Figure 4D Immunofluorescence microscopy ( top panel ) of AKT2 - , Flag - IRF3 - or Flag - IRF3 - T207A - overexpressed MEF cells after poly ( A : T ) stimulation for 6 hours . Flag ( IRF3 , red ) and Hoechst ( nuclei , blue ) . Immunoassay of nuclear - cytoplasm extractions ( bottom panel ) from HEK293T - IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours . GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively . Bar , 20 μm . Data information : * Data are representative of three independent experimentsF Survival rates ( left panel ) until 72 hours and H & E staining ( right panel ) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge . The arrows indicated the VSV - infected eye and skeletal muscle in zebrafish larvae . Bars , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent experimentsG qRT - PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later . Every dot represents three zebrafish embryos . Horizontal vertical square bracket shows the statistical analysis of comparison with ' PBS mock ' , the rest shows the comparison with ' PBS VSV ' . PBS mock , n = 15 ; PBS VSV , n = 9 ; IRF3 VSV , n = 16 ; IRF3 - T207A VSV , n = 11 ; AKT2 VSV , n = 14 ; AKT2 - T309A / S474A VSV , n = 9 ; IRF3 + AKT2 VSV , n = 9 ; IRF3 + AKT2 - T309A / S474A VSV , n = 13 ; IRF3 - T207A + AKT2 VSV , n = 10 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a one - way ANOVA test Data are representative of three independent experiments Error bars mean ± SD ) . H The survival rates of over - expressed zebrafish larvae as long as 72 hours after the VSV challenge . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 4D Immunofluorescence microscopy (top panel) of AKT2-, Flag-IRF3- or Flag-IRF3-T207A-overexpressed MEF cells after poly(A:T) stimulation for 6 hours. Flag (IRF3, red) and Hoechst (nuclei, blue). Immunoassay of nuclear-cytoplasm extractions (bottom panel) from HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours. GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively. Bar, 20 μm. Data information: * Data are representative of three independent experimentsF Survival rates (left panel) until 72 hours and H&E staining (right panel) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experimentsG qRT-PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later. Every dot represents three zebrafish embryos. Horizontal vertical square bracket shows the statistical analysis of comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=15; PBS VSV, n=9; IRF3 VSV, n=16; IRF3-T207A VSV, n=11; AKT2 VSV, n=14; AKT2-T309A/S474A VSV, n=9; IRF3 + AKT2 VSV, n=9; IRF3 + AKT2-T309A/S474A VSV, n=13; IRF3-T207A + AKT2 VSV, n=10. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are representative of three independent experiments Error bars mean ± SD).H The survival rates of over-expressed zebrafish larvae as long as 72 hours after the VSV challenge. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments


Figure 5B Survival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 hours ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent experiments respectivelyE WT and Akt2 KO mice were injected with VSV for 24 hours ( 1 × 106 PFU / g , i . v . ) . Representative images of the VSV infected cells ( GFP ) were collected by fluorescence microscope ( left panel ) and the VSV copies ( WT , n = 6 ; Akt2 KO n = 7 ) were measured by qRT - PCR ( right panel ) in the liver of mice . Bar , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a unpaired t - test Data are representative of three independent experiments respectively in which the different groups of mice were sacrificed and quantified for indicated experimental purposes Error bars mean ± SD ) . Bone marrow cells from WT and Akt2 KO mice ( CD45 . 2 ) were adoptively transferred to lethally irradiated CD45 . 1 mice and infected with VSV . representative images were analyzed Data information : * Data are representative of three independent experiments respectivelyI Survival rates of WT mice that were intraperitoneal injected with CCT128930 ( 20 mg / kg ) twice every other day before VSV infection ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent experiments

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 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 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 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

