mouse igf2bp3  (Addgene inc)

Journal: Human Cell

Article Title: N6-methyladenosine-modified SRD5A3, identified by IGF2BP3, sustains cisplatin resistance in bladder cancer

doi: 10.1007/s13577-024-01136-0

Figure Lengend Snippet: IGF2BP3 directly interacts with SRD5A3 mRNA and increases its stability. A The m6A levels of SRD5A3 in T24R and 5637R cells, and their wild-type counterparts were tested by MeRIP-qPCR. B The m6A levels of SRD5A3 in T24R and 5637R cells following IGF2BP3 knockdown or overexpression. C IGF2BP3 RNP SRD5A3 mRNA enrichment in T24R and 5637R cells following IGF2BP3 knockdown or overexpression. D A schematic diagram presenting three methylation sites on SRD5A3 and mutates these sites ( A – C ). E The luciferase activities of the WT or Mut luciferase reporters in HEK-293 T cells following IGF2BP3 knockdown. F The mRNA stability of SRD5A3, SRD5A1, and SRD5A2 in T24R and 5637R cells following IGF2BP3 knockdown after actinomycin D treatment (normalized to 0 h). G The IGF2BP3 mRNA and protein expression levels were determined by qRT-PCR and immunoblotting analysis in parental cells and CDDP-resistant cells. H SRD5A3 mRNA and protein expression levels were determined by qRT-PCR and immunoblotting analysis in T24R and 5637R cells following IGF2BP3 knockdown or overexpression. Six independent experiments yielded results. The repeated-measures ANOVA plus Sidak’s multiple comparisons test was performed in panel ( F ) and unpaired t tests were performed in other panels. * P < 0.05

Article Snippet: The membranes were incubated with rabbit polyclonal anti-SRD5A3 antibody (PA5-55480, Invitrogen, Carlsbad, CA, USA), mouse monoclonal anti-IGF2BP3 antibody (MA5-27480, Invitrogen), and mouse monoclonal anti-GAPDH antibody (MA5-15738, Invitrogen), followed by rinse with TBST and incubation with secondary antibodies.

Techniques: Knockdown, Over Expression, Methylation, Luciferase, Expressing, Quantitative RT-PCR, Western Blot

Journal: Human Cell

Article Title: N6-methyladenosine-modified SRD5A3, identified by IGF2BP3, sustains cisplatin resistance in bladder cancer

doi: 10.1007/s13577-024-01136-0

Figure Lengend Snippet: Characterization of IGF2BP3 in bladder cancer. A The UALCAN database showing an increased transcript level of IGF2BP3 in the primary bladder urothelial carcinoma ( n = 408) vs. the normal samples ( n = 19). B and C IGF2BP3 mRNA and protein expression levels were determined by qRT-PCR and immunoblotting analysis in the corresponding adjacent non-tumor bladder tissues and the bladder cancer tissues. D and E IGF2BP3 mRNA and protein expression levels were determined by qRT-PCR and immunoblotting analysis in standard SV-HUC-1 cells and bladder cancer cells (T24 and 5637). Six independent experiments yielded results. The Student’s t tests were performed in panels ( A – C ) and the ANOVA plus Tukey’s post hoc test in panel ( D – E ). * P < 0.05

Article Snippet: The membranes were incubated with rabbit polyclonal anti-SRD5A3 antibody (PA5-55480, Invitrogen, Carlsbad, CA, USA), mouse monoclonal anti-IGF2BP3 antibody (MA5-27480, Invitrogen), and mouse monoclonal anti-GAPDH antibody (MA5-15738, Invitrogen), followed by rinse with TBST and incubation with secondary antibodies.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Human Cell

Article Title: N6-methyladenosine-modified SRD5A3, identified by IGF2BP3, sustains cisplatin resistance in bladder cancer

doi: 10.1007/s13577-024-01136-0

Figure Lengend Snippet: IGF2BP3 knockdown overcomes CDDP resistance in bladder cancer in vitro. A CCK-8 assay for cell viability of T24R and 5637R cells treated with 5 μg/ml CDDP for 72 h following IGF2BP3 knockdown. B Colony formation assay for cell proliferation of T24R and 5637R cells treated with 5 μg/ml CDDP for 24 h following IGF2BP3 knockdown. C EdU staining for cell proliferation of T24R and 5637R cells treated with 5 μg/ml CDDP for 24 h following IGF2BP3 knockdown. EdU-positive stained cells were indicated by red spots, and the nuclei were indicated by blue spots. D Flow cytometric analysis for cell apoptosis of T24R and 5637R cells treated with 5 μg/ml CDDP for 24 h following IGF2BP3 knockdown. Six independent experiments yielded results. The repeated-measures ANOVA plus Sidak’s multiple comparisons test was performed in panel ( A ) and unpaired t tests were performed in other panels. * P < 0.05

Article Snippet: The membranes were incubated with rabbit polyclonal anti-SRD5A3 antibody (PA5-55480, Invitrogen, Carlsbad, CA, USA), mouse monoclonal anti-IGF2BP3 antibody (MA5-27480, Invitrogen), and mouse monoclonal anti-GAPDH antibody (MA5-15738, Invitrogen), followed by rinse with TBST and incubation with secondary antibodies.

Techniques: Knockdown, In Vitro, CCK-8 Assay, Colony Assay, Staining

Journal: Human Cell

Article Title: N6-methyladenosine-modified SRD5A3, identified by IGF2BP3, sustains cisplatin resistance in bladder cancer

doi: 10.1007/s13577-024-01136-0

Figure Lengend Snippet: IGF2BP3 knockdown overcomes CDDP resistance in bladder cancer in vivo. A Representative images of subcutaneous xenograft tumors formed by IGF2BP3-knockdown T24R cells for 35 days, and their tumor volume and tumor weight ( n = 5 for each group). B Images of subcutaneous xenograft tumors formed by T24R cells following injections of lentivirus harboring IGF2BP3-shRNA and CDDP (2 mg/kg) for 35 days, and their tumor volume and tumor weight ( n = 5 for each group). C The tumor volume and tumor weight of xenograft tumors formed by T24R cells with or without CDDP treatment. The tumor volume was analyzed by repeated-measures ANOVA plus Sidak’s multiple comparisons test. The tumor weight was analyzed by unpaired t test. * P < 0.05

Article Snippet: The membranes were incubated with rabbit polyclonal anti-SRD5A3 antibody (PA5-55480, Invitrogen, Carlsbad, CA, USA), mouse monoclonal anti-IGF2BP3 antibody (MA5-27480, Invitrogen), and mouse monoclonal anti-GAPDH antibody (MA5-15738, Invitrogen), followed by rinse with TBST and incubation with secondary antibodies.

Techniques: Knockdown, In Vivo, shRNA