Extended Data Fig. 9: Valine tRNA biogenesis and bioavailability regulates levels of mitochondrial complex I.
From: Valine tRNA levels and availability regulate complex I assembly in leukaemia
a, Blue Native (BN)-PAGE analysis (same gel, for gel source data, see Supplementary Fig. 1) and b, maximal respiration of KOPTK1 cells cultured in different levels of valine for 72 h. c, Oxygen consumption rate (OCR) and d, Maximal respiration of CUTLL1 cells cultured in different levels of valine for 72 h (mean ± SD of n=5 independent replicates; two-sided unpaired t-test). e, Relative expression of VARS mRNA by qPCR analysis following 4 days of doxycycline treatment to induce Cas13d expression. (n=2 independent replicates). f, Competition based proliferation assay to validate loss of fitness following VARS knockdown in CUTLL1 Cas13d cells. Plotted are GFP percentages measured during 20 days in culture and normalized to day 0 of doxycycline treatment. Negative control sgRosa and three independent sgRNAs targeting VARS are shown in the graphs (mean ± SD of n=3 independent replicates). g, BN-PAGE (top) analysis of CUTLL1 cells expressing inducible Cas13d and sgRNA_1 targeting VARS transcript. SDS-PAGE and immunoblots confirming VARS knockdown (same gel, for gel source data, see Supplementary Fig. 1). h, NAD+/NADH ratio of CUTLL1 cells cultured in RPMI with different levels of valine for 72 h. Representative of (n=2) independent experiments. i, MitoSox staining of CUTLL1, KOPTK1 cells cultured in RPMI with different levels of valine for 72 h and analyzed by flow cytometry (left, middle). CUTLL1 Cas13d cells transduced with three independent sgRNAs targeting VARS transcript was treated with doxycycline to induce Cas13 expression. Day 8 of doxycycline treatment, cells were stained with MitoSOX and analyzed by flow cytometry (right). Graphs show Mito-SOX mean fluorescence intensity (MFI). Representative of (n=3) independent experiments (mean ± SD of n=4 technical replicates). j, Electron micrograph from CUTLL1 cells illustrating the mitochondrial cristae structure. Arrow denotes the parameter of maximal Cristae Lumen Width (CLW) quantified in this study. k, Representative electron micrographs of CUTLL1 cells cultured in RPMI with different levels of valine for 72 h. l, Representative electron micrographs of CUTLL1-Cas13d cells transduced with either sgROSA or sgRNA targeting VARS and doxycycline treated for 8 days to induce Cas13d expression (left). Quantification of maximal cristae width in 10 randomly selected mitochondria from 15 cells (right) (n=110 cristae; mean ± SD; two-sided unpaired t-test).
