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