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Overexpression of WDR36 significantly inhibits p53 activation and calcium overload in R28 retinal precursor cells. OGD/R model was established in R28 retinal precursor cells and pcDNA3.1-WDR36 was transfected into cells to upregulate the expression of WDR36. (A) RT-qPCR to detect WDR36 mRNA; (B) Western blot to detect WDR36 protein expression; (C) CCK-8 for cell viability of R28 retinal precursor cells; (D) Commercial kit for LDH release from R28 retinal precursor cells; (E) Commercial kit for ATP levels in R28 retinal precursor cells; (F) Flow cytometry for ROS levels in R28 retinal precursor cells using DCFH-DA probe; (G) Flow cytometry for MMP in R28 retinal precursor cells using JC-1 staining, with the ratio of red to green fluorescence reflecting changes in mitochondrial membrane potential; (H) Commercial kit for MDA, SOD and GSH-Px levels; (I) Flow cytometry to detect intracellular calcium ion levels in R28 retinal precursor cells using Fluo-4 AM probe, with mean fluorescence intensity proportional to intracellular Ca 2+ concentration; (J) Western blot to detect protein expression of p53, MDM2, CHOP, p-PERK (Thr980), and p-IRE1α (Ser724) in R28 retinal precursor cells. Data are expressed as mean ± SD ( n = 3 independent experiments). * P < 0.05.

Journal: Open Life Sciences

Article Title: RNA-binding protein ELAVL1 modulates WDR36 to inhibit p53 pathway and reduce calcium overload in retinal cells under acute pressure elevation

doi: 10.1515/biol-2025-1279

Figure Lengend Snippet: Overexpression of WDR36 significantly inhibits p53 activation and calcium overload in R28 retinal precursor cells. OGD/R model was established in R28 retinal precursor cells and pcDNA3.1-WDR36 was transfected into cells to upregulate the expression of WDR36. (A) RT-qPCR to detect WDR36 mRNA; (B) Western blot to detect WDR36 protein expression; (C) CCK-8 for cell viability of R28 retinal precursor cells; (D) Commercial kit for LDH release from R28 retinal precursor cells; (E) Commercial kit for ATP levels in R28 retinal precursor cells; (F) Flow cytometry for ROS levels in R28 retinal precursor cells using DCFH-DA probe; (G) Flow cytometry for MMP in R28 retinal precursor cells using JC-1 staining, with the ratio of red to green fluorescence reflecting changes in mitochondrial membrane potential; (H) Commercial kit for MDA, SOD and GSH-Px levels; (I) Flow cytometry to detect intracellular calcium ion levels in R28 retinal precursor cells using Fluo-4 AM probe, with mean fluorescence intensity proportional to intracellular Ca 2+ concentration; (J) Western blot to detect protein expression of p53, MDM2, CHOP, p-PERK (Thr980), and p-IRE1α (Ser724) in R28 retinal precursor cells. Data are expressed as mean ± SD ( n = 3 independent experiments). * P < 0.05.

Article Snippet: Primary antibodies used were: ELAVL1 (#12582, Cell Signaling Technology), p53 (#2527, Cell Signaling Technology), phospho-p53 (Ser15) (#9284, Cell Signaling Technology), p21 (#2947, Cell Signaling Technology), MDM2 (#86934, Cell Signaling Technology), Bax (#2772, Cell Signaling Technology), cleaved Caspase-3 (#9661, Cell Signaling Technology), CHOP (#2895, Cell Signaling Technology), GRP78/BiP (#3177, Cell Signaling Technology), ATF4 (#11815, Cell Signaling Technology), p-PERK (Thr980) (#3179, Cell Signaling Technology), p-IRE1α (Ser724) (ab124945, Abcam), GAPDH (#5174, Cell Signaling Technology), and WDR36 (#H00134430-M01, Novus Biologicals).

Techniques: Over Expression, Activation Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry, Staining, Fluorescence, Membrane, Concentration Assay

ELAVL1 post-transcriptionally regulates WDR36 protein levels in R28 retinal precursor cells. (A) Potential binding sites between ELAVL1 and WDR36 mRNA predicted using the bioinformatics platform StarBase; (B) Western blot to detect ELAVL1 protein expression in the OGD/R model; (C) Western blot to detect WDR36 protein expression in R28 retinal precursor cells following ELAVL1 knockdown or overexpression; (D) RT-qPCR to detect WDR36 mRNA level; (E) Interaction between ELAVL1 and WDR36 mRNA in R28 cells verified by RNA immunoprecipitation (RIP) assay (IgG as the negative antibody control, GAPDH mRNA as the negative target gene control, enrichment normalized to Input samples, P < 0.05); (F) RIP assay to confirm the interaction between ELAVL1 and WDR36 mRNA in R28 retinal precursor cells. IgG served as negative control antibody, and GAPDH mRNA served as negative control target to verify binding specificity. Enrichment was normalized to Input samples; (G) Western blot to detect WDR36 protein expression following ELAVL1 overexpression. Data are expressed as mean ± SD ( n = 3 independent experiments). * P < 0.05.

