Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: Deletion of vhl does not prevent degeneration of ndufs4 -deficient retinal ganglion cells. (A) To assess for cell-specific recombination of floxed vhl , a flattened retina from a Vglut2-Cre;vhl loxP/loxP mouse was serially sectioned in 40-µm increments to collect tissue from the outermost to the innermost retinal layers (samples 1–5). PCR was performed on purified genomic DNA from each section to reveal the presence of vhl loxP/loxP that is non-recombined (2 loxP sites, 460 bp) and recombined (1 loxP site, 260 bp). The presence of the Vglut2-Cre transgene in the genomic DNA was also confirmed by PCR. The schematics to the right depict the beginning of the vhl locus pre- and post-recombination, showing the positions of the loxP sites flanking Exon 1 and the expected PCR product sizes resulting from a reaction in which two forward primers (F1 and F2) and one common reverse primer (Rcomm) are used. See Supplementary Fig. online for un-cropped gels. (B, C) Representative images of retinal flat mounts from control mice without the Cre transgene (left panel) and from mice with the indicated heterozygosity or homozygosity status for deletion of ndufs4 and vhl from RGCs at (B) P30 and (C) P45 time points. RGCs are immunolabeled with RNA-Binding Protein 1 (RBPMS1, green). Bar, 20 μm. In each graph below, the density of RGC somas for each genotype is quantified at distances of 0.5, 1.0, and 1.5 mm from the optic nerve head. Individual data points are overlaid on each bar. Statistical comparisons between groups are indicated above the bars, with the following significance designations: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Purification, Control, Immunolabeling, RNA Binding Assay

Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: Retinal ganglion cell-specific deletion of Hif1α and Hif2α . (A) To confirm cell-specific recombination of floxed Hif1α and Hif2α , a flattened retina from a Vglut2-Cre; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mouse was serially sectioned in 40-µm increments to collect tissue from the outermost to the innermost retinal layers (samples 1–5). PCR was performed on purified genomic DNA from each section to demonstrate the presence of Hif1 α loxP/loxP and Hif2 α loxP/loxP alleles that are non-recombined (2 loxP sites) and recombined (1 loxP site). The presence of the Vglut2-Cre transgene in the genomic DNA was also confirmed by PCR. The schematics to the right depict the beginning of the floxed Hif1α and Hif2α loci pre- and post-recombination, showing the positions of the loxP sites flanking Exon 2 of each gene. The positions of each of the two forward primers (F1 and F2) and one common reverse primer (Rcomm) used for each gene are also depicted, as well as the expected PCR product sizes for each reaction when all three primers are combined. Note that for Hif1α , the locations of the forward primers relative to each loxP site result in the post-recombination amplification product being slightly larger than for the non-recombined product. See Supplementary Fig. S2 online for un-cropped gels. (B) Retinal flat mount from a P60 Vglut2-Cre; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mouse stained for RBPMS1 demonstrates healthy RGC soma morphology and density. Bar, 20 μm. (C) Electron micrograph of an optic nerve cross section from a P60 Vglut2-Cre; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mouse demonstrates abundant RGC axons with normal myelination. Bar, 5 μm.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Purification, Amplification, Staining

Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: Neuroprotection of P60 ndufs4 -deficient retinal ganglion cell somas by hypoxia is not dependent on an intact HIF pathway . Representative images of RBPMS1-labeled retinal flat mounts from P60 mice with RGC-specific deletion of both Hif1α and Hif2α. When the mice are raised under normoxia, the density of RGC somas (immunolabeled for RBPMS1) is reduced in mice that are also homozygous for deletion of ndufs4 within RGCs (middle panel) compared to those with one intact copy (left panel). With continuous exposure to 11% O 2 beginning at P25, RGCs with homozygous deletion of ndufs4 (right panel) maintain a normal cell density. Bar, 20 μm. The graph depicts RGC soma density at 0.5, 1.0, and 1.5 mm distances from the optic nerve head, with the RGC ndufs4 genotype and the ambient O 2 concentration indicated below. Individual data points are overlaid on each bar. Statistical comparisons between groups are indicated above the bars, with the following significance designations: ns, not significant; ***, p < 0.001.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Labeling, Immunolabeling, Concentration Assay

