Figure 1 ( B ) Immunoblot analysis of the immunoprecipitated HA - SOX9 or FLAG - FBW7α and their pull - down products . The corresponding Western blotting from the whole cell extract ( WCE ) of the transfected HEK293 showed the levels of exogenous HA - SOX9 or FLAG - FBW7α proteins in the total input . Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation . Blots are representative from 3 independent experiments . ( C ) FBW7α immunoblot eluted from the agarose bead - bound peptide , which had been incubated with the recombinant SCF - FBW7α for 1 hour at 37 ◦ C . The agarose bead - bound peptide contains either the non - phosphorylated SOX9 amino acid sequence spanning from residue number 231 to 245 ( SOX9 peptide ) , or those with the threonine corresponding to residue number 236 and 240 being phosphorylated ( pSOX9 peptide ) . The input ( 10 % ) showed the level of the supplemented recombinant SCF - FBW7α in the in vitro binding reaction . Shown blot is representative from 2 independent experiments . ( D ) Representative Western blotting set from 3 independent repeats of pT236 - SOX9 from lysates of HEK293 transfected with either HA - EV or HA - SOX9 . Each lysate were immediately divided and left untreated or subjected to lambda phosphatase ( λ - PPase ) treatment for 1 hour at 37 ◦ C prior to gel electrophoresis . SOX9 blot showed the presence of SOX9 protein in both the untreated and the phosphatase - treated SOX9 - transfected cell lysates . ( E ) Bead - immobilized in vitro translated ( IVT ) HA - SOX9 wild type ( WT ) or T236 / 240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 minutes at 37 ◦ C prior to elution and gel electrophoresis . The SOX9 blot showed total SOX9 protein eluted from the beads from each reaction . Shown immunoblots are single representative experiment from 3 independent repeats . ( F ) GSK3 - mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF - FBW7α in vitro . Bead - immobilized IVT HA - SOX9 were either untreated or phosphorylated with GSK3 as described in ( E ) prior to further incubation with recombinant SCF - FBW7α for in vitro binding assay . Immunoblot showed FBW7α protein in the in vitro binding reaction ( input ) as well as those which had been eluted from the untreated and GSK3 - treated immobilized SOX9 . The set of blots shown is representative from 3 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Immunoblot analysis of the immunoprecipitated HA-SOX9 or FLAG- FBW7α and their pull-down products. The corresponding Western blotting from the whole cell extract (WCE) of the transfected HEK293 showed the levels of exogenous HA-SOX9 or FLAG-FBW7α proteins in the total input. Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation. Blots are representative from 3 independent experiments.(C) FBW7α immunoblot eluted from the agarose bead-bound peptide, which had been incubated with the recombinant SCF-FBW7α for 1 hour at 37◦C. The agarose bead-bound peptide contains either the non-phosphorylated SOX9 amino acid sequence spanning from residue number 231 to 245 (SOX9 peptide), or those with the threonine corresponding to residue number 236 and 240 being phosphorylated (pSOX9 peptide). The input (10%) showed the level of the supplemented recombinant SCF-FBW7α in the in vitro binding reaction. Shown blot is representative from 2 independent experiments.(D) Representative Western blotting set from 3 independent repeats of pT236-SOX9 from lysates of HEK293 transfected with either HA-EV or HA-SOX9. Each lysate were immediately divided and left untreated or subjected to lambda phosphatase (λ-PPase) treatment for 1 hour at 37◦C prior to gel electrophoresis. SOX9 blot showed the presence of SOX9 protein in both the untreated and the phosphatase-treated SOX9-transfected cell lysates.(E) Bead-immobilized in vitro translated (IVT) HA-SOX9 wild type (WT) or T236/240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 minutes at 37◦C prior to elution and gel electrophoresis. The SOX9 blot showed total SOX9 protein eluted from the beads from each reaction. Shown immunoblots are single representative experiment from 3 independent repeats.(F) GSK3-mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF-FBW7α in vitro. Bead-immobilized IVT HA-SOX9 were either untreated or phosphorylated with GSK3 as described in (E) prior to further incubation with recombinant SCF-FBW7α for in vitro binding assay. Immunoblot showed FBW7α protein in the in vitro binding reaction (input) as well as those which had been eluted from the untreated and GSK3-treated immobilized SOX9. The set of blots shown is representative from 3 independent experiments.


Figure 2 ( A ) Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α , β or γ . HEK293 cells were transfected with 20 nM of non - targeting scramble RNA or those specifically targeting FBW7α , β or γ for 72 hours prior to chase with addition of 100 ng / mL cycloheximide . Immunoblot of Cyclin E , an established SCF - FBW7 substrate , was used to assess the efficacy of RNAi - mediated depletion of FBW7 protein with GAPDH served as protein loading control . ( B ) Immunofluorescence staining of D324MED medulloblastoma cells showing nuclear co - localization of HA - SOX9 WT ( AlexaFluor 568 ; Red ) and FLAG - FBW7α ( AlexaFluor 488 ; Green ) . The cell nuclei were counter - stained with DAPI ( blue ) . Image set is representative for multiple image fields taken at 40x objective magnification . Scale bar indicate 20 µm . ( C ) Western blotting of HEK293 endogenous SOX9 proteins 24 hours following forced expression of 100 , 250 , 500 , and 1000 ng of plasmid expressing FLAG - FBW7α . Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ . The set of blots shown is representative from 3 independent experiments . ( D ) Expression of FLAG - FBW7α enhanced HA - SOX9 - WT protein turnover , which can be attenuated with addition of 10 µM MG - 132 ( MG ) in HEK293 cells . By contrast , forced expression of FLAG - FBW7α did not enhance HA - SOX9 - T236A and - T236A / T240A protein turnover over the course of 4 hours protein chase . Protein loading for each sample was indicated by the level of β - actin protein . The set of blots shown is representative from 4 independent experiments . ( E ) Treatment of HEK293 cells with increasing concentration of BIO , a GSK3α / β inhibitor , increased HA - SOX9 - WT protein level in dose - dependent manner . HA - SOX9 - WT and FLAG - FBW7α were co - expressed in HEK293 overnight prior to treatment with various concentration of BIO for 4 hours . Whole cell lysates were collected for Western blotting with protein loading for each sample adjusted to GAPDH protein level . The set of blots shown is representative from 2 independent experiments . ( F ) The RNAi depletion of GSK3ß decreased FBW7α - induced HA - SOX9 - WT turnover in HEK293 cells . The HA - SOX9 - WT protein turnover was examined in the presence of 100 ng / mL cycloheximide , 48 hours following si - GSK3ß ( 20 nM ) transfection , 24 hours after co - expression of HA - SOX9 - WT and FLAG - FBW7α . Immunoblots of GSK3ß demonstrated depletion of GSK3ß protein with the siRNA . Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ . The set of blots shown is representative from 3 independent experiments . ( G ) Forced FLAG - FBW7α - WT expression promoted poly - ubiquitylation of endogenous SOX9 in HEK293 , which accumulated further in the presence of 10 µM MG132 . Expression of non - functional FBW7α lacking the F - box domain ( ΔF ) , or those containing R465A mutation did not induce SOX9 poly - ubiquitylation in vivo . Ubiquitylation assay was performed under denaturing condition to disrupt non - covalently linked ubiquitin as described in the Materials Methods . Expression of various FLAG - FBW7α constructs and endogenous SOX9 protein were examined in the whole cell extract ( WCE ) . GAPDH protein was used to indicate total protein loading with protein loading . The set of blots shown is representative from 4 independent experiments . ( H ) Reconstitution of SOX9 ubiquitylation reaction by FLAG - FBW7α in vitro . Immobilized - IVT HA - SOX9 WT were incubated with complete ubiquitylation reaction mixture containing either the eluted FLAG - FBW7α - WT or the ΔF mutant for 60 minutes at 37 ◦ C . Reaction mixture lacking the UbE1 , an E1 ubiquitin - activating enzyme , or UbcH3 , an E2 ubiquiting - conjugating enzyme , served as negative control for the reaction . SOX9 ubiquitylation was assessed following elution of the protein from the immobilized beads under denaturing condition as described in Materials Methods . The set of blots shown is representative from 3 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α, β or γ. HEK293 cells were transfected with 20 nM of non-targeting scramble RNA or those specifically targeting FBW7α, β or γ for 72 hours prior to chase with addition of 100 ng /mL cycloheximide. Immunoblot of Cyclin E, an established SCF-FBW7 substrate, was used to assess the efficacy of RNAi-mediated depletion of FBW7 protein with GAPDH served as protein loading control.(B) Immunofluorescence staining of D324MED medulloblastoma cells showing nuclear co-localization of HA-SOX9 WT (AlexaFluor 568; Red) and FLAG-FBW7α (AlexaFluor 488; Green). The cell nuclei were counter-stained with DAPI (blue). Image set is representative for multiple image fields taken at 40x objective magnification. Scale bar indicate 20 µm.(C) Western blotting of HEK293 endogenous SOX9 proteins 24 hours following forced expression of 100, 250, 500, and 1000 ng of plasmid expressing FLAG-FBW7α. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.(D) Expression of FLAG-FBW7α enhanced HA-SOX9-WT protein turnover, which can be attenuated with addition of 10 µM MG-132 (MG) in HEK293 cells. By contrast, forced expression of FLAG-FBW7α did not enhance HA-SOX9-T236A and -T236A/T240A protein turnover over the course of 4 hours protein chase. Protein loading for each sample was indicated by the level of β-actin protein. The set of blots shown is representative from 4 independent experiments.(E) Treatment of HEK293 cells with increasing concentration of BIO, a GSK3α/β inhibitor, increased HA-SOX9-WT protein level in dose-dependent manner. HA-SOX9-WT and FLAG-FBW7α were co-expressed in HEK293 overnight prior to treatment with various concentration of BIO for 4 hours. Whole cell lysates were collected for Western blotting with protein loading for each sample adjusted to GAPDH protein level. The set of blots shown is representative from 2 independent experiments.(F) The RNAi depletion of GSK3ß decreased FBW7α-induced HA-SOX9-WT turnover in HEK293 cells. The HA-SOX9-WT protein turnover was examined in the presence of 100 ng/mL cycloheximide, 48 hours following si-GSK3ß (20 nM) transfection, 24 hours after co-expression of HA-SOX9-WT and FLAG-FBW7α. Immunoblots of GSK3ß demonstrated depletion of GSK3ß protein with the siRNA. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.(G) Forced FLAG-FBW7α-WT expression promoted poly-ubiquitylation of endogenous SOX9 in HEK293, which accumulated further in the presence of 10 µM MG132. Expression of non-functional FBW7α lacking the F-box domain (ΔF), or those containing R465A mutation did not induce SOX9 poly-ubiquitylation in vivo. Ubiquitylation assay was performed under denaturing condition to disrupt non-covalently linked ubiquitin as described in the Materials Methods. Expression of various FLAG-FBW7α constructs and endogenous SOX9 protein were examined in the whole cell extract (WCE). GAPDH protein was used to indicate total protein loading with protein loading. The set of blots shown is representative from 4 independent experiments.(H) Reconstitution of SOX9 ubiquitylation reaction by FLAG-FBW7α in vitro. Immobilized-IVT HA-SOX9 WT were incubated with complete ubiquitylation reaction mixture containing either the eluted FLAG-FBW7α-WT or the ΔF mutant for 60 minutes at 37◦C. Reaction mixture lacking the UbE1, an E1 ubiquitin-activating enzyme, or UbcH3, an E2 ubiquiting-conjugating enzyme, served as negative control for the reaction. SOX9 ubiquitylation was assessed following elution of the protein from the immobilized beads under denaturing condition as described in Materials Methods. The set of blots shown is representative from 3 independent experiments.