Journal: Open Life Sciences

Article Title: RNA-binding protein ELAVL1 modulates WDR36 to inhibit p53 pathway and reduce calcium overload in retinal cells under acute pressure elevation

doi: 10.1515/biol-2025-1279

Figure Lengend Snippet: ELAVL1 post-transcriptionally regulates WDR36 protein levels in R28 retinal precursor cells. (A) Potential binding sites between ELAVL1 and WDR36 mRNA predicted using the bioinformatics platform StarBase; (B) Western blot to detect ELAVL1 protein expression in the OGD/R model; (C) Western blot to detect WDR36 protein expression in R28 retinal precursor cells following ELAVL1 knockdown or overexpression; (D) RT-qPCR to detect WDR36 mRNA level; (E) Interaction between ELAVL1 and WDR36 mRNA in R28 cells verified by RNA immunoprecipitation (RIP) assay (IgG as the negative antibody control, GAPDH mRNA as the negative target gene control, enrichment normalized to Input samples, P < 0.05); (F) RIP assay to confirm the interaction between ELAVL1 and WDR36 mRNA in R28 retinal precursor cells. IgG served as negative control antibody, and GAPDH mRNA served as negative control target to verify binding specificity. Enrichment was normalized to Input samples; (G) Western blot to detect WDR36 protein expression following ELAVL1 overexpression. Data are expressed as mean ± SD ( n = 3 independent experiments). * P < 0.05.

Article Snippet: Primary antibodies used were: ELAVL1 (#12582, Cell Signaling Technology), p53 (#2527, Cell Signaling Technology), phospho-p53 (Ser15) (#9284, Cell Signaling Technology), p21 (#2947, Cell Signaling Technology), MDM2 (#86934, Cell Signaling Technology), Bax (#2772, Cell Signaling Technology), cleaved Caspase-3 (#9661, Cell Signaling Technology), CHOP (#2895, Cell Signaling Technology), GRP78/BiP (#3177, Cell Signaling Technology), ATF4 (#11815, Cell Signaling Technology), p-PERK (Thr980) (#3179, Cell Signaling Technology), p-IRE1α (Ser724) (ab124945, Abcam), GAPDH (#5174, Cell Signaling Technology), and WDR36 (#H00134430-M01, Novus Biologicals).

Techniques: Binding Assay, Western Blot, Expressing, Knockdown, Over Expression, Quantitative RT-PCR, RNA Immunoprecipitation, Control, Negative Control

ELAVL1 inhibits p53 activation and calcium overload in R28 retinal precursor cells by targeting WDR36. The OGD/R model was established in R28 retinal precursor cells and pcDNA3.1-ELAVL1 and si-WDR36 were co-transfected into the cells. (A) Western blot to detect ELAVL1 and WDR36 protein expression after transfection; (B) CCK-8 for cell viability of R28 retinal precursor cells; (C) Commercial kit for LDH release from R28 retinal precursor cells; (D) Commercial kit for ATP levels in R28 retinal precursor cells; (E) Flow cytometry for ROS levels in R28 retinal precursor cells; (F) Flow cytometry for MMP in R28 retinal precursor cells; (G) Commercial kit for MDA, SOD and GSH-Px levels; (H) Flow cytometry to detect intracellular calcium ion levels in R28 retinal precursor cells; (I) Western blot to detect protein expression of p53, MDM2, CHOP, p-PERK (Thr980), and p-IRE1α (Ser724) in R28 retinal precursor cells. Data are expressed as mean ± SD ( n = 3 independent experiments). * P < 0.05.

