Additional file 1 of Identifying Hmga2 preserving visual function by promoting a shift of Müller glia cell fate in mice with acute retinal injury
Creators: name: Yin, Zhiyuan
name: Ge, Lingling
name: Cha, Zhe
name: Gao, Hui
name: A, Luodan
name: Zeng, Yuxiao
name: Huang, Xiaona
name: Cheng, Xuan
name: Yao, Kai
name: Tao, Zui
name: Xu, Haiwei
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology

description: Additional file 1: Figure S1. Sodium iodate induced acute retinal injury in MG lineage tracing mice. a Experimental design for SI treatment and multiple analysis time points in MG lineage tracing mice. b MG were marked with tdTomato red fluorescence in MG lineage tracing mice. A whole retinal cross section and a slightly expanded view are displayed (inner side). c The nucleus of tdTomato positive cells are co-labeled with the MG marker Sox9 (c1), not co-labeled with the ganglion cell marker-Rbpms (c2), photoreceptor marker-Recoverin (c3), and cone marker-arrestin (c4). d Ratio of Sox9, Rbpms, Recoverin and arrestin co-labeling with tdTomato cells in each retina section (n = 3). e A comparison of retinal structures between control and SI-treated groups using DAPI staining. f The mean number of photoreceptor rows along central sections of the retina between control and SI-treated retinas (n = 3). g ERG waveforms in scotopic condition between control and SI-treated retinas. h A comparison of a-wave (h1) and b-wave (h2) amplitudes in scotopic conditions between control and SI-treated retinas (n = 5). ONL Outer nuclear layer, INL Inner nuclear layer, GCL Ganglion cell layer. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001. one-way ANOVA test was used in (d, f, h). Figure S2. The most obvious transcriptional alterations of Müller glia occurred at 3 days post injection. a A feature expression dot map of the principal retinal class markers over 10 clusters of retinal cells. b UMAP image of 54,236 retinal cells colored by annotation of 10 transcriptionally different clusters. c The number of DEGs in the SI-3d and SI-7d groups compared with control in MG. d Comparing representative DEGs of MG from the SI-3d group compared with the control group in MG. e Enriched GO terms of DEGs in the SI-3d group compared with the control group in MG. Figure S3. Potential genes for MG reprogramming process among DEGs from bulk-RNA seq and scRNA seq. a UniProt database annotations for keywords of module 1. Line thickness represents the degree of data support; a minimum interaction score of 0.5 is needed. b Protein-protein interaction network of screened 10 DEGs of scRNA seq from Venn diagram. c Relative expression of screened 10 DEGs from scRNA seq in each group, the colors correspond to relative expression. All 10 DEGs had |log2 (fold changes)| values between 1 and 2 at 3 dpi. Figure S4. Transcriptional expression of Hmga2 and other related genes in control and SI-injury groups. a RT-qPCR analysis of Hmga2, Igfbp3, Tgfb1, and Ccnd1 transcription levels compared with bulk-RNA seq (n = 3). b A feature dot map of Hmga2 average expression and percent expression in 10 clusters of retinal cells from the control, SI-3d and SI-7d groups. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001. One-way ANOVA test was used in (a). Figure S5. Co-labeling of EGFP with Sox9 and Hmga2 protein in control and experimental AAVs. a Experimental design for Hmga2 overexpression by AAV prior to SI treatment, with multiple time points for analysis in mice. b Virus-infected MG are labeled with EGFP green fluorescence in the uninjured retina, and an entire retinal cross section is shown. c Immunohistochemical co-labeling of Sox9 and EGFP protein in control and experimental AAV-infected retinas. White hollow arrows identify EGFP positive and Sox9 negative cells. d Ratio of Sox9 and EGFP double positive cells in control and experimental AAV-infected retinal sections (n = 5). e Immunohistochemical co-labeling of Hmga2 and EGFP protein in control and experimental AAV-infected retinas. f Ratio of Hmga2 and EGFP double positive cells in control and experimental AAV-infected retinal sections (n = 5). ONL, outer nuclear layer, INL, inner nuclear layer, GCL, ganglion cell layer, *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001, Unpaired t-test was used in (d, f). Figure S6. The morphology of arrestin and EGFP double positive cells in ONL. a Representative images of arrestin and EGFP double positive cells in ONL at Hmga2-SI treated group. Insets display the expression of each fluorescent reporter (White Square). ONL Outer nuclear layer, INL Inner nuclear layer, GCL Ganglion cell layer. Figure S7. Hmga2 expression in MG did not promote MG proliferation a Immunohistochemical co-labeling of EGFP and Ccnd1 protein in the central retina that received SI treatment and Hmga2-SI treatment at various time points. White arrowheads identify where the MG is Ccnd1 positive. Insets display the expression of each fluorescent reporter (White Square). b Ratio of Ccnd1 positive MG in each section of the retina with SI treatment and Hmga2-SI treatment at various time points (n = 3). c Ratio of Ki67 positive MG in each section of the retina with SI treatment and Hmga2-SI treatment at various time points (n = 3). d Two EdU injection schemes during Hmga2 overexpression and SI treatment, and multiple time points for analysis in mice. e Ratio of EdU positive MG labeled by the first method in each section of the retinas with SI treatment and Hmga2-SI treatment at various time points (n = 3). f Ratio of EdU positive MG labeled by the second method in each section of the retinas in the retinas with SI treatment and Hmga2-SI treatment at various time points (n = 3). ONL, outer nuclear layer, INL, inner nuclear layer, GCL, ganglion cell layer, *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001, Unpaired t-test was used in (b, c, e, f). Figure S8. Full-length gels and blot of Fig. 1c. a Full-length gels and blot of GFAP in control, SI-1d, SI-3d, SI-5d and SI-14d groups. The green box represents the cropping position. b Full-length gels and blot of β-actin in control, SI-1d, SI-3d, SI-5d and SI-14d groups. The red box represents the cropping position. Table S1. Primary and secondary antibodies used in immunohistochemistry. Table S2. Primary and secondary antibodies used in western blot. Table S3. Primer sequences for RT-qPCR analysis. Table S4. Comparison of the transcriptional expression of Hmga2 and other related genes in the retina with SI treatment and Hmga2-SI treatment.