Figure 3 ( B ) Immunoblotting of HA - SOX9 - WT protein following expression of various FLAG - FBW7α constructs including wild type ( WT ) , or those with R465A or R479A mutation in Daoy . The cells were transfected with equal amount of HA - SOX9 - WT and FLAG - FBW7α and the whole cell lysates were collected 24 hours following the transfection . GAPDH proteins were shown to indicate total protein loading for each sample . The set of blots shown is representative from 2 independent experiments . ( C ) Co - immunoprecipitation of HA - SOX9 with either wild type ( WT ) or R465A - FLAG - FBW7α . Unlike the wild type protein , FLAG - FBW7α containing arginine 465 mutation did not significantly co - immunoprecipitate with HA - SOX9 ( IP with anti HA antibody ) and vice versa ( IP with anti FLAG antibody ) . The expressions of HA - SOX9 and FLAG - FBW7α - WT or R465A mutant were assessed in the whole cell extract ( WCE ) of the transfected HEK293 with GAPDH protein used to indicate total protein loading . ( D ) Comparison of FBW7 mRNA expression across the medulloblastoma subgroups ( WNT , SHH , Group 3 , and 4 ) in a cohort containing 423 medulloblastoma cases . Expression of FBW7 in both normal fetal ( n = 5 ) and adult cerebellum ( n = 13 ) were used for comparison against the medulloblastoma . ( E ) Overall survival analysis of medulloblastoma patient based upon FBW7 expression level . Analysis was performed on 383 out of the 423 cases from which the survival data were available . Cohort was divided into high - ( blue line ) and low - FBW7 ( red line ) expressing subgroup using FBW7 median expression as group classifier . Statistical analysis was determined by using a LogRank test . ( F ) Correlation analysis between SOX9 protein / mRNA and FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples . SOX9 protein level was scored following immunohistochemistry staining , while SOX9 and FBW7 mRNAs expression by the RNA Scope as described in Materials Methods . A modest negative correlation was determined by Kendall ' s correlation test ( τ = - 0 . 223 , p0 . 001 ) between SOX9 protein / mRNA and FBW7 expression . Affiliation of samples with medulloblastoma subgroups was indicated by different colored circle ( blue = WNT , red = SHH , yellow = Group 3 , green = Group 4 , grey = Undetermined ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Immunoblotting of HA-SOX9-WT protein following expression of various FLAG-FBW7α constructs including wild type (WT), or those with R465A or R479A mutation in Daoy. The cells were transfected with equal amount of HA-SOX9-WT and FLAG-FBW7α and the whole cell lysates were collected 24 hours following the transfection. GAPDH proteins were shown to indicate total protein loading for each sample. The set of blots shown is representative from 2 independent experiments.(C) Co-immunoprecipitation of HA-SOX9 with either wild type (WT) or R465A- FLAG-FBW7α. Unlike the wild type protein, FLAG-FBW7α containing arginine 465 mutation did not significantly co-immunoprecipitate with HA-SOX9 (IP with anti HA antibody) and vice versa (IP with anti FLAG antibody). The expressions of HA-SOX9 and FLAG- FBW7α- WT or R465A mutant were assessed in the whole cell extract (WCE) of the transfected HEK293 with GAPDH protein used to indicate total protein loading.(D) Comparison of FBW7 mRNA expression across the medulloblastoma subgroups (WNT, SHH, Group 3, and 4) in a cohort containing 423 medulloblastoma cases. Expression of FBW7 in both normal fetal (n= 5) and adult cerebellum (n = 13) were used for comparison against the medulloblastoma.(E) Overall survival analysis of medulloblastoma patient based upon FBW7 expression level. Analysis was performed on 383 out of the 423 cases from which the survival data were available. Cohort was divided into high- (blue line) and low- FBW7 (red line) expressing subgroup using FBW7 median expression as group classifier. Statistical analysis was determined by using a LogRank test.(F) Correlation analysis between SOX9 protein/mRNA and FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples. SOX9 protein level was scored following immunohistochemistry staining, while SOX9 and FBW7 mRNAs expression by the RNA Scope as described in Materials Methods. A modest negative correlation was determined by Kendall's correlation test (τ = - 0.223, p0.001) between SOX9 protein/mRNA and FBW7 expression. Affiliation of samples with medulloblastoma subgroups was indicated by different colored circle (blue=WNT, red=SHH, yellow=Group 3, green=Group 4, grey=Undetermined).