Journal: Scientific Reports

Article Title: Hypoxia-mediated rescue of retinal ganglion cells deficient in mitochondrial complex I is independent of the hypoxia-inducible factor pathway

doi: 10.1038/s41598-024-75916-x

Figure Lengend Snippet: The partial rescue of ndufs4 - deficient retinal ganglion cells by hypoxia at P90 is maintained in the absence of an intact HIF pathway . (A, B) Representative images of RBPMS1-labeled retinal flat mounts (A) or optic nerve cross sections (B) from P90 mice with RGC-specific homozygous deletion of both Hif1α and Hif2α. The left panels depict tissue in which there is also heterozygous deletion of ndufs4 from RGCs, while in the right panels there is homozygous deletion of ndufs4 from RGCs. From P25 to P90, the mice were raised under 21% O 2 (top panels) or 11% O 2 (bottom panels). Bar, 20 μm. The graphs to the right quantify RGC soma densities at three distances from the optic nerve head (A) and RGC axon densities across entire optic nerve cross sections (B). Individual data points are overlaid on each bar. Statistical comparisons between groups are indicated above the bars, with the following significance designations: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Electron micrographs of optic nerve cross sections from P90 Vglut2-Cre; ndufs4 loxP/loxP ; Hif1 α loxP/loxP ; Hif2 α loxP/loxP mice raised under normoxia (top panels) or hypoxia (bottom panels). Continuous hypoxia reduced RGC axon loss and surrounding fibrosis. The thickening and duplication of myelin sheaths on surviving axons (higher magnification images) were less common in hypoxia-treated mice, but scattered examples were still observed. Black bars, 10 μm. White bars, 0.5 μm.

Article Snippet: Retinas were isolated, blocked in 5% goat serum in PBS with 0.3% Triton X-100, incubated with rabbit polyclonal anti-RBPMS1 primary antibody (1:500; Novus, NBP2-20112) in block for 5 days at 4 °C, and then incubated with anti-rabbit Alexa Fluor 488 (1:500; Invitrogen) overnight at 4 °C.

Techniques: Labeling

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: Intravitreal injection of SCGF-β protects RGCs after optic nerve crush. A Human SCGF-β or PBS was intravitreally injected into the adult mouse eye right after optic nerve crush. The retina was explanted and immunostained 5 days post cush. B RBPMS + RGCs under SCGF-β administration showed significantly higher survival, compared with the PBS treatment. * P < 0.05. ONC, optic nerve crush. Error bar denotes SD

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Injection

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: RGCs enhance iPSC’s SCGF-β release via IL-12(p70). A - B IL-12(p70) was detected in RGC supernatant, not in RGC medium, through multiplexed antibody-based assays and confirmed by ELISA. C - D Compared to the control (“No IL-12(p70)” group), significant upregulation of SCGF-β (via ELISA ) and CLEC11A mRNA (via qRT-PCR ) were observed in iPSCs treated with 2.5, 5, and 10 ng/mL of IL-12(p70). The expression peaked in the 5 ng/mL IL-12(p70)-treated group. E RGCs were cultured in the RGC medium with different dosages of IL-12(p70) administration. A significantly greater cell viability was not found until treated with 40 ng/mL of IL-12(p70). * P < 0.05. Error bar denotes SD. NS, nonsignificant

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: iPSC-derived SCGF-β promotes RGC survival via upregulation of ngn2. A - B RT-qPCR results showed a significant increase of ngn2 mRNA in RGCs cocultured with iPSCs or treated with SCGF-β. C - D Overexpression of ngn2 in RGCs cultured in the RGC medium for 1 week significantly increased RGC viability. E Knockdown of ngn2 in RGCs cultured for 1 week does not significantly alter RGC survival, whether treated with SCGF-β or not. * P < 0.05. Error bar denotes SD

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Derivative Assay, Quantitative RT-PCR, Over Expression, Cell Culture, Knockdown

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: Overexpression of ngn2 protects endogenous and transplanted RGCs in vivo. A AAV2-ngn2-EGFP or AAV2-EGFP particles were intravitreally injected 2 weeks into adult mouse eyes 2 weeks before optic nerve crush. Retina explants were harvested and immunostained 2 weeks after the optic nerve crush. Surviving RGCs were labeled with RBPMS (red). B A significantly greater RBPMS + RGC number was found on retina explants from the AAV-ngn2 treated group. C The EGFP and mCherry were used to label the entire transplanted and ngn2-overexpressing donor mouse RGCs, respectively. Ngn2-mCherry-overexpressing mouse RGCs that were transplanted into adult rat eyes shows significantly higher survival rates, compared with the negative control RGC transplant 1 week after transplantation in vivo. D & E Difference between transplanted RGC survival rates and average neurite lengths with and without ngn2 overespression was quantified . Donor RGCs overexpressing ngn2-mCherry grew significantly longer neurites than those from negative control-RGC transplantation in vivo. * P < 0.05, paired t-test. ONC, optic nerve crush; NC, negative control; OE, overexpression. Error bar denotes SD