Figure 5B Survival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 hours (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectivelyE WT and Akt2 KO mice were injected with VSV for 24 hours (1 × 106 PFU/g, i.v.). Representative images of the VSV infected cells (GFP) were collected by fluorescence microscope (left panel) and the VSV copies (WT, n=6; Akt2 KO n=7) were measured by qRT-PCR (right panel) in the liver of mice. Bar, 100 μm. Data information: *P < 0.05, **P < 0.01; using a unpaired t-test Data are representative of three independent experiments respectively in which the different groups of mice were sacrificed and quantified for indicated experimental purposes Error bars mean ± SD).Bone marrow cells from WT and Akt2 KO mice (CD45.2) were adoptively transferred to lethally irradiated CD45.1 mice and infected with VSV. representative images were analyzed Data information: * Data are representative of three independent experiments respectivelyI Survival rates of WT mice that were intraperitoneal injected with CCT128930 (20 mg/kg) twice every other day before VSV infection (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively


Figure 6immunoblot analysis of IRF3 , LaminB1 and GAPDH in the PIC nuclear and cytoplasmic fractions ( C ) from WT and Akt2 KO mice treated with TMPD for 2 weeks . GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively . Data information : * Data are representative of three independent experimentsH & E - staining ( E left panel ) and immunofluorescence of IgG deposits ( E right panel ) in the kidney sections were collected from WT and Akt2 KO bone marrow - reconstituted CD45 . 1 mice Bar , 50 μm . Data information : * Data are representative of three independent experimentsJ . qRT - PCR analysis of the relative mRNA expression levels of AKT2 ( left panel ) and IFNA ( middle panel ) or IFNβ1 ( right panel ) in CD14 positive monocytes isolated from SLE patients ( n = 9 ) or healthy donors ( n = 9 ) . Data information : * P < 0 . 05 , * * P < 0 . 01 and * * * * P < 0 . 0001 ; using unpaired t - test or correlation analyses Data are pooled from different individuals Error bars mean ± SD ) . K Immunofluorescence of IRF3 ( Red ) , AKT2 ( Green ) , DAPI ( Blue , nucleus ) in CD14 + cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with Image J software . Cells ( n = 39 ) were collected 3 patients . Bar , 2 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 and * * * * P < 0 . 0001 ; using correlation analyses Data are pooled from different individuals or representative of three independent

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Figure 6immunoblot analysis of IRF3, LaminB1 and GAPDH in the PIC nuclear and cytoplasmic fractions (C) from WT and Akt2 KO mice treated with TMPD for 2 weeks. GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of three independent experimentsH&E-staining (E left panel) and immunofluorescence of IgG deposits (E right panel) in the kidney sections were collected from WT and Akt2 KO bone marrow-reconstituted CD45.1 mice Bar, 50 μm. Data information: * Data are representative of three independent experimentsJ. qRT-PCR analysis of the relative mRNA expression levels of AKT2 (left panel) and IFNA (middle panel) or IFNβ1 (right panel) in CD14 positive monocytes isolated from SLE patients (n=9) or healthy donors (n=9). Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using unpaired t-test or correlation analyses Data are pooled from different individuals Error bars mean ± SD).K Immunofluorescence of IRF3 (Red), AKT2 (Green), DAPI (Blue, nucleus) in CD14+ cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with Image J software. Cells (n=39) were collected 3 patients. Bar, 2 μm. Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using correlation analyses Data are pooled from different individuals or representative of three independent experiments


Figure 1Representative images of GINs cultures showing GABA and TUJ1 expression . Scale bar = 100 μm . The proportion of GABA + cells of TUJ1 + neurons from both CTRL and sMDD iPSCs derived GINs at day 35 . ( CTRL , n = 120 fields were counted from 5 cell lines ; sMDD , n = 134 fields were counted from 6 cell lines ) . Representative images and schematic morphometric Sholl analysis from CTRL and sMDD GINs at day 35 . Sholl intersection number of CTRL and sMDD GINs at day 35 ( Each red box is from 5 CTRL cell lines , n = 25 independently differentiations from 5 cell lines , each point represents mean value of neurons from five independent experiments , n = 226 neurons from CTRL groups . Each blue box is from 6 sMDD cell lines , n = 30 differentiations independently from 6 cell lines , each point represents mean value of neurons from five independent experiments , n = 260 neurons from sMDD groups ) . Two - way ANOVA for different distance from soma , * * * * p ＜ 0 . 0001 for 10 , 30 and 50 μm . Representative images of GINs from CTRL and sMDD at day 65 . Quantification of primary branch numbers of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65 , n ≥ 11 , Nested t - test , * * p = 0 . 0016 for CTRL versus sMDD . Quantification of longest neurite length of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65 , n ≥ 11 , Nested t - test , * * p = 0 . 0075 for CTRL versus sMDD . Representative images of GINs subtype calretinin ( CR ) expression . The proportion of CR + cells of TUJ1 + neurons from both CTRL and sMDD iPSCs derived GINs at days 35 , 40 and 50 , respectively . ( Red bar from 5 CTRL cell lines , blue bar from 6 sMDD cell lines ) . n = 5 in each cell line . Two - way ANOVA for timepoints , Representative images of GINs subtype somatostatin ( SST ) expression co - staining with mature neuron marker MAP2 in CTRL and sMDD GINs at day 65 . The proportion of SST + neurons of MAP2 + cells from CTRL and sMDD iPSCs derived GINs at day 65 . n = 43 in CTRL groups and n = 37 in sMDD groups .