Figure 4 ( A ) Overall survival analysis of mice bearing Daoy cell line expressing SOX9 with doxycycline ( dox ) - inducible FBW7α ( Daoy - SOX9 / T - FBW7α ) . The dox - preconditioned or the control cells ( 105cells ) were orthotopically xenografted to the cerebellum of 6 - weeks old Athymic Nude - Foxn1nu mice , which were continuously fed with either the dox - containing chow ( red line ) or the regular chow food ( green line ) , respectively . Micexenografted with dox - preconditioned Daoy - SOX9 / T - FBW7α and fed continuously with dox - containing chow ( red line ) survived longer ( p = 0 . 0285 ) than the control group ( green line ) ( B ) Representative of Haemotoxylin Eosin ( Hamp ; amp ; E ) staining of medulloblastoma tumors arising from the orthotopic xenograft experiment described in ( A ) . Both the control ( no dox - treated ) and the dox - treated mice developed primary intracerebellar tumors ( green and red arrows , respectively ) . SOX9 - expressing Daoy tumors disseminated to the spinal cord ( 3 / 4 ; green asterisks ) , while the group continuously receiving dox - containing chow showed no metastatic spread ( 0 / 4 ) . ( C ) Representative bright field images of migrating Daoy - SOX9 / T - FBW7α in the absence or presence of Dox . Daoy - SOX9 / T - FBW7α cells were either maintain in regular culture or pre - conditioned with dox prior to experiments . The cells ( 2 x 105cells / insert ) were seeded and let to migrate across 5 μm filter pore toward the laminin - coated trans - well for 4 hours prior to fixation and staining with crystal violet . Histogram quantification of cell migration were performed by measuring the crystal violet absorbance of the stained - migrating cells and presented as mean + standard deviation from 2 independent experiments each containing 2 technical replicates . ( D ) Representative H & amp ; amp ; E staining of primary ( arrow ) and disseminated tumors ( asterisk ) arising from orthotopic xenograft of parental and SOX9 - expressing medulloblastoma initiating cells ( MIC and MIC - Sox9 , respectively ) . Quantification of primary and metastatic tumors arising from both of MIC ( n = 16 ) and MIC - SOX9 ( n = 8 ) is shown in the histogram ( p = 0 . 0000136 ) . ( E ) Quantitative comparison of MIC and MIC - SOX9 cells to migrate across 5 μm filter pore toward the laminin - coated trans - well for 4 hours . Quantification of cell migration were performed as described in ( C ) and presented as mean + standard error mean from 6 replicates . ( F ) Transwell migration of SOX9 - expressing MIC with dox - inducible FBW7α or dominant negative ∆ F FBW7α . The cells were either maintain in regular culture or pre - conditioned with dox prior to assessment of in vitro transwell migration as described in ( C ) . Data are mean + standard deviation from 2 independent experiments each containing 2 technical replicates . ( G ) Representative bright field images of ( F ) ( H ) Correlation between clinical metastatic staging and SOX9 protein across the TMA of 142 cases of human medulloblastoma . The SOX9 protein levels were determined by IHC in Figure 3F . Tumors with SOX9 protein level frequently presented ( p = 0 . 038 , Fisher ' s exact test , ) with the M3 metastatic staging , spread to the spinal cord ( red bars ) . Analysis was carried out by comparing patients with high SOX9 protein rank ( Rank 5 - 7 ) versus patients with low SOX9 protein rank ( Rank 1 - 3 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Overall survival analysis of mice bearing Daoy cell line expressing SOX9 with doxycycline (dox) -inducible FBW7α ( Daoy-SOX9/T-FBW7α). The dox-preconditioned or the control cells (105cells) were orthotopically xenografted to the cerebellum of 6-weeks old Athymic Nude-Foxn1nu mice, which were continuously fed with either the dox-containing chow (red line) or the regular chow food (green line), respectively. Micexenografted with dox-preconditioned Daoy-SOX9/T-FBW7α and fed continuously with dox-containing chow (red line) survived longer (p= 0.0285) than the control group (green line)(B) Representative of Haemotoxylin Eosin (Hamp;amp;E) staining of medulloblastoma tumors arising from the orthotopic xenograft experiment described in (A). Both the control (no dox-treated) and the dox-treated mice developed primary intracerebellar tumors (green and red arrows, respectively). SOX9-expressing Daoy tumors disseminated to the spinal cord (3/4; green asterisks), while the group continuously receiving dox-containing chow showed no metastatic spread (0/4).(C) Representative bright field images of migrating Daoy-SOX9/T-FBW7α in the absence or presence of Dox. Daoy-SOX9/T-FBW7α cells were either maintain in regular culture or pre-conditioned with dox prior to experiments. The cells (2 x 105cells / insert) were seeded and let to migrate across 5 μm filter pore toward the laminin-coated trans-well for 4 hours prior to fixation and staining with crystal violet. Histogram quantification of cell migration were performed by measuring the crystal violet absorbance of the stained-migrating cells and presented as mean + standard deviation from 2 independent experiments each containing 2 technical replicates.(D) Representative H&amp;E staining of primary (arrow) and disseminated tumors (asterisk) arising from orthotopic xenograft of parental and SOX9-expressing medulloblastoma initiating cells (MIC and MIC-Sox9, respectively). Quantification of primary and metastatic tumors arising from both of MIC (n=16) and MIC-SOX9 (n=8) is shown in the histogram (p=0.0000136).(E) Quantitative comparison of MIC and MIC-SOX9 cells to migrate across 5 μm filter pore toward the laminin-coated trans-well for 4 hours. Quantification of cell migration were performed as described in (C) and presented as mean + standard error mean from 6 replicates.(F) Transwell migration of SOX9-expressing MIC with dox-inducible FBW7α or dominant negative ∆F FBW7α. The cells were either maintain in regular culture or pre-conditioned with dox prior to assessment of in vitro transwell migration as described in (C). Data are mean + standard deviation from 2 independent experiments each containing 2 technical replicates.(G) Representative bright field images of (F)(H) Correlation between clinical metastatic staging and SOX9 protein across the TMA of 142 cases of human medulloblastoma. The SOX9 protein levels were determined by IHC in Figure 3F. Tumors with SOX9 protein level frequently presented (p=0.038, Fisher's exact test,) with the M3 metastatic staging, spread to the spinal cord (red bars). Analysis was carried out by comparing patients with high SOX9 protein rank (Rank 5-7) versus patients with low SOX9 protein rank (Rank 1-3).


Figure 5 ( A ) Scatter plot depicting the relationship between transcriptional changes measured as log2 FC between EV to SOX9 - WT ( x - axis ) and EV to SOX9 - T236 / T240A ( y - axis ) . Each dot represents one transcript , with colored dots indicating transcripts upregulated ( log2 FC > 0 . 585 ; p < 0 . 05 ; red dots ) or downregulated ( log2 FC < - 0 . 585 ; p < 0 . 05 ; blue dots ) in both comparisons . The transcript corresponding to the SOX9 gene is highlighted as a red filled dot . Indicated r - value indicates Spearman ' s rank correlation coefficient between the two FC profiles . ( B ) Box - and - Whisker plot displaying the sample specific expression of three FBW7 isoforms ; FBW7α ( NM _ 033632 ) , FBW7β ( NM _ 018315 ) and FBW7γ ( NM _ 001013415 ' ) . Each box represents the expression distribution over three replicates . ( D ) Heat map of the row - wise z - scores of 16 EMT hallmark genes in SOX9 - WT and - T236 / T240A samples . Heat map was generated using the GenePattern software ( Reich et al . , 2006 ) . ( E ) Quantitative PCR ( qPCR ) of SNAI2 ( SLUG ) , VIM and POU3F1 ( OCT6 ) ( normalized to GAPDH levels ) in MB002 cells stably transduced with EV ( set to 1 ) , SOX9 - WT and SOX9 - T236 / 240A . Cells were induced for 8 or 24 hours with doxycycline . ( F ) Western blot of SOX9 , HA - SOX9 , pSOX9 - T236 , SNAI2 ( SLUG ) , VIM and POU3F1 ( OCT6 ) in MB002 cells stably transduced with EV , SOX9 - WT and SOX9 - T236 / 240A . Cells were induced for 24 and 48 hours with doxycycline and GAPDH were used as a loading control . ( G ) Heat map of the row - wise z - scores of 14 pro - metastasis genes differentially expressed between SOX9 - WT and - T236 / T240A samples and previously identified as SOX9 target genes ( Kadaja et al . , 2014 , Larsimont et al . , 2015 , Oh et al . , 2014 ) . Heat map was generated using the GenePattern software ( Reich et al . , 2006 ) .

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Figure 5(A) Scatter plot depicting the relationship between transcriptional changes measured as log2 FC between EV to SOX9-WT (x-axis) and EV to SOX9-T236/T240A (y-axis). Each dot represents one transcript, with colored dots indicating transcripts upregulated (log2 FC>0.585; p<0.05; red dots) or downregulated (log2 FC<-0.585; p<0.05; blue dots) in both comparisons. The transcript corresponding to the SOX9 gene is highlighted as a red filled dot. Indicated r-value indicates Spearman's rank correlation coefficient between the two FC profiles.(B) Box-and-Whisker plot displaying the sample specific expression of three FBW7 isoforms; FBW7α (NM_033632), FBW7β (NM_018315) and FBW7γ(NM_001013415'). Each box represents the expression distribution over three replicates.(D) Heat map of the row-wise z-scores of 16 EMT hallmark genes in SOX9-WT and -T236/T240A samples. Heat map was generated using the GenePattern software (Reich et al., 2006).(E) Quantitative PCR (qPCR) of SNAI2 (SLUG), VIM and POU3F1 (OCT6) (normalized to GAPDH levels) in MB002 cells stably transduced with EV (set to 1), SOX9-WT and SOX9-T236/240A. Cells were induced for 8 or 24 hours with doxycycline.(F) Western blot of SOX9, HA-SOX9, pSOX9-T236, SNAI2 (SLUG), VIM and POU3F1 (OCT6) in MB002 cells stably transduced with EV, SOX9-WT and SOX9-T236/240A. Cells were induced for 24 and 48 hours with doxycycline and GAPDH were used as a loading control.(G) Heat map of the row-wise z-scores of 14 pro-metastasis genes differentially expressed between SOX9-WT and -T236/T240A samples and previously identified as SOX9 target genes (Kadaja et al., 2014, Larsimont et al., 2015, Oh et al., 2014). Heat map was generated using the GenePattern software (Reich et al., 2006).