Journal: Open Life Sciences

Article Title: RNA-binding protein ELAVL1 modulates WDR36 to inhibit p53 pathway and reduce calcium overload in retinal cells under acute pressure elevation

doi: 10.1515/biol-2025-1279

Figure Lengend Snippet: ELAVL1 inhibits p53 activation and calcium overload in R28 retinal precursor cells by targeting WDR36. The OGD/R model was established in R28 retinal precursor cells and pcDNA3.1-ELAVL1 and si-WDR36 were co-transfected into the cells. (A) Western blot to detect ELAVL1 and WDR36 protein expression after transfection; (B) CCK-8 for cell viability of R28 retinal precursor cells; (C) Commercial kit for LDH release from R28 retinal precursor cells; (D) Commercial kit for ATP levels in R28 retinal precursor cells; (E) Flow cytometry for ROS levels in R28 retinal precursor cells; (F) Flow cytometry for MMP in R28 retinal precursor cells; (G) Commercial kit for MDA, SOD and GSH-Px levels; (H) Flow cytometry to detect intracellular calcium ion levels in R28 retinal precursor cells; (I) Western blot to detect protein expression of p53, MDM2, CHOP, p-PERK (Thr980), and p-IRE1α (Ser724) in R28 retinal precursor cells. Data are expressed as mean ± SD ( n = 3 independent experiments). * P < 0.05.

Article Snippet: Primary antibodies used were: ELAVL1 (#12582, Cell Signaling Technology), p53 (#2527, Cell Signaling Technology), phospho-p53 (Ser15) (#9284, Cell Signaling Technology), p21 (#2947, Cell Signaling Technology), MDM2 (#86934, Cell Signaling Technology), Bax (#2772, Cell Signaling Technology), cleaved Caspase-3 (#9661, Cell Signaling Technology), CHOP (#2895, Cell Signaling Technology), GRP78/BiP (#3177, Cell Signaling Technology), ATF4 (#11815, Cell Signaling Technology), p-PERK (Thr980) (#3179, Cell Signaling Technology), p-IRE1α (Ser724) (ab124945, Abcam), GAPDH (#5174, Cell Signaling Technology), and WDR36 (#H00134430-M01, Novus Biologicals).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, CCK-8 Assay, Flow Cytometry

ELAVL1 ameliorates retinal damage in the acute IOP elevation model by targeting WDR36. An acute IOP elevation-induced retinal injury mouse model was established and gene intervention was performed using overexpression or knockdown rAAV vectors targeting ELAVL1 and WDR36. AAV vectors were delivered via intravitreal injection 3 weeks before model establishment. (A) Western blot to detect the protein expression of ELAVL1 and WDR36 in the retinal tissues of mice in each group; (B) HE staining to detect retinal tissue damage; (C) TUNEL staining to detect apoptotic cells in retinal tissues of mice in each group, with TUNEL-positive cells counted in the ganglion cell layer; (D) Western blot to detect protein expression of p53, MDM2, CHOP, p-PERK (Thr980), p-IRE1α (Ser724) in retinal tissues of mice in each group. Data are expressed as mean ± SD ( n = 8 mice per group). * P < 0.05.

Journal: Open Life Sciences

Article Title: RNA-binding protein ELAVL1 modulates WDR36 to inhibit p53 pathway and reduce calcium overload in retinal cells under acute pressure elevation

doi: 10.1515/biol-2025-1279

Figure Lengend Snippet: ELAVL1 ameliorates retinal damage in the acute IOP elevation model by targeting WDR36. An acute IOP elevation-induced retinal injury mouse model was established and gene intervention was performed using overexpression or knockdown rAAV vectors targeting ELAVL1 and WDR36. AAV vectors were delivered via intravitreal injection 3 weeks before model establishment. (A) Western blot to detect the protein expression of ELAVL1 and WDR36 in the retinal tissues of mice in each group; (B) HE staining to detect retinal tissue damage; (C) TUNEL staining to detect apoptotic cells in retinal tissues of mice in each group, with TUNEL-positive cells counted in the ganglion cell layer; (D) Western blot to detect protein expression of p53, MDM2, CHOP, p-PERK (Thr980), p-IRE1α (Ser724) in retinal tissues of mice in each group. Data are expressed as mean ± SD ( n = 8 mice per group). * P < 0.05.

Article Snippet: Primary antibodies used were: ELAVL1 (#12582, Cell Signaling Technology), p53 (#2527, Cell Signaling Technology), phospho-p53 (Ser15) (#9284, Cell Signaling Technology), p21 (#2947, Cell Signaling Technology), MDM2 (#86934, Cell Signaling Technology), Bax (#2772, Cell Signaling Technology), cleaved Caspase-3 (#9661, Cell Signaling Technology), CHOP (#2895, Cell Signaling Technology), GRP78/BiP (#3177, Cell Signaling Technology), ATF4 (#11815, Cell Signaling Technology), p-PERK (Thr980) (#3179, Cell Signaling Technology), p-IRE1α (Ser724) (ab124945, Abcam), GAPDH (#5174, Cell Signaling Technology), and WDR36 (#H00134430-M01, Novus Biologicals).

Techniques: Over Expression, Knockdown, Injection, Western Blot, Expressing, Staining, TUNEL Assay