Extended Data Fig. 5: Additional data for Fig. 6.
Panels (a–e, g–i) are from HEK293T cells. Lanes shown with lines are biological replicates. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 10−3. Tukey’s Honestly Significant Difference test (HSD), two-sided. Data are presented as mean values ± SD. a) Q-modifiable tRNA levels by Northern blot with and without emetine inhibition of translation. b) Q-modifiable tRNA levels and preQ1-modification by Northern blot using NHS treatment method with and without cycloheximide inhibition of translation. c) SUnSET translation activity assay with puromycin antibody under mock, emetine, or cycloheximide conditions with and without preQ1 treatment. Left: Western blot using anti-puromycin and β-actin antibodies (top) and the same blot stained with Coomassie blue (bottom). Right: quantification of Western blots in left panel. β-actin is the loading control. n = 3 biological replicates. p values from left to right: 1.31e-8, 9.78e-9, 0.833, 5.45e-9, 4.21e-9. d) GO term of genes enriched in the polysome (red mRNA transcripts in Fig. 4h) with preQ1. e) Northern blot using NHS reaction method to detect preQ1-tRNA using preQ1 treated cells with and without 4µ8C, the inhibitor of IRE1 ribonuclease activity for different times as indicated. Total RNA without preQ1 and 4µ8C treatment was used as the no NHS reaction control. f) Conservation of IRE1 protein from Pfam. g) Western blot (left) and quantitation (right) of IRE1 phosphorylation under queuine/preQ1 and TG treatment. n = 3 biological replicates. Fisher’s Least Significant Difference (LSD) test, two-sided. p values from left to right: 4.75e-3, 0.516, 1.28e-3. h) Blue-Native gel followed by Western blot (left) and quantitation (right) of IRE1 oligomerization under queuine, preQ1, or TG treatment. n = 3 biological replicates. p values from left to right: 0.238, 0.891, 0.955. i) Western blot (top) and quantitation (bottom) of eIF2α phosphorylation with the indicated queuine, preQ1, and positive control thapsigargin (TG) treatments. n = 3 biological replicates. p values from left to right: 0.0862, 0.446, 7.72e-3, 1.77e-6. j) RT-PCR followed by PAGE gel electrophoresis (left) and quantitation (right) of XBP1 pre-mRNA splicing with the indicated queuine, preQ1, and positive control thapsigargin (TG) treatments. n = 3 biological replicates. p values from left to right: 0.0684, 0.946, 2.01e-12, 1.77e-6, 0.691, 3.23e-3, 2.04e-13. Source numerical data and unprocessed blots are available in.
