Fig 2: PHGDH KD promotes differentiation of ECSLCs through differential-ubiquitination of ß3-tubulin.NT2/D1 cells, with scrambled control or PHGDH shRNA, were subjected to a microscopic analysis to compare morphology; and immunofluorescence for ß3-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 ß3-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-ß3-tubulin antibody followed by WB analysis for specific ubiquitination of ß3-tubulin. e NT2/D1 cells, with scrambled control or PHGDH shRNA, were treated with MG132 and subjected to WB analysis for ß3-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 ß3-tubulin. *p = 0.05; **p = 0.01; ***p = 0.001