Fig 2: Extra-mitochondrial prosurvival MCL-1 protein regulates SUFU activity(a) CRISPR-Cas9 mediated deletion of the MCL-1 TM sequence. (b) Biochemical evaluation of MCL-1 proteins in CRISPR-Cas9 edited cells. Re-localization of MCL-1 from the OMM to the cytoplasm (MCL-1ΔTM) or MCL-1 elimination (KO) does no alter the distribution of SUFU. Quantification of relative SUFU/TOM20 levels is provided. (c) Enhanced HH response in MCL1ΔTM cells. Increasing amounts of conditioned medium (CM) containing SHH-N protein was applied to CRISPR-Cas9-edited C3H10T1/2 cells expressing wt, ΔTM or no MCL-1 (n=3 independent measurements). (d) Biochemical characterization of GLI target gene expression in CRISPR-Cas9-edited Mcl1ΔTM C3H10T1/2 cells. (e) CRISPR-Cas9 mediated deletion of the BCL-2 TM sequence. (f) Biochemical evaluation of BCL-2 proteins in CRISPR-Cas9 edited cells. Re-localization of BCL-2 to the cytoplasm (BCL-2ΔTM) does not alter the abundance of SUFU associated with the OMM. (g) Enhanced HH response in Bcl2ΔTM cells. Increasing amounts of SHH-N CM was applied to CRISPR-Cas9-edited C3H10T1/2 cells expressing wt or ΔTM BCL-2 protein (n=3 independent measurements). (h) Biochemical characterization of GLI target gene expression in Bcl2ΔTM C3H10T1/2 cells in response to SHH-N containing CM. (i) Model of extramitochondrial prosurvival BCL-2-dependent transcriptional regulation. All error bars represent mean ± s.d. Source data are available in Supplementary Table 4. Unprocessed blots are provided in Supplementary Fig. 7.

Fig 3: mTORC1/S6K1 activates Gli2 to promote PTHrP transcription.(a) Chondrocytes from control and TSC1CKO mice with or without rapamycin treatment were lysed and the interactions between S6K1 and Gli2 or Gli1 were analysed by IP-western analysis. (b) IP-western analysis showing the phosphorylation of Gli2 in primary cultured chondrocytes with or without rapamycin treatment. (c) Analysis of subcellular fractionation of Gli2 and Gli1 in cultured chondrocytes from control and TSC1CKO mice. (d) Analysis of subcellular fractionation of Gli2 and Gli1 in normal ATDC5 cells and ATDC5 cells transfected with the S6K1 expressing plasmid. (e) Immunofluorescence of Gli2 in cultured chondrocytes from control and TSC1CKO mice with or without rapamycin treatment showed the translocation of Gli2. Scale bar, 25 µm. (f) Analysis of subcellular fractionation of Gli2 and Gli1 in normal ATDC5 treated with IGF-1 and rapamycin. (g) Immunoprecipitation analysis showing the interactions of SuFu and Gli1/2, SuFu and S6K1 in normal chondrocytes or TSC1 null chondrocytes treated with rapamycin or not. (h) Immunoprecipitation analysis showing the interactions of SuFu, Gli2 and its mutants S234A, S234E. (i) Western analysis showing the expression of Gli1, PTHrP, Gli2 in ATDC5 cells transiently transfected with Gli2 and its mutants. (j) Western analysis showing the expression of PTHrP, Gli1, Gli2, pS6 and S6K1 in ATDC5 cells and ATDC5 cells transiently transfected with S6K1 expression vector. (k) Immunofluorescence of PTHrP in cultured chondrocytes from control and TSC1CKO mice with rapamycin treatment. Scale bar, 25 µm.

Fig 4: miR-194 is upregulated, whereas SUFU is downregulated in GC. (A and B) Reverse transcription-quantitative polymerase chain reaction was performed using 62 GC tissues and corresponding adjacent normal tissues, in order to analyse the relative expression levels of miR-194. P<0.0001. (C) Relative expression levels of SUFU in the HFE145 human normal gastric epithelial cell line, and in the AGS, MKN28, BGC-823 and SGC-7901 human GC cell lines. One-way analysis of variance followed by Dunnett's test was used for statistical analysis. *P<0.05. (D) Spearman's correlation analysis between miR-194 and SUFU expression in 30 paired GC specimens. GC, gastric cancer; miR-194, microRNA-194; SUFU, SUFU negative regulator of Ηedgehog signaling.

Fig 5: (A) Inhibition of miR-194 caused ß-catenin to translocate to the cell membrane and caused SUFU to translocate to the cytoplasm. BGC-823 cells were transfected with a miR-194 inhibitor for 48 h or were incubated with 1 µM XAV-939 for 16 h. Subsequently, subcellular ß-catenin and SUFU localisation was assessed by immunofluorescence staining. Scale bar, 10 µm. (B) Reverse transcription-quantitative polymerase chain reaction analysis of miR-194 was performed on BGC-823 cells 48 h following miR-194 inhibitor transfection to confirm successful transfection. Student's t-test was performed to analyse results. (C) SUFU expression was successfully knocked down using SUFU siRNA; BGC-823 cells were transfected with NC or SUFU siRNAs, and 48 h post-transfection, the cell lysates were immunoblotted with a SUFU antibody. (D) Suppression of SUFU alleviated the cytoplasmic translocation of ß-catenin induced by miR-194 inhibition. BGC-823 cells were transfected with a miR-194 inhibitor or a miR-194 inhibitor and SUFU siRNA, and 48 h post-transfection, the cell lysates were fractionated into cytoplasmic and nuclear portions. Cell lysates were then immunoblotted with ß-catenin and SUFU antibodies. (E) Densitometric analyses of western blots from three independent experiments, with normalization to the control, were performed with ImageJ software. The relative expression levels of the control condition were designated as 1. One-way analysis of variance followed by Dunnett's test was used for statistical analysis, *P<0.05. DMSO, dimethyl sulfoxide; GC, gastric cancer; HDAC1, histone deacetylase; miR-194, microRNA-194; NC, negative control; NSC, non-specific; si/siRNA, small interfering RNA; SUFU, SUFU negative regulator of ?edgehog signaling.