increases its stability, which leads to increased MYH9 protein expression and EMT. These cells then migrate, proliferate, aggregate and form the epiretinal or subretinal membrane, which contracts and ultimately leads to tractional retinal detachment.

Fig 4: H19 induction of Wnt signalling action. A, Increasing Let‐7c, decreasing H19 and ESR1 all decreased the TCF‐4 promoter activity. B, Decreasing H19 in 293T cells of Let‐7c increasing, ESR1 inhibiting, or Axitinib addition made no significant difference in three groups. C, Inhibiting Let‐7c and restoring ESR1 greatly abolished the H19 inhibition suppressed Wnt activity. In groups treated with Axitinib, Let‐7c inhibition failed to reactivate TCF‐4 promoter. D,E, H19 inhibition did not consequently restore Let‐7c level, failing to degrade Let‐7c expression after connection. F, The Let‐7c‐specific binding site towards to H19 MS2 was effectively grouped with H19. G, Let‐7c did not affect the H19 expression. H,I, Let‐7c degraded the H19 binding site of wild‐type mRNA effectively and failed in groups of mutant mRNA. J,K, Western blotting revealed the H19 controlling of Let‐7c contributed to ESR1 and Wnt activity deregulation, and ESR1 mainly functioned through Wnt signalling

Fig 5: ERLR triggers EMT by directly binding to the MYH9 protein.A An RNA pulldown assay was performed using phRPE cells and biotinylated ERLR or antisense-ERLR. Proteins coprecipitated with biotin-labeled ERLR or antisense-ERLRs were detected by WB. One of the representative results is shown (N = 3 independent experiments /group). B–F PhRPE cells were transfected with two ERLR siRNAs (SiERLR-1 and SiERLR-2), two TCF4 siRNAs (siTCF4-1 and siTCF4-2) or a negative control siRNA (Si-NC) and then treated with or without TGF-β1 (10 ng/ml) for 48 h. B, D The expression of MYH9 protein was detected by WB. One of the representative results is shown. C, E The band intensities in the WB results were analyzed and normalized to β-actin (N = 3 independent experiments /group) in B, D, respectively. **P < 0.01 and *P < 0.05 by one-way ANOVA and post hoc Bonferroni’s test. F The expression of MYH9 mRNA was detected by qRT-PCR and was normalized to that of GAPDH (N = 3 independent experiments /group). No significant difference by one-way ANOVA and post hoc Bonferroni’s test. G–I: PhRPE cells were transfected with two MYH9 siRNAs (SiMYH9-1 and SiMYH9-2) or a negative control siRNA (Si-NC) and then were treated with or without TGF-β1 (10 ng/ml) for 48 h. G Expression levels of MYH9 and EMT-related markers were detected by RT-PCR, and the relative fold changes were normalized to GAPDH (N = 3 independent experiments /group). **P < 0.01 and *P < 0.05 by one-way ANOVA and post hoc Bonferroni’s test. H EMT-related markers were detected by WB. One of the representative results is shown. I The band intensities in WB results were analyzed and normalized to the intensity of β-actin (N = 3 independent experiments/group). **P < 0.01 and *P < 0.05 by one-way ANOVA and post hoc Bonferroni’s test. J–L PhRPE cells were transfected with ERLR-overexpressing lentivirus (lv-ERLR) or control lentivirus (lv-NC) and were then transfected with two MYH9 siRNAs (SiMYH9-1 and SiMYH9-2) or a negative control siRNA (Si-NC) and cultured for 48 h. J. Expression levels of MYH9 and EMT-related markers were detected by RT-PCR, and the relative fold changes were normalized to GAPDH (N = 3 independent experiments/group). **P < 0.01 and *P < 0.05 by one-way ANOVA and post hoc Bonferroni’s test. K EMT-related markers were detected by WB. One of the representative results is shown. L The band intensities in WB results were analyzed and normalized to the intensity of β-actin (N = 3 independent experiments /group). **P < 0.01 and *P < 0.05 by one-way ANOVA and post hoc Bonferroni’s test. All data are presented as means ± SEM.