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Figure 1Representative images of GINs cultures showing GABA and TUJ1 expression. Scale bar = 100 μm.The proportion of GABA+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at day 35. (CTRL, n=120 fields were counted from 5 cell lines; sMDD, n=134 fields were counted from 6 cell lines).Representative images and schematic morphometric Sholl analysis from CTRL and sMDD GINs at day 35. Sholl intersection number of CTRL and sMDD GINs at day 35 (Each red box is from 5 CTRL cell lines, n=25 independently differentiations from 5 cell lines, each point represents mean value of neurons from five independent experiments, n=226 neurons from CTRL groups. Each blue box is from 6 sMDD cell lines, n=30 differentiations independently from 6 cell lines, each point represents mean value of neurons from five independent experiments, n=260 neurons from sMDD groups). Two-way ANOVA for different distance from soma, ****p＜0.0001 for 10, 30 and 50 μm.Representative images of GINs from CTRL and sMDD at day 65.Quantification of primary branch numbers of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65, n≥11, Nested t-test, **p=0.0016 for CTRL versus sMDD. Quantification of longest neurite length of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65, n≥11, Nested t-test, **p=0.0075 for CTRL versus sMDD.Representative images of GINs subtype calretinin (CR) expression. The proportion of CR+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at days 35, 40 and 50, respectively. (Red bar from 5 CTRL cell lines, blue bar from 6 sMDD cell lines). n=5 in each cell line. Two-way ANOVA for timepoints,Representative images of GINs subtype somatostatin (SST) expression co-staining with mature neuron marker MAP2 in CTRL and sMDD GINs at day 65. The proportion of SST+ neurons of MAP2+ cells from CTRL and sMDD iPSCs derived GINs at day 65. n=43 in CTRL groups and n=37 in sMDD groups.


Figure 2Representative electrophysiological traces of AP at a holding potential of - 70 mV from GINs in sMDD ( blue ) and CTRL ( red ) groups . Average number of APs evoked during 500ms stepwise depolarization ( CTRL , n = 35 neurons from 3 lines ; sMDD , n = 55 neurons from 3 lines ) . D - F . Amplitude and half width of the first AP generated in response to a 10 - pA injection ( CTRL , n = 35 neurons from 3 lines ; sMDD , n = 55 neurons from 3 lines ) . Nested t - test , * * p ＜ 0 . 01 ; * * * p ＜ 0 . 001 . Mean ratio ± SEM . G . Traces of Na + / K + currents recorded from GINs in sMDD and CTRL groups . Quantification showed average of peak values of Na + currents ( CTRL , n = 46 neurons from 3 lines ; sMDD , n = 38 neurons from 3 lines ) . Nested t - test , * * p ＜ 0 . 01 . H . Average fast potassium currents of GINs in sMDD and CTRL groups ( CTRL , n = 43 neurons from 3 lines ; sMDD , n = 36 neurons from 3 lines ) .

