Fig. 4: TORC1-mediated protein synthesis promotes ATFS-1 activation.

a, b Transcript levels of heat shock protein-6 (hsp-6) (a) and of activated transcription factor stress-1 (atfs-1) (b) as determined by qRT-PCR in wild-type and rsks-1(ok1255) worms. N = 4 biologically independent experiments. Error bars mean ± SD (two-tailed Student’s t-test). RFU, relative fluorescence units. c hsp-6_pr::gfp and aak-2(rr48);hsp-6_pr::gfp worms raised on control, let-363, or atfs-1 (RNAi). Scale bar 0.1 mm. Experiments were repeated three biologically independent times with similar results. d Quantification of GFP intensity in hsp-6_pr::gfp (N = 10) and aak-2(rr48);hsp-6_pr::gfp worms raised on control (N = 8 worms), let-363 (N = 8 worms), or atfs-1 (RNAi) (N = 7 worms). Error bars mean ± SD (two-tailed Student’s t-test). a.u., arbitrary units. e, f Transcript levels of heat shock protein-6 (hsp-6) as determined by qRT-PCR in wild-type and aak-2(rr48) worms N = 3 biologically independent experiments (e) and in wild type, agd383, and agd383;atfs-1(et18) strains N = 5 biologically independent experiments (f). Error bars mean ± SD (two-tailed Student’s t-test). RFU, relative fluorescence units. g ChIP of hsp-6 promoter in wild-type, rsks-1(ok1255), and aak-2(rr48) worms as measured by qRT-PCR. N = 2 biologically independent experiments. RFU, relative fluorescence units. h SDS-Page immunoblots of wild-type and rsks-1(ok1255) worms, raised on control or lonp-1(RNAi). Tubulin (Tub) was used as a loading control. N = 3 biologically independent experiments with similar results. i atfs-1(et18);hsp-6_pr::gfp worms raised on control or rsks-1(RNAi). Scale bar 0.1 mm. Experiments were repeated three biologically independent times with similar results. j TMRE staining (red) of worms expressing myo-3_pr::gfp or myo-3_pr:: ^mtgfp. Scale bar 10 µm. Experiments were repeated three biologically independent times with similar results. k Quantification of TMRE intensity in muscle cells of worms expressing myo-3_pr::gfp or myo-3_pr:: ^mtgfp. N = 34 worms (myo-3_pr:: gfp), N = 24 worms (myo-3_pr:: ^mtgfp). Error bars mean ± SD (two-tailed Student’s t-test). a.u., arbitrary units. l Quantification of mtDNA in myo-3_pr:: gfp and myo-3_pr:: ^mtgfp worms as determined by qPCR. N = 6 biologically independent experiments. Error bars mean ± SD (two-tailed Student’s t-test). m, n Transcript levels of translocase of inner mitochondrial membrane-23 (timm-23) (m) and of heat shock protein-6 (hsp-6) (n) as determined by qRT-PCR in myo-3_pr::gfp and in myo-3_pr:: ^mtgfp worms. N = 4 biologically independent experiments. Error bars mean ± SD (two-tailed Student’s t-test). RFU, relative fluorescence units. o TMRE staining of worms expressing myo-3_pr:: ^mtgfp raised on control or atfs-1(RNAi). Scale bar 10 µm. Experiments were repeated three biologically independent times with similar results. p Quantification of TMRE intensity in muscle cells of worms expressing myo-3_pr:: ^mtgfp raised on control or atfs-1(RNAi). N = 18 worms (control), N = 13 worms (atfs-1(RNAi)). Error bars mean ± SD (two-tailed Student’s t-test). a.u., arbitrary units. q TMRE staining of wild-type and atfs-1(null) worms expressing myo-3_pr:: ^mtgfp. Scale bar 10 µm. Experiments were repeated three biologically independent times with similar results. r Quantification of TMRE intensity in muscle cells of wild-type, and atfs-1(null) worms. N = 15 worms (wild type), N = 24 worms (atfs-1(null)). Error bars mean ± SD (two-tailed Student’s t-test). a.u., arbitrary units. s TMRE staining of wild-type and atfs-1^R/R worms expressing myo-3_pr:: ^mtgfp. Scale bar 10 µm. Experiments were repeated three biologically independent times with similar results. Source data are provided as a Source Data file.