Figure 6 ( A ) Cisplatin dose response curves of ( i ) MB002 , ( ii ) Daoy , and ( iii ) MIC in the absence ( EV ) or presence of SOX9 by Alamar blue . Cells were pre - conditioned with doxycycline to induce expression of SOX9 ( or EV ) prior to treatment with increasing concentrations of cisplatin . The IC50 were calculated following 5 ( MB002 and MIC ) or 3 days ( Daoy ) of treatment . Data are mean + standard deviation from 3 independent repeats , each containing 5 technical replicates . ( B ) Cisplatin dose response curves of SOX9 - expressing ( i ) Daoy and ( ii ) MIC in the absence or presence of FBW7α . Experiments and data analysis were performed as described in ( A ) ( C ) Overall survival analysis of mice bearing Daoy or Daoy - expressing dox - inducible SOX9 treated with cisplatin . The dox - preconditioned cells ( 105 cells ) were orthotopically xenografted to Nude - Foxn1nu mice and left for 1 week to prior to being treated with vehicle control or cisplatin ( 2mg / kg ) intraperitoneally for every other day for a total of 6 doses . ( D ) Heat map of the row - wise z - scores of 11 genes associated with cisplatin resistance in MB002 expressing Sox9 - WT or Sox9 - T236 / T240A . Heat map was generated using the GenePattern software . ( E ) Quantitative analysis of ATP7A , DUSP2 , and TTK mRNAs in MB002 following expression of SOX9 - WT or SOX9 - T236 / 240A . Total RNA were collected 24 hours following doxycycline treatment , from which cDNA were generated for qPCR . Data are mean mRNA level ( normalized to B2M transcript ) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2 - way ANOVA with Bonferroni ' s post - test . ( F ) Time course western blotting of HA - SOX9 , ATP7A , DUSP2 , ERK1 / 2 pThr202 / Tyr204 and total ERK1 / 2 in MB002 cells following doxycycline induction of either EV , SOX9 - WT or SOX9 - T236 / 240A . GAPDH was used as a loading control .

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Figure 6(A) Cisplatin dose response curves of (i) MB002, (ii) Daoy, and (iii) MIC in the absence (EV) or presence of SOX9 by Alamar blue. Cells were pre-conditioned with doxycycline to induce expression of SOX9 (or EV) prior to treatment with increasing concentrations of cisplatin. The IC50 were calculated following 5 (MB002 and MIC) or 3 days (Daoy) of treatment. Data are mean + standard deviation from 3 independent repeats, each containing 5 technical replicates.(B) Cisplatin dose response curves of SOX9-expressing (i) Daoy and (ii) MIC in the absence or presence of FBW7α. Experiments and data analysis were performed as described in (A)(C) Overall survival analysis of mice bearing Daoy or Daoy-expressing dox-inducible SOX9 treated with cisplatin. The dox-preconditioned cells (105 cells) were orthotopically xenografted to Nude-Foxn1nu mice and left for 1 week to prior to being treated with vehicle control or cisplatin (2mg/kg) intraperitoneally for every other day for a total of 6 doses.(D) Heat map of the row-wise z-scores of 11 genes associated with cisplatin resistance in MB002 expressing Sox9-WT or Sox9-T236/T240A. Heat map was generated using the GenePattern software.(E) Quantitative analysis of ATP7A, DUSP2, and TTK mRNAs in MB002 following expression of SOX9-WT or SOX9-T236/240A. Total RNA were collected 24 hours following doxycycline treatment, from which cDNA were generated for qPCR. Data are mean mRNA level (normalized to B2M transcript) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2-way ANOVA with Bonferroni's post-test.(F) Time course western blotting of HA-SOX9, ATP7A, DUSP2, ERK1/2 pThr202/Tyr204 and total ERK1/2 in MB002 cells following doxycycline induction of either EV, SOX9-WT or SOX9-T236/240A. GAPDH was used as a loading control.


Figure 7 ( A ) Western blotting of pT236 - and total SOX9 following treatment of Daoy with a panel of inhibitors targeting PI3K / AKT / mTOR pathway . The whole cell lysates were collected for gel electrophoresis following 6 hours treatment with 1 μM of AZD5363 , 8186 , or 2014 . The set of blots shown is a representative from 3 independent repeats . ( B ) Western blotting of endogenous SOX9 protein turnover in the presence of PI3K / mTOR dual inhibitor , AZD2014 . Daoy were transfected with either siScr or siFBW7 for 48 hours prior to further treatment with 1 µM AZD2014 . The cycloheximide chase of SOX9 protein turnover was examined 2 hours following the inhibitor treatment . Immunobloting of Cyclin E protein was used to indicate the efficacy of siFBW7 transfection . Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ . The set of blots shown is representative from 3 independent experiments . ( C ) Quantification of resazurin - based Daoy and MB002 cell viability following treatment with Cisplatin ( blue bars ) , AZD2014 ( red bars ) , or their combination ( green bars ) . Daoy were treated for 72 hours , while MB002 were subjected to 5 day course of treatment . The cellviability were calculated relative to the vehicle - treated cells . The synthetic lethality combination index ( CI ) for each treatment was calculated from the mean of 3 independent experiments using the Compusyn software . Error bars indicate standard deviation . ( D ) Time course western blotting of endogenous SOX9 and apoptotic markers including cleaved PARP and caspase 3 following MB002 treatment with cisplatin , AZD2014 , or their combination . β - actin were used as a loading control . The set of blots shown is representative from 3 independent experiments . ( E ) Western blotting profiling of MB002 expressing EV , SOX9 - WT , or SOX9 - T236 / 240A following 48 hours treatment with cisplatin , AZD2014 , and their combination . The set of blots shown is representative from 3 independent experiments . ( F ) Quantification of cell viability of MB002 expressing EV , SOX9 - WT , or SOX9 - T236 / 240A following 5 days treatment with either of 10 μM cisplatin ( black bars ) , 0 . 5 µM AZD2014 ( blue bars ) , or their combination ( red bars ) . Data are mean + standard deviation from 3 independent experiments , from which statistical significance were analysed by 2 - way ANOVA multiple comparisons with Bonferroni ' s post - test . ( G ) Immunoblots of ATP7A and DUSP2 protein following doxycycline treatment of Daoy - SOX9 / T - FBW7α . The cells were either untreated or incubated with 5 µg / mL doxycycline for 72 hours prior to being harvested for gel electrophoresis . The set of blots shown is representative from 2 independent experiments .

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Figure 7(A) Western blotting of pT236- and total SOX9 following treatment of Daoy with a panel of inhibitors targeting PI3K/AKT/mTOR pathway. The whole cell lysates were collected for gel electrophoresis following 6 hours treatment with 1 μM of AZD5363, 8186, or 2014. The set of blots shown is a representative from 3 independent repeats.(B) Western blotting of endogenous SOX9 protein turnover in the presence of PI3K/mTOR dual inhibitor, AZD2014. Daoy were transfected with either siScr or siFBW7 for 48 hours prior to further treatment with 1 µM AZD2014. The cycloheximide chase of SOX9 protein turnover was examined 2 hours following the inhibitor treatment. Immunobloting of Cyclin E protein was used to indicate the efficacy of siFBW7 transfection. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.(C) Quantification of resazurin-based Daoy and MB002 cell viability following treatment with Cisplatin (blue bars), AZD2014 (red bars), or their combination (green bars). Daoy were treated for 72 hours, while MB002 were subjected to 5 day course of treatment. The cellviability were calculated relative to the vehicle-treated cells. The synthetic lethality combination index (CI) for each treatment was calculated from the mean of 3 independent experiments using the Compusyn software. Error bars indicate standard deviation.(D) Time course western blotting of endogenous SOX9 and apoptotic markers including cleaved PARP and caspase 3 following MB002 treatment with cisplatin, AZD2014, or their combination. β-actin were used as a loading control. The set of blots shown is representative from 3 independent experiments.(E) Western blotting profiling of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 48 hours treatment with cisplatin, AZD2014, and their combination. The set of blots shown is representative from 3 independent experiments.(F) Quantification of cell viability of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 5 days treatment with either of 10 μM cisplatin (black bars), 0.5 µM AZD2014 (blue bars), or their combination (red bars). Data are mean + standard deviation from 3 independent experiments, from which statistical significance were analysed by 2-way ANOVA multiple comparisons with Bonferroni's post-test.(G) Immunoblots of ATP7A and DUSP2 protein following doxycycline treatment of Daoy-SOX9/T-FBW7α. The cells were either untreated or incubated with 5 µg/mL doxycycline for 72 hours prior to being harvested for gel electrophoresis. The set of blots shown is representative from 2 independent experiments.


