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: Cardiovascular Diagnosis and Therapy

Article Title: The m 6 A demethylase fat mass and obesity-associated protein mitigates pyroptosis and inflammation in doxorubicin-induced heart failure via the toll-like receptor 4/NF-κB pathway

doi: 10.21037/cdt-23-326

Figure Lengend Snippet: m 6 A methylation is markedly increased in Dox-induced HF, and FTO lowers m 6 A-RNA methylation. (A) Quantification of m 6 A methylation levels in cardiomyocytes after Dox treatment. (B) RNA m 6 A dot blot detection of methylation levels in cardiomyocytes. (C) Quantification of m 6 A methylation levels in cardiomyocytes after overexpression of FTO. (D) RNA m 6 A dot blot to measure methylation levels in cardiomyocytes. Data were expressed as mean ± standard deviation. Unpaired t -test was adopted for the comparison of two groups of data, with Tukey’s test for post hoc test. *, P<0.05 compared with the control group; # , P<0.05 compared with the oe-NC group. Cell experiments were done three times. Dox, doxorubicin; m 6 A, N6-methyladenosine; NC, negative control; FTO, fat mass and obesity-associated protein; HF, heart failure.

Article Snippet: In detail, the cells were submerged in the RIP lysis buffer and reacted with antibodies against FTO [1:50, #31687, Cell Signaling Technologies (CST), Beverly, MA, USA], YTH N6-methyladenosine RNA binding protein 1 (YTHDF1; 1:50, #57530, CST), YTHDF2 (1:100, #71283, CST), YTHDF3 (1:100, #24206, CST), or rabbit IgG (1:100, ab172730, Abcam) overnight at 4 °C, followed by washing with RIP washing buffer at 4 °C for 10 min and then with RIP lysis buffer at 4 °C for 5 min.

Techniques: Methylation, Dot Blot, Over Expression, Standard Deviation, Comparison, Control, Negative Control

Journal: Cardiovascular Diagnosis and Therapy

Article Title: The m 6 A demethylase fat mass and obesity-associated protein mitigates pyroptosis and inflammation in doxorubicin-induced heart failure via the toll-like receptor 4/NF-κB pathway

doi: 10.21037/cdt-23-326

Figure Lengend Snippet: FTO decreases TLR4 expression by mediating the m 6 A modification of TLR4. (A) TLR4 expression in cardiomyocytes after overexpression of FTO tested with RT-qPCR and western blotting. (B) The m 6 A modification level of TLR4 mRNA examined with the Me-RIP assay. (C) RIP assay to determine TLR4 expression after overexpression of FTO. (D) TLR4 expression after knockdown of YTHDF1, YTHDF2, and YTHDF3 assessed with RT-qPCR and western blotting. (E) mRNA stability measurement by determining the TLR4 decay rate. Data were expressed as mean ± standard deviation. Normally distributed data were compared between two groups with the unpaired t -test and among multiple groups with one-way analysis of variance. Tukey’s test was used for post hoc analysis. *, P<0.05 compared with the control group; # , P<0.05 compared with the oe-NC group; & , P<0.05 compared with the si-NC group. Cell experiments were done three times. NC, negative control; FTO, fat mass and obesity-associated protein; TLR4, toll-like receptor 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Dox, doxorubicin; RIP, RNA immunoprecipitation; m 6 A, N6-methyladenosine; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Me-RIP, methylated-RIP.

Article Snippet: In detail, the cells were submerged in the RIP lysis buffer and reacted with antibodies against FTO [1:50, #31687, Cell Signaling Technologies (CST), Beverly, MA, USA], YTH N6-methyladenosine RNA binding protein 1 (YTHDF1; 1:50, #57530, CST), YTHDF2 (1:100, #71283, CST), YTHDF3 (1:100, #24206, CST), or rabbit IgG (1:100, ab172730, Abcam) overnight at 4 °C, followed by washing with RIP washing buffer at 4 °C for 10 min and then with RIP lysis buffer at 4 °C for 5 min.

Techniques: Expressing, Modification, Over Expression, Quantitative RT-PCR, Western Blot, Knockdown, Standard Deviation, Control, Negative Control, RNA Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction, Methylation