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Over Expression, In Vivo, Injection, Labeling, Negative Control, Transplantation Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Crystallin β-b2 promotes retinal ganglion cell protection in experimental autoimmune uveoretinitis

doi: 10.3389/fncel.2024.1379540

Figure Lengend Snippet: Expression and localization of TUBB3 (green), RBPMS (red) and glial fibrillary acidic protein (GFAP; red) in naïve retinas or in retinas with EAU and prophylactic treatment with 2 μL rcrybb2 (3 μg/2 μL) or PBS as control. Some mice were also treated with a single intralenticular injection of PBS as a positive control. Mice were immunized 3 days later and eyes were collected 21 days after immunization. The expression of retinal (A,D) TUBB3 (green), (B,E) RBPMS (red) and (C,F) GFAP (red) was determined by immunofluorescence staining of sections (7 μm) from mice that were naive, with EAU (untreated), EAU + PBS (vitreous), EAU + PBS (lens), or EAU + rcrybb2 (vitreous) on day 21 p.i. Positive cells were counted in 3 different section per eye ( N = 5 eyes per group) and data are expressed as mean ± SD. Scale bar: 100 μm. (G,H,I) The number of retinal RBPMS+ cells (red) in naïve, EAU (untreated) and EAU(rcrybb2)-treated retinas was determined by flatmount staining ( N = 4 per group). Scale bar: 50 μm. (D–F,J) ANOVA with Tukey’s post-hoc test: ∗ p < 0.05. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: The flat mounts were stained overnight with a polyclonal rabbit anti-RBPMS antibody or a monoclonal rabbit anti-CD31/PECAM1 and monoclonal mouse anti-TUBB3 (RBPMS: 1:200 dilution, GTX118619, CD31/PECAM1: 1:200 dilution, A19014, Abclonal, Wuhan, China, TUBB3: 1:200 dilution, MA1-118X, Thermo Fisher, Germany) in PBST-X (0.3% Triton X-100 in PBS).

Techniques: Expressing, Control, Injection, Positive Control, Immunofluorescence, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Crystallin β-b2 promotes retinal ganglion cell protection in experimental autoimmune uveoretinitis

doi: 10.3389/fncel.2024.1379540

Figure Lengend Snippet: Expression of TUBB3+, RBPMS+, and GFAP+ cells in the retina.

Article Snippet: The flat mounts were stained overnight with a polyclonal rabbit anti-RBPMS antibody or a monoclonal rabbit anti-CD31/PECAM1 and monoclonal mouse anti-TUBB3 (RBPMS: 1:200 dilution, GTX118619, CD31/PECAM1: 1:200 dilution, A19014, Abclonal, Wuhan, China, TUBB3: 1:200 dilution, MA1-118X, Thermo Fisher, Germany) in PBST-X (0.3% Triton X-100 in PBS).

Techniques: Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Crystallin β-b2 promotes retinal ganglion cell protection in experimental autoimmune uveoretinitis

doi: 10.3389/fncel.2024.1379540

Figure Lengend Snippet: Expression and localization of TUBB3, RBPMS and CD31/PECAM1 in naïve retinas and in retinas with EAU. The expression of TUBB3 (green), RBPMS (red) in (A) naïve retinas and (B) retinas with EAU was determined by flatmount staining. The results shows that TUBB3 is expressed in retinal ganglion cells (RGCs) but also in (C) CD31+ endothelial cells. Scale bar: 50 μm.

Article Snippet: The flat mounts were stained overnight with a polyclonal rabbit anti-RBPMS antibody or a monoclonal rabbit anti-CD31/PECAM1 and monoclonal mouse anti-TUBB3 (RBPMS: 1:200 dilution, GTX118619, CD31/PECAM1: 1:200 dilution, A19014, Abclonal, Wuhan, China, TUBB3: 1:200 dilution, MA1-118X, Thermo Fisher, Germany) in PBST-X (0.3% Triton X-100 in PBS).

Techniques: Expressing, Staining

Journal: iScience

Article Title: The RBPMS CreERT2-tdTomato mouse line for studying retinal and vascular relevant diseases

doi: 10.1016/j.isci.2023.108111

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-RBPMS , Thermo Fisher Scientific , Cat# PA5-31231; RRID: AB_2548705.

Techniques: Recombinant, Lysis, Protease Inhibitor, Plasmid Preparation, Software