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 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 2Representative electrophysiological traces of AP at a holding potential of -70 mV from GINs in sMDD (blue) and CTRL (red) groups. Average number of APs evoked during 500ms stepwise depolarization (CTRL, n=35 neurons from 3 lines; sMDD, n=55 neurons from 3 lines).D-F. Amplitude and half width of the first AP generated in response to a 10-pA injection (CTRL, n=35 neurons from 3 lines; sMDD, n=55 neurons from 3 lines). Nested t-test, **p＜0.01; ***p＜0.001. Mean ratio ± SEM.G. Traces of Na+/K+ currents recorded from GINs in sMDD and CTRL groups. Quantification showed average of peak values of Na+ currents (CTRL, n=46 neurons from 3 lines; sMDD, n=38 neurons from 3 lines). Nested t-test, **p＜0.01. H. Average fast potassium currents of GINs in sMDD and CTRL groups (CTRL, n=43 neurons from 3 lines; sMDD, n=36 neurons from 3 lines).


Figure 3Representative images of calcium imaging in CTRL and sMDD GINs at each timepoint . Scale bar = 20 μm . The representative trajectory of average intensity changes over time from CTRL ( red line ) and sMDD ( blue line ) neurons in response to 67 mM KCL . CTRL , n = 6 neurons ; sMDD , n = 6 neurons . Traces are from a representative experiment ( the whole quantification result is shown in D ) . Quantification of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per cell line ( n = 155 neurons derived from 5 CTRL cell lines , n = 198 neurons derived from 6 sMDD cell lines ) . Nested t - test , * * p = 0 . 0022 for CTRL versus sMDD . Immunostaining for the GABA interneuron marker GAD67 , neural progenitor marker SOX2 and ventral prosencephalic progenitor marker NKX2 . 1 at day 30 in CTRL and sMDD ventral forebrain organoids . Representative images of calcium imaging in CTRL and sMDD ventral forebrain organoids at different states . Quantification of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per cell line ( n = 156 neurons derived from 4 CTRL cell lines , n = 88 neurons derived from 4 sMDD cell lines ) . Nested t - test , * * * p = 0 . 0005 for CTRL versus sMDD .

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Figure 3Representative images of calcium imaging in CTRL and sMDD GINs at each timepoint. Scale bar = 20 μm.The representative trajectory of average intensity changes over time from CTRL (red line) and sMDD (blue line) neurons in response to 67 mM KCL. CTRL, n=6 neurons; sMDD, n=6 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=155 neurons derived from 5 CTRL cell lines, n=198 neurons derived from 6 sMDD cell lines). Nested t-test, **p=0.0022 for CTRL versus sMDD.Immunostaining for the GABA interneuron marker GAD67, neural progenitor marker SOX2 and ventral prosencephalic progenitor marker NKX2.1 at day 30 in CTRL and sMDD ventral forebrain organoids.Representative images of calcium imaging in CTRL and sMDD ventral forebrain organoids at different states.Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=156 neurons derived from 4 CTRL cell lines, n=88 neurons derived from 4 sMDD cell lines). Nested t-test, ***p=0.0005 for CTRL versus sMDD.


Figure 4Bar plot showing the number of genes associated with known mental illnesses , based on PsyGeNET . " Total " presents all of the genes related to a given disease . " 100 % association " presents the genes only positively correlated with the disease . " 100 % no association " presents the genes only negatively correlated with the disease . " Both " means genes that both positive and negative results were found . AUD , BD , CUD , MDD and SCZ represent alcohol use disorder , bipolar disorder , cocaine use disorder , major depressive disorder and schizophrenia , respectively . Heatmap illustrating the expression level of DEGs enriched in selected Reactome pathways , gene ontology ( GO ) terms , and Kyoto Encyclopedia of Genes and Genomes ( KEGG ) pathways , which is normalized by row . Calcium imaging was performed on GFP + neurons in the knockdown group . The representative image shows the rest state of GFP + cells and the state of cells after KCl stimulation . The pseudo - color showed Rhod - 4 calcium fluorescent dye , and the selected cells are GFP + neurons . The representative trajectory of average intensity changes over time in Vehicle virus and KD virus groups . CTRL , n = 10 neurons ; sMDD , n = 10 neurons . Traces are from a representative experiment ( the whole quantification result is shown in I ) . Quantification of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per virus group . n = 89 for Vehicle virus , n = 118 for KD virus , respectively from 4 batches . T - test , * * * * 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 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 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