Figure 1Heatmap of the differentially expressed cholesterol - related genes between normal brain tissues ( n = 28 ) and glioblastomas ( n = 217 ) from the Rembrandt dataset . Gene expression values are z - transformed and colored red for high expression and blue for low expression , as indicated in the scale bar . Volcano plot showing the fold - change ( log2 ) in cholesterol - related gene levels based on GBM vs normal brain tissue . Data were obtained from the Rembrandt dataset . Representative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas ( n = 64 ) . Scale bar = 30 μm . Quantification of CYP46A1 IHC staining in normal brain ( n = 6 ) and different pathological grades of gliomas ( n = 58 ) . Representative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case . Scale bar = 30 μm . CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification . Data are shown as the mean ± the standard error of the mean ( SEM ; n = 667 ) . * * * P < 0 . 0001 . Statistical significance was determined by one ‐ way ANOVA . CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset . Shown are means and SEM ( n = 245 ) . * * * P < 0 . 0001 . Statistical significance was determined by one ‐ way ANOVA . Kaplan - Meier analysis for patient OS and PFS based on high vs low expression of CYP46A1 in LGG and GBM . Data were obtained from the CGGA dataset . P - values were obtained from the log - rank test .

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Figure 1Heatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 28) and glioblastomas (n = 217) from the Rembrandt dataset. Gene expression values are z-transformed and colored red for high expression and blue for low expression, as indicated in the scale bar. Volcano plot showing the fold-change (log2) in cholesterol-related gene levels based on GBM vs normal brain tissue. Data were obtained from the Rembrandt dataset.Representative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas (n = 64). Scale bar = 30 μm. Quantification of CYP46A1 IHC staining in normal brain (n = 6) and different pathological grades of gliomas (n = 58).Representative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case. Scale bar = 30 μm.CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification. Data are shown as the mean ± the standard error of the mean (SEM; n = 667). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset. Shown are means and SEM (n = 245). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.Kaplan-Meier analysis for patient OS and PFS based on high vs low expression of CYP46A1 in LGG and GBM. Data were obtained from the CGGA dataset. P-values were obtained from the log-rank test.


Figure 2qRT - PCR analysis of CYP46A1 mRNA levels in LN229 and GBM # P3 cells transfected with lentivirus expressing CYP46A1 ( lenti - CYP46A1 ) or control sequence ( lenti - Ctrl ) . GAPDH was used as an internal control . Shown are means and SEM ( n = 3 ) . LN229 : * * * P < 0 . 0001 ; GBM # P3 : * * * P < 0 . 0001 . Statistical significance was determined by two - sided Student ' s t ‐ test . Western blot analysis to confirm CYP46A1 overexpression in GBM cells . Growth curves for GBM cells in vitro infected with lenti - Ctrl or lenti - CYP46A1 derived from trypan blue staining . Shown are means and SEM ( n = 3 ) . LN229 : * * P = 0 . 003 ; GBM # P3 : * * P = 0 . 002 . Statistical significance was determined by two - sided Student ' s t ‐ test . Colony forming assay for GBM cells infected with lenti - Ctrl or lenti - CYP46A1 . Shown are means and SEM ( n = 3 ) . LN229 : * P = 0 . 013 ; GBM # P3 : * P = 0 . 012 . Statistical significance was determined by two - sided Student ' s t ‐ test . Representative H & E staining of orthotopic tumours derived from GBM cells infected with lenti - Ctrl or lenti - CYP46A1 . Scale bar = 2 mm . Kaplan - Meier survival curve of tumour bearing mice injected with GBM cells infected with lenti - Ctrl or lenti - CYP46A1 ( n = 5 per group ) . A log - rank test was used to assess statistical significance . IHC for CYP46A1 , PCNA and cleaved caspase - 3 protein levels in the intracranial tumours . Scale bar = 100 μm .

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Figure 2qRT-PCR analysis of CYP46A1 mRNA levels in LN229 and GBM#P3 cells transfected with lentivirus expressing CYP46A1 (lenti-CYP46A1) or control sequence (lenti-Ctrl). GAPDH was used as an internal control. Shown are means and SEM (n = 3). LN229: ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by two-sided Student's t‐test.Western blot analysis to confirm CYP46A1 overexpression in GBM cells.Growth curves for GBM cells in vitro infected with lenti-Ctrl or lenti-CYP46A1 derived from trypan blue staining. Shown are means and SEM (n = 3). LN229: **P = 0.003; GBM#P3: **P = 0.002. Statistical significance was determined by two-sided Student's t‐test.Colony forming assay for GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Shown are means and SEM (n = 3). LN229: *P = 0.013; GBM#P3: *P = 0.012. Statistical significance was determined by two-sided Student's t‐test.Representative H&E staining of orthotopic tumours derived from GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Scale bar = 2 mm.Kaplan-Meier survival curve of tumour bearing mice injected with GBM cells infected with lenti-Ctrl or lenti-CYP46A1 (n = 5 per group). A log-rank test was used to assess statistical significance.IHC for CYP46A1, PCNA and cleaved caspase-3 protein levels in the intracranial tumours. Scale bar = 100 μm.


Figure 324OHC levels in spent media from LN229 ( left ) and GBM # P3 ( right ) cells transduced with lenti - Ctrl or lenti - CYP46A1 measured using targeted LC - MS / MSIC50 curves for 24OHC in LN229 and GBM # P3 cells using the CCK - 8 assay . Flow cytometry to detect annexin V - FITC and PI staining to determine the percentage of LN229 and GBM # P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h . Data are shown as the mean ± SEM ( n = 3 ) . Colony forming ability of GBM cell lines treated with 24OHC ( 0 - 20 μM ) for 14 days . Western blot analysis of the apoptosis marker c - PARP , c - caspase 3 and PCNA in lysates ( 20 µg ) from GBM cells treated with 24OHC ( 0 - 20 μM ) for 72 h . Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC ( 0 - 20 μM ) . Scale bar = 100 μm . Graphic representation of the quantification of tumoursphere formation . Data are shown as the mean ± SEM ( n = 3 ) . GBM # P3 : * P = 0 . 0192 , * * * P = 0 . 0006 , * * * P = 0 . 0002 ; GBM # BG7 : * * P = 0 . 0017 , * * * P < 0 . 0001 , * * * P < 0 . 0001 ; GBM # BG5 : * * P = 0 . 0027 , * * * P = 0 . 0001 , * * * P < 0 . 0001 .

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Figure 324OHC levels in spent media from LN229 (left) and GBM#P3 (right) cells transduced with lenti-Ctrl or lenti-CYP46A1 measured using targeted LC-MS/MSIC50 curves for 24OHC in LN229 and GBM#P3 cells using the CCK-8 assay.Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3).Colony forming ability of GBM cell lines treated with 24OHC (0 - 20 μM) for 14 days.Western blot analysis of the apoptosis marker c-PARP, c-caspase 3 and PCNA in lysates (20 µg) from GBM cells treated with 24OHC (0 - 20 μM) for 72 h.Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation. Data are shown as the mean ± SEM (n = 3). GBM#P3: *P = 0.0192, ***P = 0.0006, ***P = 0.0002; GBM#BG7: **P = 0.0017, ***P < 0.0001, ***P < 0.0001; GBM#BG5: **P = 0.0027, ***P = 0.0001, ***P < 0.0001.


Figure 4Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen ™ Amplex ™ Red Cholesterol Assay Kit and normalized to total protein . Shown are means and SEM ( n = 3 ) . LN229 : * * P = 0 . 002 ; GBM # P3 : * * P = 0 . 011 . Statistical significance was determined by two - sided Student ' s t ‐ test . Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC ( 0 - 20 μM ) for 72 h . Scale bar = 30 μm . Growth curves for LN229 cells treated with DMSO , 0 . 5 μg / mL cholesterol or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol using trypan blue staining . Flow cytometry to detect annexin V - FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol for 72 h . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * P = 0 . 0021 , * * P = 0 . 0046 ; GBM # P3 : * * P = 0 . 0012 , * P = 0 . 0193 . Western blot analysis of c - PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol for 72 h . Tumoursphere formation assays for GBM # P3 and GBM # BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol . Scale bar = 100 μm . Data are shown as the mean ± SEM ( n = 3 ) . GBM # P3 : * * P = 0 . 0032 , * P = 0 . 031 ; GBM # BG7 : * * * P = 0 . 0008 , * P = 0 . 0242 . CCK - 8 assay to determine relative cell survival in LN229 or GBM # P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * P = 0 . 0051 , * * * P = 0 . 0002 ; GBM # P3 : * * P = 0 . 0018 , * * * P < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen™ Amplex™ Red Cholesterol Assay Kit and normalized to total protein. Shown are means and SEM (n = 3). LN229: **P = 0.002; GBM#P3: **P = 0.011. Statistical significance was determined by two-sided Student's t‐test.Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC (0 - 20 μM) for 72 h. Scale bar = 30 μm.Growth curves for LN229 cells treated with DMSO, 0.5 μg/mL cholesterol or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol using trypan blue staining.Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0021, **P = 0.0046; GBM#P3: **P = 0.0012, *P = 0.0193.Western blot analysis of c-PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h.Tumoursphere formation assays for GBM#P3 and GBM#BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol. Scale bar = 100 μm. Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0032, *P = 0.031; GBM#BG7: ***P = 0.0008, *P = 0.0242.CCK-8 assay to determine relative cell survival in LN229 or GBM#P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0051, ***P = 0.0002; GBM#P3: **P = 0.0018, ***P < 0.0001.


