Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) qRT/PCR showing that mGSRR cells display increased Cdh2 and decreased Cdh1 mRNA expression compared with mGS. Two-tailed Student’s t test. (B) Western blot showing expression of Slug, Snail1, and Zeb1 are gradually increased upon repeated irradiation in mGS cells. (C) Western blot showing that Snail overexpression induces elevation of N-cad, Olig2, and Zeb1, and suppression of Tuj1 in mGS cells. (D) Wnt/β-catenin transcriptional activity is suppressed in mGSRR and mGS with Snail1 overexpression (OE) compared with mGS cells. ***P < 0.001, Tukey’s HSD test. (E) Clonogenic survival assay shows mGS Snail1 OE cells have a higher survival rate than mGS cells. Two-tailed Student’s t test. *P < 0.05, **P < 0.01. (F) Western blot showing increased N-cad, β-catenin, Slug, and Zeb1 expression 2 days after mouse recombinant IGF1 (100 ng/mL), but not TGF-β1 (10 ng/mL) treatment in mGS cells. (G) Western blot showing IGF1 overexpression increases N-cad, β-catenin, Zeb1, and IGF1R expression in mGS cells. (H) Survival curves for mice implanted with 1000 cells (GS with IGF1 expression vector) and subjected to whole-brain irradiation (2 Gy/day, days 3 to 7, 10 Gy total). (I) Left: schematic showing experimental design for clonogenic survival assay with repeated irradiation. Single mGS cells seeded in agarose medium were exposed to repeated irradiation (5 doses of 4 Gy, every 3 days) with or without drug rescue. IGF1R (AEW541 0.5 μM; PPP 0.2 μM) and TGF-β1 (LY2157299 10 μM, SB431542 10 μM) inhibitors were used. Right: quantification of percentage of surviving colonies shows that IGF1R inhibitors selectively decreased survival rate. Drugs alone had no effect on colony formation (data not shown). ***P < 0.001, Dunnett’s test. (J) Mice implanted orthotopically with mGSRR cells had a survival benefit after whole-brain irradiation (2 Gy × 5 days) with adjuvant PPP (15 mg/kg, i.p. twice a day from day 3–7) in contrast to vehicle control, only IR or PPP alone (8 mice/group; log-rank test). All blots show representative images (n = 3 or more).

Article Snippet: Cells were treated with recombinant mouse IGF1 (100 ng/mL, R&D Systems, #791-MG), recombinant mouse TGF-β1 (10 ng/mL, R&D Systems, #7666-MB), SB216763 (1–5 μM, Selleckchem, S1075), or CHIR99021 (1–5 μM, Selleckchem, S1263).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Irradiation, Over Expression, Activity Assay, Clonogenic Cell Survival Assay, Recombinant, Plasmid Preparation, Adjuvant

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet: In vitro differential suppression of podocyte IGF1R activity reveals that partial inhibition is beneficial but near total loss is highly detrimental (A) Representative Western blot shows >90% reduction of IGF1R protein in NC-IGF1RKD cells but no reduction of IR protein expression. Bar graphs show densitometry expressed as the mean fold change +/− SEM, t-test, ∗∗∗∗ p < 0.0001, n = 18 independent experiments. (B) Phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min was significantly reduced in NC-IGF1RKD podocytes. Data are expressed as the mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, ∗∗ p < 0.005, n = 3 independent experiments. (C) Western blot shows that IGF1R expression is reduced by ∼70% in wild-type podocytes exposed to 100 nM picropodophyllin for 24 h. Data are expressed as the mean ± SEM, t-test, ∗ p < 0.05, n = 3 independent experiments. No significant change in IR expression was observed. (D) Western blot shows the phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min in podocytes exposed to 100 nM picropodophyllin for 24 h. Data expressed as the mean ± SEM, ∗ p < 0.05, n = 3 independent experiments. (E) ∼50% of NC-IGF1RKD cells survive 7 days after gene excision. Treatment of wild-type podocytes with 100 nM picropodophyllin for 24 h has no effect on cell survival. Data are expressed as the mean ± SEM, t-test, ∗∗ p < 0.005, n = 3–4 independent experiments. See also Figures S5 and .

Article Snippet: For acute insulin and IGF1 stimulation, Conditionally immortalised wild-type mouse podocyte cell were serum starved for 4 h then 10 nM and 100 nM of insulin (Biotechne, Cat# 3435) or 10 ng/mL and 100 ng/mL IGF1 (Novus, Cat# NBP2-35081) was applied to the cells for 10 min.

Techniques: In Vitro, Activity Assay, Inhibition, Western Blot, Expressing, Phospho-proteomics, Comparison

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet:

Article Snippet: For acute insulin and IGF1 stimulation, Conditionally immortalised wild-type mouse podocyte cell were serum starved for 4 h then 10 nM and 100 nM of insulin (Biotechne, Cat# 3435) or 10 ng/mL and 100 ng/mL IGF1 (Novus, Cat# NBP2-35081) was applied to the cells for 10 min.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, RNAscope, Western Blot, Cell Culture, Software, Plasmid Preparation