Figure 4Bar plot showing the number of genes associated with known mental illnesses, based on PsyGeNET. "Total" presents all of the genes related to a given disease. "100% association" presents the genes only positively correlated with the disease. "100% no association" presents the genes only negatively correlated with the disease. "Both" means genes that both positive and negative results were found. AUD, BD, CUD, MDD and SCZ represent alcohol use disorder, bipolar disorder, cocaine use disorder, major depressive disorder and schizophrenia, respectively.Heatmap illustrating the expression level of DEGs enriched in selected Reactome pathways, gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which is normalized by row.Calcium imaging was performed on GFP+ neurons in the knockdown group. The representative image shows the rest state of GFP+ cells and the state of cells after KCl stimulation. The pseudo-color showed Rhod-4 calcium fluorescent dye, and the selected cells are GFP+ neurons.The representative trajectory of average intensity changes over time in Vehicle virus and KD virus groups. CTRL, n=10 neurons; sMDD, n=10 neurons. Traces are from a representative experiment (the whole quantification result is shown in I). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per virus group. n=89 for Vehicle virus, n=118 for KD virus, respectively from 4 batches. T-test, ****p<0.0001.


Figure 5Calcium imaging was performed on GFP + neurons in the overexpression group . The representative image shows the rest state of GFP + cells and the state of cells after KCl stimulation . The pseudo - color showed Rhod - 4 calcium fluorescent dye , and the selected cells are GFP + neurons . The representative trajectory of average intensity changes over time in Vehicle virus and OE virus groups . CTRL , n = 10 neurons ; sMDD , n = 10 neurons . Traces are from a representative experiment ( the whole quantification result is shown in D ) . Quantification of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per virus group . n = 166 for Vehicle virus , n = 81 for OE virus , respectively from 3 batches . Representative images of calcium imaging in CTRL ( from RC01001 - C ) and sMDD ( from SA004 ) GINs at resting and stimulus state with different treatments . Quantification of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown with different treatments including BDNF , Trzd , PNU , Ro at concentrations of 1 μM and 10 μM . T - test , * * p = 0 . 0042 for sMDD + BDNF versus sMDD ; one - way ANOVA , * * * * p ＜ 0 . 0001 for sMDD + 10 μM Trzd versus sMDD . n ≥ 7 neurons from each group . Dot plots showing the expression level and percentage of genes selected from functional gene sets across CTRL , sMDD and sMDD with Trzd GINs clusters . Top : Violin plots showing the expression level of CAMK2N1 and CAMK2N2 in three groups . Bottom : Scatter plot showing the correlations of foldchanges according to the expression of genes encoding CaMK family proteins between CTRL GINs and sMDD GINs and between sMDD with Trzd GINs and sMDD GINs . A linear regression line is added to the plot based on these two variables . sMDD + Trzd vs . sMDD , n = 14 genes ; CTRL vs . sMDD , n = 14 genes .

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 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 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 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

Figure 5Calcium imaging was performed on GFP+ neurons in the overexpression group. The representative image shows the rest state of GFP+ cells and the state of cells after KCl stimulation. The pseudo-color showed Rhod-4 calcium fluorescent dye, and the selected cells are GFP+ neurons.The representative trajectory of average intensity changes over time in Vehicle virus and OE virus groups. CTRL, n=10 neurons; sMDD, n=10 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per virus group. n=166 for Vehicle virus, n=81 for OE virus, respectively from 3 batches.Representative images of calcium imaging in CTRL (from RC01001-C) and sMDD (from SA004) GINs at resting and stimulus state with different treatments.Quantification of peak [Ca2+] (Fmax-F0)/F0 shown with different treatments including BDNF, Trzd, PNU, Ro at concentrations of 1 μM and 10 μM. T-test, **p=0.0042 for sMDD+BDNF versus sMDD; one-way ANOVA, ****p＜0.0001 for sMDD+10 μM Trzd versus sMDD. n≥7 neurons from each group.Dot plots showing the expression level and percentage of genes selected from functional gene sets across CTRL, sMDD and sMDD with Trzd GINs clusters.Top: Violin plots showing the expression level of CAMK2N1 and CAMK2N2 in three groups. Bottom: Scatter plot showing the correlations of foldchanges according to the expression of genes encoding CaMK family proteins between CTRL GINs and sMDD GINs and between sMDD with Trzd GINs and sMDD GINs. A linear regression line is added to the plot based on these two variables. sMDD+Trzd vs. sMDD, n=14 genes; CTRL vs. sMDD, n=14 genes.