Figure 5Heatmap of differentially expressed transcripts in RNA - seq data from GBM # P3 cells treated with DMSO or 20 μM 24OHC for 72 h . Gene expression data was mean z - transformed for display , and colored red for high expression and blue for low expression . GSEA plot showing normalized enrichment scores ( NES ) for cholesterol homeostasis , positive regulation of cholesterol efflux , SREBP targets and stem cell proliferation signatures using RNA - seq data from 24OHC and DMSO treated cells . Volcano plot highlighting up - regulated ( red ) and down - regulated ( blue ) genes based on RNA - seq data from GBM # P3 cells after 24OHC treatment . LXR targets , SREBP targets and stemness transcription factors are specifically included . qRT - PCR analysis of 24OHC - regulated genes in GBM # P3 cells after treatment with 20 μM 24OHC for 72 h . GAPDH was used as an internal control . Data are shown as the mean ± SEM ( n = 3 ) . Cell viability assay performed on GBM # P3 cells after 3 days of treatment with DMSO or 5 - 10 μM LXR - 623 in the presence or absence of 10 μM 24OHC . Data are shown as the mean ± SEM ( n = 3 ) . Western blot analysis of cholesterol metabolism - related proteins in LN229 or GBM # P3 after treatment with 24OHC ( 0 - 20 μM ) . Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC ( 0 - 40 μM ) as assessed by a trypan blue test . Data are shown as the mean ± SEM ( n = 3 ) . Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days . Representative images of filipin staining in LN229 ( up ) or LN18 ( down ) cells treated with different concentration of fatostatin ( 0 - 20 μM ) . Scale bar = 30 μm .

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Figure 5Heatmap of differentially expressed transcripts in RNA-seq data from GBM#P3 cells treated with DMSO or 20 μM 24OHC for 72 h. Gene expression data was mean z-transformed for display, and colored red for high expression and blue for low expression.GSEA plot showing normalized enrichment scores (NES) for cholesterol homeostasis, positive regulation of cholesterol efflux, SREBP targets and stem cell proliferation signatures using RNA-seq data from 24OHC and DMSO treated cells.Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included.qRT-PCR analysis of 24OHC-regulated genes in GBM#P3 cells after treatment with 20 μM 24OHC for 72 h. GAPDH was used as an internal control. Data are shown as the mean ± SEM (n = 3).Cell viability assay performed on GBM#P3 cells after 3 days of treatment with DMSO or 5 -10 μM LXR-623 in the presence or absence of 10 μM 24OHC. Data are shown as the mean ± SEM (n = 3).Western blot analysis of cholesterol metabolism-related proteins in LN229 or GBM#P3 after treatment with 24OHC (0 - 20 μM).Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC (0 - 40 μM) as assessed by a trypan blue test. Data are shown as the mean ± SEM (n = 3).Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days.Representative images of filipin staining in LN229 (up) or LN18 (down) cells treated with different concentration of fatostatin (0 - 20 μM). Scale bar = 30 μm.


Figure 6Intracellular concentrations of cholesterol in GBM cells after treatment with 20 μM EFV or DMSO and normalized to total protein . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * P = 0 . 013 ; GBM # P3 : * * P = 0 . 0023 . Filipin staining of GBM cells after treatment with EFV ( 0 - 20 μM ) . Scale bar = 30 μm . IC50 curves for EFV in GBM cells determined using OD readings ( 450nm ) from the CCK - 8 assay . 24OHC levels in spent media from LN229 ( left ) or GBM # P3 ( right ) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC - MS / MS . Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV ( 0 - 20 μM ) . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * P = 0 . 0056 , * * * P < 0 . 0001 ; LN18 : * * * P < 0 . 0001 . Flow cytometry to detect annexin V - FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV ( 0 - 20 μM ) for 72 h . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * * P = 0 . 0006 , * * * P < 0 . 0001 ; GBM # P3 : * * * P < 0 . 0001 . Tumoursphere formation assays for GSCs treated with different concentrations of EFV ( 0 - 20 μM ) . Scale bar = 100 μm . Graphic representation of the quantification of tumoursphere formation ( right ) . Data are shown as the mean ± SEM ( n = 3 ) . GBM # P3 : * * P = 0 . 0011 , * * * P = 0 . 0003 , * * * P < 0 . 0001 ; GBM # BG7 : * * * P < 0 . 0001 ; GBM # BG5 : * * P = 0 . 0037 , * * P = 0 . 0015 , * * * P = 0 . 0002 . Western blot analysis of cholesterol homeostasis related proteins ( LDLR , ABCA1 , P - SREBP1 and N - SREBP1 ) , c - PARP , PCNA and SOX2 in GBM cells treated with EFV ( 0 - 20 μM ) for 72 h . The survival curves of tumour - bearing mice implanted with LN229 or GBM # P3 cells after EFV or PBS treatment ( n = 5 per group ) . A log - rank test was used to assess statistical significance . Bioluminescent images and the corresponding quantification of tumours in mice implanted with GBM # P3 cells at days 7 ( n = 5 per group , P = 0 . 58 ) and 21 ( n = 5 per group , P = 0 . 005 ) . Statistical significance was determined by two - sided Student ' s t ‐ test . Body weight of tumour - bearing mice after 3 weeks of EFV or PBS treatment . Data are shown as the mean ± SEM . Statistical significance was determined by two - sided Student ' s t ‐ test .

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Figure 6Intracellular concentrations of cholesterol in GBM cells after treatment with 20 μM EFV or DMSO and normalized to total protein. Data are shown as the mean ± SEM (n = 3). LN229: *P = 0.013; GBM#P3: **P = 0.0023.Filipin staining of GBM cells after treatment with EFV (0 - 20 μM). Scale bar = 30 μm.IC50 curves for EFV in GBM cells determined using OD readings (450nm) from the CCK-8 assay.24OHC levels in spent media from LN229 (left) or GBM#P3 (right) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC-MS/MS.Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV (0 - 20 μM). Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0056, ***P < 0.0001; LN18: ***P < 0.0001.Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV (0 - 20 μM) for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0006, ***P < 0.0001; GBM#P3: ***P < 0.0001.Tumoursphere formation assays for GSCs treated with different concentrations of EFV (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation (right). Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0011, ***P = 0.0003, *** P < 0.0001; GBM#BG7: ***P < 0.0001; GBM#BG5: **P = 0.0037, **P = 0.0015, ***P = 0.0002.Western blot analysis of cholesterol homeostasis related proteins (LDLR, ABCA1, P-SREBP1 and N-SREBP1), c-PARP, PCNA and SOX2 in GBM cells treated with EFV (0 - 20 μM) for 72 h.The survival curves of tumour-bearing mice implanted with LN229 or GBM#P3 cells after EFV or PBS treatment (n = 5 per group). A log-rank test was used to assess statistical significance.Bioluminescent images and the corresponding quantification of tumours in mice implanted with GBM#P3 cells at days 7 (n = 5 per group, P = 0.58) and 21 (n = 5 per group, P = 0.005). Statistical significance was determined by two-sided Student's t‐test. Body weight of tumour-bearing mice after 3 weeks of EFV or PBS treatment. Data are shown as the mean ± SEM. Statistical significance was determined by two-sided Student's t‐test.


Figure 1A - L HeLa cells stably expressing FLAG - tagged UNC13D ( F - UNC13D ) ( A and B ) , UNC13D stable knock - down ( KD ) ( C , E , G , I and K ) or knock out ( KO ) ( D , F , H , J and L ) HeLa cells , and their control cells were infected with VACV ( A , C and D ) , HSV ( E and F ) , or SeV ( K and L ) , or they were transfected with plasmid DNA ( pDNA ) ( 1 μg / mL ) ( B , G - J ) for the indicated time , before immunoblotting with anti - Flag , - UNC13D , - p - TBK1 , - p - IRF3 , - STING , - p - P65 , - p - IκB - α , - actin or - tubulin antibody . UNC13D stable knock - down ( KD ) HeLa cells and their control cells were transfected with plasmid DNA ( 1 μg / mL ) for the indicated time . UNC13D , IFN - β and IL6 mRNA levels were measured by qPCR ( M - O ) . UNC13D stable knock - down ( KD ) HeLa cells and their control cells were transfected with plasmid DNA ( 1 μg / mL ) infected with SeV ( Q ) for the indicated time . The secretion of IFN - β was measured by bioassay ( P and Q ) . R , S UNC13D knock - out ( KO ) HeLa cells and their control cells were infected with GFP - HSV for 18 h or 24 h . GFP expression was visualized ( left ) and the green fluorescence intensity was determined by ImageJ .

