Extended Data Fig. 5: Induction of reductive stress by NRF2 activation in tRCC cells.
a, Western blot showing TFE3 and NRF2 protein levels across a panel of ccRCC and tRCC cell lines. Note, A498 (ccRCC) cells have been previously reported to have high NRF2 activation, possibly due to SQSTM1 overexpression and/or CUL3 mutation79. b, Immunofluorescence showing subcellular localization of NRF2 in tRCC cell lines vs. a ccRCC cell line (786-O). Scale bar: 50 μm. c, IGV snapshot showing TFE3 fusion signal at NFE2L2 and SQSTM1 loci in tRCC cell lines. d, Western blot of p62 expression in ccRCC and tRCC cell lines. e, SQSTM1 mRNA level in ccRCC or tRCC tumours from three independent studies (TCGA, Motzer et al., Elias et al. (PDX)). Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5 times the interquartile range. f, Western blot showing NRF2 and KEAP1 levels following KEAP1 knockout in ccRCC cell line (786-O) and tRCC cell lines (UOK109, s-TFE and FU-UR-1). g, Western blot showing doxycycline-inducible V5-tagged NRF2 overexpression in ccRCC and tRCC cells. h, Quantification of clonogenic capacity (Crystal Violet assay) after NRF2 overexpression in ccRCC or tRCC cell lines. Data are shown as mean -+ s.d. for n = 3 biological replicates. i, Representative wells for colony formation in ccRCC and tRCC cells upon KEAP1 knockout. j, Quantification of clonogenic capacity after knockout of KEAP1 in ccRCC or tRCC cell lines. Data are shown as mean -+ s.d. for n = 3 biological replicates. k, Quantification of NADH to NAD+ ratio following NRF2 overexpression in ccRCC cell line and tRCC cell lines. l, Representative images displaying NADH/NAD+ ratio in tRCC vs. ccRCC cells (images pseudocolored by NADH/NAD+ ratio). Scale bar: 20 μm. m, Western blot confirming LbNOX overexpression in UOK109 and s-TFE cells transduced with lentiviral vector encoding doxycycline-inducible LbNOX-V5. n, Quantification of clonogenic capacity after doxycycline-inducible expression of NRF2, with or without co-expression of the NADH-oxidizing enzyme LbNOX in UOK109 and s-TFE cells. Data are shown as mean -+ s.d. for n = 3 biological replicates. For (e) and (k), statistical significance was determined by two-sided Mann–Whitney test. For (h), (j) and (n), statistical significance was determined by two-tailed Student’s t-test.
