Fig. 5: PHGDH KD promotes differentiation of ECSLCs through differential-ubiquitination of β3-tubulin. | Cell Death & Disease

Fig. 5: PHGDH KD promotes differentiation of ECSLCs through differential-ubiquitination of β3-tubulin.

From: Phosphoglycerate dehydrogenase inhibition induces p-mTOR-independent autophagy and promotes multilineage differentiation in embryonal carcinoma stem-like cells

Fig. 5

NT2/D1 cells, with scrambled control or PHGDH shRNA, were subjected to a microscopic analysis to compare morphology; and immunofluorescence for β3-tubulin and analyzed by confocal microscopy; or b qRT-PCR analysis for differentiation markers from early-stage ectoderm (BMP4, NES, TUBB3), neuronal lineage (TUBB3, NEUROG1), early-stage endoderm (GATA4, GATA6, SOX7, SOX17), and early-stage mesoderm (Nodal, T, TBX6). c (i) NT2/D1 cells, with scrambled control or PHGDH shRNA, were subjected to western blot (WB) analysis for β3-tubulin protein levels. (ii) NT2/D1 cells, with scrambled control or PHGDH siRNA, were subjected to qRT-PCR analysis for TUBB3 mRNA levels 48 and 72 h post transfection. d NT2/D1 cells, with scrambled control or PHGDH shRNA, were subjected to (i) WB analysis for total levels of ubiquitin, and (ii) immunoprecipitation (IP) using anti-β3-tubulin antibody followed by WB analysis for specific ubiquitination of β3-tubulin. e NT2/D1 cells, with scrambled control or PHGDH shRNA, were treated with MG132 and subjected to WB analysis for β3-tubulin. f NT2/D1 cells, with (i) scrambled control or (ii) PHGDH shRNA were treated with the translation inhibitor cyclohexamide (CHX) for 1 h, and then subjected to WB analysis for β3-tubulin. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

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