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Figure 1A - L HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (A and B), UNC13D stable knock-down (KD) (C, E, G, I and K) or knock out (KO) ( D, F, H, J and L) HeLa cells, and their control cells were infected with VACV (A, C and D), HSV (E and F), or SeV (K and L), or they were transfected with plasmid DNA (pDNA) (1 μg/mL) (B, G-J) for the indicated time, before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -p-P65, -p-IκB-α, -actin or -tubulin antibody.UNC13D stable knock-down (KD) HeLa cells and their control cells were transfected with plasmid DNA (1 μg/mL) for the indicated time. UNC13D, IFN-β and IL6 mRNA levels were measured by qPCR (M-O).UNC13D stable knock-down (KD) HeLa cells and their control cells were transfected with plasmid DNA (1 μg/mL) infected with SeV (Q) for the indicated time. The secretion of IFN-β was measured by bioassay (P and Q).R, S UNC13D knock-out (KO) HeLa cells and their control cells were infected with GFP-HSV for 18 h or 24 h. GFP expression was visualized (left) and the green fluorescence intensity was determined by ImageJ.


Figure 2A HEK293T cells were transfected with the indicated plasmids ( 5 μg each ) for 24 h . Whole cell lysates ( WCL ) were examined , and cell lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies , followed by immunoblotting ( IB ) with anti - HA and - Flag antibody . B - E HeLa cells stably expressing FLAG - tagged UNC13D ( F - UNC13D ) ( B ) , UNC13D stable knock - down ( KD ) ( D ) or knock - out ( KO ) ( C and E ) HeLa cells , and their control cells were stimulated with cGAMP ( 100 nM ) for 30 min ( B and C ) or for the indicated time ( D and E ) before immunoblotting with anti - Flag , - UNC13D , - p - TBK1 , - p - IRF3 , - STING , - GAPDH , - actin or - tubulin antibody . F - H HEK293T cells were transfected with the indicated plasmids ( 5 μg each ) for 24 h ( F and G ) . UNC13D knock - out ( KO ) and control HeLa cells were stimulated with cGAMP ( 100 nM ) for 30 min ( H ) . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with anti - HA antibodies ( F and G ) , IgG and anti - STING antibodies ( H ) , followed by immunoblotting ( IB ) with anti - HA , - Flag antibody or the indicated antibodies . UNC13D knock - out ( KO ) HeLa cells and HeLa cells stably expressing FLAG - tagged UNC13D ( F - UNC13D ) and control HeLa cells were treated with cGAMP ( 100 nM ) for 30 min or SeV for 12 h . Confocal microscopy was used to determine the localization of endogenous STING ( red ) , ERGIC marker ERGIC - 53 ( green ) and nuclei ( blue ) . UNC13D knock - out ( KO ) HeLa cells and HeLa cells stably expressing FLAG - tagged UNC13D ( F - UNC13D ) and control HeLa cells were treated with cGAMP ( 100 nM ) for 30 min or SeV for 12 h . The co - localization of STING and ERGIC was analyzed using ImageJ ( J ) . K , L UNC13D stable knock - down ( KD ) HeLa cells and control HeLa cells were treated with cGAMP ( 100 nM ) for 30 min or SeV for 12 h . The aggregation of endogenous STING ( red ) was examined via confocal microscopy ( K ) . Scale bar : 25 μm . The red fluorescence intensity was determined by ImageJ ( L ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (IB) with anti-HA and -Flag antibody.B - E HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (B), UNC13D stable knock-down (KD) (D) or knock-out (KO) (C and E) HeLa cells, and their control cells were stimulated with cGAMP (100 nM) for 30 min (B and C) or for the indicated time (D and E) before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -GAPDH, -actin or -tubulin antibody.F- H HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h (F and G). UNC13D knock-out (KO) and control HeLa cells were stimulated with cGAMP (100 nM) for 30 min (H). Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies (F and G), IgG and anti-STING antibodies (H), followed by immunoblotting (IB) with anti-HA, -Flag antibody or the indicated antibodies.UNC13D knock-out (KO) HeLa cells and HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. Confocal microscopy was used to determine the localization of endogenous STING (red), ERGIC marker ERGIC-53 (green) and nuclei (blue).UNC13D knock-out (KO) HeLa cells and HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. The co-localization of STING and ERGIC was analyzed using ImageJ (J).K, L UNC13D stable knock-down (KD) HeLa cells and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. The aggregation of endogenous STING (red) was examined via confocal microscopy (K). Scale bar: 25 μm. The red fluorescence intensity was determined by ImageJ (L).


Figure 3A UNC13D knock - out HeLa cells stably expressing FLAG - tagged UNC13D were stimulated with cGAMP ( 100 nM ) or left unstimulated for the indicated time . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with IgG and anti - Flag antibodies , followed by immunoblotting ( IB ) with the indicated antibodies . UNC13D knock - out HeLa cells stably expressing FLAG - tagged UNC13D were pretreated with BFA ( 20 μM ) or left untreated for 2 h , followed by stimulation with cGAMP ( 100 nM ) ( or left unstimulated ) for 30 min . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with IgG and anti - Flag antibodies , followed by immunoblotting ( IB ) with the indicated antibodies ( E ) . F HEK293T cells were transfected for 24h with IFN - β - luciferase reporter plasmid , pRL - TK and the indicated expression plasmids , followed by dual - luciferase reporter assays . The expression of plasmids was examined by western blotting with anti - Flag , - HA or - tubulin antibody . H Wild - type HeLa cells were pretreated with BFA ( 20 μM ) or left untreated for 2 h before stimulation with plasmid DNA ( 1 μg / ml ) for 18h or cGAMP ( 100 nM ) for 30 min ( or left unstimulated ) . Cell lysates were separated by native ( top ) or SDS ( bottom ) PAGE and analyzed by immunoblotting ( IB ) with anti - p - TBK1 , - p - IRF3 , - STING or - tubulin antibody . UNC13D stable knock - down ( KD ) HeLa cells and control cells were transfected with plasmid DNA ( pDNA ) ( 1 μg / mL ) for the indicated time . Cell lysates were separated by native ( top ) or SDS ( bottom ) PAGE and analyzed by immunoblotting ( IB ) with anti - UNC13D , - p - TBK1 , - p - IRF3 , - STING or - tubulin antibody . UNC13D knock - out ( KO ) ( J ) HeLa cells and control cells were transfected with plasmid DNA ( pDNA ) ( 1 μg / mL ) for the indicated time . Cell lysates were separated by native ( top ) or SDS ( bottom ) PAGE and analyzed by immunoblotting ( IB ) with anti - UNC13D , - p - TBK1 , - p - IRF3 , - STING or - tubulin antibody . K , L HEK293T cells were transfected with HA - tagged UNC13D , VN / VC - STING or vector for 24 h , followed by stimulation with cGAMP ( 100 nM ) ( or left unstimulated ) for 30 min . Nucleus marker DAPI ( blue ) , VN / VC - STING ( green ) and HA - UNC13D ( red ) were examined via confocal microscopy ( K ) . The green fluorescence intensity was determined by ImageJ ( L ) . n = 5 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A UNC13D knock-out HeLa cells stably expressing FLAG-tagged UNC13D were stimulated with cGAMP (100 nM) or left unstimulated for the indicated time. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with IgG and anti-Flag antibodies, followed by immunoblotting (IB) with the indicated antibodies.UNC13D knock-out HeLa cells stably expressing FLAG-tagged UNC13D were pretreated with BFA (20 μM) or left untreated for 2 h, followed by stimulation with cGAMP (100 nM) (or left unstimulated) for 30 min. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with IgG and anti-Flag antibodies, followed by immunoblotting (IB) with the indicated antibodies (E).F HEK293T cells were transfected for 24h with IFN-β-luciferase reporter plasmid, pRL-TK and the indicated expression plasmids, followed by dual-luciferase reporter assays. The expression of plasmids was examined by western blotting with anti-Flag, -HA or -tubulin antibody.H Wild-type HeLa cells were pretreated with BFA (20 μM) or left untreated for 2 h before stimulation with plasmid DNA (1 μg/ml) for 18h or cGAMP (100 nM) for 30 min (or left unstimulated). Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-p-TBK1, -p-IRF3, -STING or -tubulin antibody.UNC13D stable knock-down (KD) HeLa cells and control cells were transfected with plasmid DNA (pDNA) (1 μg/mL) for the indicated time. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -p-TBK1, -p-IRF3, -STING or -tubulin antibody.UNC13D knock-out (KO) (J) HeLa cells and control cells were transfected with plasmid DNA (pDNA) (1 μg/mL) for the indicated time. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -p-TBK1, -p-IRF3, -STING or -tubulin antibody.K, L HEK293T cells were transfected with HA-tagged UNC13D, VN/VC-STING or vector for 24 h, followed by stimulation with cGAMP (100 nM) (or left unstimulated) for 30 min. Nucleus marker DAPI (blue), VN/VC-STING (green) and HA-UNC13D (red) were examined via confocal microscopy (K). The green fluorescence intensity was determined by ImageJ (L). n = 5 biological replicates.


