Fig. 3: Cyst(e)ine promotes GPX4 protein synthesis partly through Rag-mTORC1-4EBP signaling.

a UMRC6 cells were cultured in media with indicated concentrations of cystine for 24 h followed by Western blotting to analyze protein levels. b UMRC6 cells were treated with indicated concentrations of Rapamycin (Rapa) or Torin1 for 24 h. GPX4 and mTOR signaling protein levels were assessed by Western blotting. c UMRC6 cells were treated with indicated concentrations of AZD8055 for 24 h. GPX4 and mTOR signaling protein levels were assessed by Western blotting. d UMRC6 cells were treated with 1 μM Torin1 or AZD8055 for 24 h followed by polyribosome fractionation and RT-PCR to analyze GPX4 and ACTB (Actin) mRNA distribution profiles during protein translation. n = 3 biologically independent repeats. Error bars are mean ± SD. P value was determined by two-tailed unpaired Student’s t test. e Western blotting analysis of indicated protein levels in UMRC6 control (sgC) and RagA/RagB double-knockout (A/B-DKO) cell lines. LE long exposure. f sgC and A/B-DKO cells were cultured in cystine-free media for 24 h followed by 200 μM cystine stimulation for 8 h and Western blotting analysis. g, h Wild-type (shown as “/”) and 4EBP1-4A–overexpressing (shown as “4 A”) UMRC6 or 786-O cells were treated with or without 100 ng/ml Doxycycline (Dox.) for 30 h followed by Western blotting analysis. i Western blotting analysis of protein expression in control (sgCon) and 4EBP1/2-double-knockout (DKO-1 and DKO-2) UMRC6 cells. j Western blotting analysis of protein expression in indicated cell lines treated with or without 1 μM Torin1 for 24 h. k Western blotting analysis of protein expression in indicated cell lines cultured in complete media or cystine-free media for 24 h. l Representative images from immunohistochemical staining of kidney tumor tissue microarray for GPX4 and P-4EBP1. m Summary and statistical analysis of immunohistochemical staining results for GPX4 and P-4EBP1 in tumor tissue microarray.