pone.0239325.g003.tif (1.46 MB)

Characterization of induced neural stem cells with doxycycline-inducible IDH1R132H.

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posted on 18.09.2020, 17:30 by Kamila Rosiak-Stec, Dagmara Grot, Piotr Rieske

Induced expression of IDH1R132H upon doxycycline treatment. (A) Sanger sequencing electropherograms of the mutated nucleotide in codon 132 (R132H) in iNSc tet and iNSc tet-IDH1R132H in the presence or absence of doxycycline (1 μg/mL, 96 h, Dox). (B) IDH1 (non-mutation-specific) mRNA expression in iNSc tet and iNSc tet-IDH1R132H stimulated or not with doxycycline (1 μg/mL, 96 h). Asterisks over bars indicate statistical significance towards untreated iNSc tet (not shown). Statistical significance calculated by One-way ANOVA with Dunnett’s multiple comparison test. Error bars indicate SEM, n = 3. **, p<0.01; ns, not significant. (C) Representative images of IDH1R132H (red, IDH1m) and IDH1WT (green) expression in iNSc tet-IDH1R132H treated with doxycycline (1 μg/mL, 96 h) and MGG152 neurospheres with endogenous IDH1R132H. Cell nuclei were counterstained with the nuclear DNA marker DAPI (blue). Inset boxes show lower magnifications of MGG152 neurospheres. Scale bars represent 50 μm. (D) Western blot analysis of IDH1R132H and IDH1WT expression in iNSc tet, iNSc tet-IDH1R132H untreated or treated with doxycycline (1 μg/mL) for indicated periods of time, and MGG152 neurospheres. Equal loading was verified by Ponceau S staining of the membrane. (E) Quantification of blot in D displaying ratio of band density of IDH1R132H over Actin. Statistical significance calculated by One-way ANOVA with Dunnett’s multiple comparison test. Error bars indicate SEM, n = 3. *, p<0.05; ns, not significant.

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