Figure 4HEK293T cells were transfected with the indicated plasmids ( 5 μg each ) for 24 h . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with the indicated antibodies , followed by immunoblotting ( IB ) with anti - HA or Flag antibody . HEK293T cells were transfected with the indicated plasmids ( 5 μg each ) for 24 h . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with the indicated antibodies , followed by immunoblotting ( IB ) with anti - HA or Flag antibody . HEK293T cells were transfected with the indicated plasmids ( 5 μg each ) for 24 h . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with the indicated antibodies , followed by immunoblotting ( IB ) with anti - HA or Flag antibody . HEK293T cells were transfected with the indicated plasmids ( 5 μg each ) for 24 h . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with the indicated antibodies , followed by immunoblotting ( IB ) with anti - HA or Flag antibody . HEK293T cells were transfected with the indicated plasmids ( 5 μg each ) for 24 h . Whole cell lysates ( WCL ) were examined , and cell lysates were immunoprecipitated ( IP ) with the indicated antibodies , followed by immunoblotting ( IB ) with anti - HA or Flag antibody .

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Figure 4HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with the indicated antibodies, followed by immunoblotting (IB) with anti-HA or Flag antibody.HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with the indicated antibodies, followed by immunoblotting (IB) with anti-HA or Flag antibody.HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with the indicated antibodies, followed by immunoblotting (IB) with anti-HA or Flag antibody.HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with the indicated antibodies, followed by immunoblotting (IB) with anti-HA or Flag antibody.HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with the indicated antibodies, followed by immunoblotting (IB) with anti-HA or Flag antibody.


Figure 5D UNC13D knock - out ( KO ) HeLa cells , UNC13D knock - out HeLa cells stably expressing FLAG - tagged wild - type UNC13D ( UNC13D RE ) and FHL3 mutation ( FHL3 mut ) , and control cells were stimulated with cGAMP ( 100 nM ) ( or left unstimulated ) for 30 min . Cell lysates were separated by native ( top ) or SDS ( bottom ) PAGE and analyzed by immunoblotting ( IB ) with anti - UNC13D , - Flag , - p - IRF3 , - STING or - tubulin antibody .

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Figure 5D UNC13D knock-out (KO) HeLa cells, UNC13D knock-out HeLa cells stably expressing FLAG-tagged wild-type UNC13D (UNC13D RE) and FHL3 mutation (FHL3 mut), and control cells were stimulated with cGAMP (100 nM) (or left unstimulated) for 30 min. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -Flag, -p-IRF3, -STING or -tubulin antibody.


Figure 1Western blots for exercise - associated phosphorylated proteins in ( top ) contracted ( + ) or sham ( - ) rat muscle and ( bottom ) muscle from mice that underwent a treadmill running protocol ( + ) or remained at rest ( - ) . Total AMPK and 14 - 3 - 3 were used as loading controls . Volcano plots of phosphopeptide median Log2 fold - change ( contraction / sham surgery ) plotted against the - Log10 p - value in rat contraction ( left ) or mouse running ( right ) . Colored dots represent significantly - regulated phosphopeptides ( p < 0 . 05 , n = 5 , moderated t - test ) . Gray dots represent phosphopeptides that were detected but not significantly altered in exercise .

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Figure 1Western blots for exercise-associated phosphorylated proteins in (top) contracted (+) or sham (-) rat muscle and (bottom) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total AMPK and 14-3-3 were used as loading controls.Volcano plots of phosphopeptide median Log2 fold-change (contraction/sham surgery) plotted against the -Log10 p-value in rat contraction (left) or mouse running (right). Colored dots represent significantly-regulated phosphopeptides (p < 0.05, n=5, moderated t-test). Gray dots represent phosphopeptides that were detected but not significantly altered in exercise.


Figure 3Western blots for STIM1 phosphorylated at S257 or S521 in ( left ) contracted ( + ) or sham ( - ) rat muscle and ( right ) muscle from mice that underwent a treadmill running protocol ( + ) or remained at rest ( - ) . Total STIM1 and 14 - 3 - 3 were used as loading controls . 14 - 3 - 3 data for mouse running was re - used from Figure 1B . Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in L6 rat myoblasts with siRNA - mediated knockdown of endogenous STIM1 ( siSTIM1 ) or transfected with scramble control siRNA ( Control ) . Cells were treated with the AMPK activator A - 769662 ( + ) or vehicle ( - ) for 30 min . Total STIM1 was used to demonstrate STIM1 knockdown efficiency . Total ACC and 14 - 3 - 3 were used as loading controls . Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in wild type ( WT ) or AMPK β1 / β2 knockout ( AMPKβ KO ) mouse endothelial fibroblasts were isolated and treated with A - 769662 ( + ) or vehicle ( - ) for 30 min . AMPKβ was used to demonstrate knockout , and AMPKɑ was used to demonstrate specificity for AMPKβ . Total ACC and STIM1 were used as loading controls . Western blots for total ACC and STIM1 and phosphorylated STIM1 ( S257 and S521 ) and ACC ( S79 ) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP - STIM1 . Representative Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 and S521 ) in response to a 15 min treatment with increasing doses of thapsigargin ( . 01 , . 1 , 1 or 10 µM ) or . 5 mM AICAR . DMSO was used as a vehicle control . 14 - 3 - 3 was used as a loading control . n = 3 independent experiments . Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in wild type ( WT ) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin ( + ) or vehicle ( - ) for 15 min .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Western blots for STIM1 phosphorylated at S257 or S521 in (left) contracted (+) or sham (-) rat muscle and (right) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total STIM1 and 14-3-3 were used as loading controls. 14-3-3 data for mouse running was re-used from Figure 1B.Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in L6 rat myoblasts with siRNA-mediated knockdown of endogenous STIM1 (siSTIM1) or transfected with scramble control siRNA (Control). Cells were treated with the AMPK activator A-769662 (+) or vehicle (-) for 30 min. Total STIM1 was used to demonstrate STIM1 knockdown efficiency. Total ACC and 14-3-3 were used as loading controls.Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK β1/β2 knockout (AMPKβ KO) mouse endothelial fibroblasts were isolated and treated with A-769662 (+) or vehicle (-) for 30 min. AMPKβ was used to demonstrate knockout, and AMPKɑ was used to demonstrate specificity for AMPKβ. Total ACC and STIM1 were used as loading controls.Western blots for total ACC and STIM1 and phosphorylated STIM1 (S257 and S521) and ACC (S79) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP-STIM1.Representative Western blots for phosphorylated ACC (S79) and STIM1 (S257 and S521) in response to a 15 min treatment with increasing doses of thapsigargin (.01, .1, 1 or 10 µM) or .5 mM AICAR. DMSO was used as a vehicle control. 14-3-3 was used as a loading control. n=3 independent experiments.Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin (+) or vehicle (-) for 15 min.


Figure 4Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . The mClover2 : tagRFP - T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker . Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . STIM1 OASF expressed in live L6 myoblasts before ( - ) or after ( + ) treatment with A - 769662 . Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . L6 myoblasts with S257 mutated to A or E .

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Figure 4Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. The mClover2:tagRFP-T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker.Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. STIM1 OASF expressed in live L6 myoblasts before (-) or after (+) treatment with A-769662.Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. L6 myoblasts with S257 mutated to A or E.


Figure 5L6 myoblasts were transfected with the genetic cytosolic Ca2 + sensor GCaMP6s . Cells were also transfected with non - targeted siRNA ( Control ) or siRNA directed at endogenous STIM1 ( siSTIM1 ) . Some cells with endogenous STIM1 knockdown were also transfected with full - length wild type ( WT ) or mutant ( S257A , S257E or L251S ) STIM1 - mRuby3 . Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts , L6 myoblasts were transfected with the genetic cytosolic Ca2 + sensor GCaMP6s . Cells were also transfected with non - targeted siRNA ( Control ) or siRNA directed at endogenous STIM1 ( siSTIM1 ) . Some cells with endogenous STIM1 knockdown were also transfected with full - length wild type ( WT ) or mutant ( S257A , S257E or L251S ) STIM1 - mRuby3 . Ca2 + levels over time ( fold of 0 mM Ca2 + + EGTA basal ) and ( C ) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2 + and treated with 2 µM thapsigargin during the indicated times .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts,L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Ca2+ levels over time (fold of 0 mM Ca2+ + EGTA basal) and (C) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2+ and treated with 2 µM thapsigargin during the indicated times.