Fig 2: Only subtle changes in gene expression in Müller glia are observed after Ptbp1 deletion(A) Representative SOX9 and GFP immunostaining images of the retina from 3 genotypes at 4 weeks after 4-OHT i.p. injection (n = 4 mice/genotype).(B) Representative images of immunohistochemical analysis for GFAP expression in the retinas of the three genotypes after 4 weeks of 4-OHT i.p. injection (n = 3 mice/genotype).(C) Dot plots showing some mildly changed gene expression of Müller glia across the three genotypes in scRNA-seq dataset.ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars, 100 µm.

Fig 3: PANDAR induced BCL-XS mRNA reduction rescued by PTBP1. (a) PTBP1 western blot analysis of biotin-PANDAR pull-down samples from U-2 OS cell lysates revealed an enrichment of PTBP1 for PANDAR full length RNA. No PTBP1 enrichment was observed for the pull-down with non binding RNA control. (b) RT-qPCR analysis of BCL-XS mRNA level upon PANDAR overexpression in comparison to simultaneous overexpression of PTBP1 in U-2 OS cells. Shown are mean ± s. d.; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, n. s. = not significant; e. v. = empty vector control; Rq = relative quantity.

Fig 4: TRMP regulates p27 protein levels by competing p27 mRNA for PTBP1 binding.a Lysates from A549 cells were incubated with either sense or antisense biotin-labeled DNA oligomers corresponding to TRMP, followed by the pull-down experiments using streptavidin-coated beads. The pull-downed proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining. The separated proteins were analyzed by mass spectrometry. PTBP1 was identified as a potential TRMP-binding protein. The PTBP1 peptide sequences obtained by MS are shown in Supplementary Figure S4A. b Lysates from HEK293T cells expressing Flag or Flag-PTBP1 were immunoprecipitated with anti-Flag antibody. Eluted products from the immunoprecipitates were used for examining TRMP and PTBP1 levels by semi-quantitative RT-PCR and western blot analyses, respectively. The same eluted products were also subjected to real-time RT-PCR analysis to examine TRMP levels. Data shown are mean ± SD (n = 3). c Lysates from A549 cells were incubated with either sense or antisense biotin-labeled DNA oligomers corresponding to TRMP, followed by the pull-down experiments using streptavidin-coated beads. The pull-downed complexes were analyzed by western blotting with anti-PTBP1 and anti-GAPDH antibodies. The same complexes were also subjected to real-time RT-PCR analysis to confirm the enrichment of TRMP by the antisense DNA oligomer, as shown in Supplementary Figure S4B. d Lysates from A549 cells were incubated with in vitro synthesized biotin-labeled TRMP or its antisense RNA, followed by the pull-down experiments using streptavidin-coated beads. The pull-downed complexes were analyzed by western blotting with anti-PTBP1 and anti-GAPDH antibodies. e In vitro synthesized TRMP was incubated with purified recombinant Flag-PTBP1 bound with M2 beads. After incubation and extensive washing, the beads-bound RNAs were eluted as templates for RT-PCR analysis. f HEK293T cells expressing control shRNA, TRMP shRNA#1, or TRMP shRNA#2 were transfected with or without Flag-PTBP1 as indicated. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. RNAs present in the input and immunoprecipitates were analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3). The input and immunoprecipitates were also analyzed by western blotting with anti-Flag antibody. * and *** indicate p < 0.05 and p < 0.001, respectively. g HEK293T cells were transfected with plasmids expressing Flag-PTBP1 and TRMP RNA in the indicated combination. Twenty-four hours later, cell lysates were immunoprecipitated with anti-Flag antibody, followed by real-time RT-PCR analysis to examine p27 mRNA levels in the immunoprecipitates. Data shown are mean ± SD (n = 3). ** indicates p < 0.01. h Purified Flag-PTBP1 proteins bound with M2 beads were incubated with in vitro synthesized p27 5'UTR (-447 to -1) in the absence or presence of different doses of in vitro transcribed TRMP. After incubation and extensive washing, the beads-bound RNAs were eluted as templates for RT-PCR analysis. i Shown is the luciferase reporter plasmid containing the p27 5'-UTR (-447 to -1) used in this study. j HEK293T cells expressing control shRNA, TRMP shRNA#1, or TRMP shRNA#2 were transfected with the p27 5'-UTR luciferase reporter plasmid. Twenty-four hours after transfection, the reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3). ** indicates p < 0.01. k HEK293T cells over-expressing either control RNA or TRMP were transfected with the p27 5'-UTR luciferase reporter plasmid. Twenty-four hours after transfection, the reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3). ** indicates p < 0.01. l A549 cells were infected with lentiviruses encoding control shRNA, PTBP1 shRNA, TRMP shRNA#1, and TRMP shRNA#2 in the indicated combinations. Forty-eight hours after infection, cell lysates were subjected to western blot analysis with anti-p27, anti-PTBP1, and anti-GAPDH antibodies. The band intensities were quantified by using ImageJ software. The ratio of p27 to GAPDH is presented in Supplementary Figure S4D

Fig 5: miR-326 promotes autophagy via the PTBP1/PI3K signaling pathway. A The potential binding sites for miR-326 on the 3'UTR of PTBP1. B 293T cells were co-transfected with miR-326 mimic or miRNA control and with a luciferase reporter vector containing WT or mutant 3'UTR of PTBP1. C The expression level of PTBP1 negatively correlated with the level of miR-326 in OM-MSCs. D Compared with those in Hemin-exposed normoxia OM-MSCs, the expression levels of p-PI3K/PI3K and PTBP1 were robustly decreased in Hemin-exposed hypoxia OM-MSCs. E Overexpressed PTBP1 increased the expression of PTBP1 and p-PI3K/PI3K. F Under miR-326 mimics treatment, overexpressed PTBP1 increased the expression of p21, P53, p16INK4A, and P62 protein and decreased the expression of LC3II/I and Bcelin-1 protein. G Treatment with PI3K activator partially reversed the downregulation of p-PI3K/PI3K and P63 and the upregulation of LC3II/I and Bcelin-1 induced by the miR-326 mimics. Data are expressed as the mean ± SEM (n = 3) (*P <0.05, **P <0.01, ***P <0.001, ns. no significance)