Figure 6Representative images of single neuron from CTRL , CTRL + Trzd , sMDD and sMDD + Trzd GINs . Representative line chart of Sholl intersection number over distance from soma in CTRL , CTRL + Trzd , sMDD and sMDD + Trzd GINs at day 45 . CTRL , n = 81 neurons from 5 cell lines ; CTRL + Trzd , n = 81 neurons from 5 cell lines ; sMDD , n = 73 neurons from 6 cell lines ; sMDD + Trzd , n = 71 neurons from 6 cell lines . Quantification of maximum intersections from CTRL , CTRL + Trzd , sMDD and sMDD + Trzd GINs via Sholl analysis . CTRL , n = 81 neurons from 5 cell lines ; CTRL + Trzd , n = 81 neurons from 5 cell lines ; sMDD , n = 73 neurons from 6 cell lines ; sMDD + Trzd , n = 71 neurons from 6 cell lines . Quantification of sum of intersections from CTRL , CTRL + Trzd , sMDD and sMDD + Trzd GINs via Sholl analysis . CTRL , n = 81 neurons from 5 cell lines ; CTRL + Trzd , n = 81 neurons from 5 cell lines ; sMDD , n = 73 neurons from 6 cell lines ; sMDD + Trzd , n = 71 neurons from 6 cell lines . One - way ANONA , * * * * p ＜ 0 . 0001 for CTRL versus sMDD ; * * * p = 0 . 0002 for sMDD versus sMDD + Trzd . The representative trajectory of average intensity changes over time from CTRL , CTRL + Trzd , sMDD and sMDD + Trzd GINs in response to 67 mM KCL . CTRL , n = 20 neurons ; CTRL + Trzd , n = 20 neurons ; sMDD , n = 20 neurons ; sMDD + Trzd , n = 21 neurons . Tracs are from a representative experiment ( the whole quantification result is shown in Fig EV5E ) . Quantification of peak [ Ca2 + ] ( Fmax - F0 ) / F0 shown per group , CTRL , CTRL + Trzd , sMDD and sMDD + Trzd all from 3 cell lines . T - test , * p = 0 . 0495 for CTRL versus sMDD , * * p = 0 . 0075 for sMDD versus sMDD + Trzd . Representative images of calcium imaging in sMDD and sMDD + Trzd ventral forebrain organoids at different states . Quantification of the effect of Trzd on peak [ Ca2 + ] ( Fmax - F0 ) / F0 in ventral forebrain organoids ( CTRL from IMR90 - 4 , n = 40 cells ; CTRL + Trzd from IMR90 - 4 , n = 58 cells ; sMDD from SA007 , n = 49 cells ; sMDD + Trzd from SA007 , n = 64 cells ) . Two - tailed test , * * * * p ＜ 0 . 0001 for CTRL versus sMDD ; * * * p = 0 . 0007 for sMDD versus sMDD + Trzd . Representative electrophysiological traces of AP at a holding potential of - 70 mV from GINs in sMDD and sMDD + Trzd groups . Average number of APs evoked during 500ms stepwise depolarization ( CTRL , n = 35 neurons from 3 lines ; CTRL + Trzd , n = 30 neurons from 3 lines ; sMDD , n = 55 neurons from 3 lines ; sMDD + Trzd , n = 44 neurons from 3 lines ) . Traces of Na + / K + currents were recorded from GINs in sMDD and sMDD + Trzd groups . Average peak values of Na + currents ( CTRL , n = 46 neurons from 3 lines ; CTRL + Trzd , n = 32 neurons ; sMDD , n = 38 neurons from 3 lines ; sMDD + Trzd , n = 36 neurons from 3 lines ) .

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

Figure 6Representative images of single neuron from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs.Representative line chart of Sholl intersection number over distance from soma in CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs at day 45. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines. Quantification of maximum intersections from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs via Sholl analysis. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines.Quantification of sum of intersections from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs via Sholl analysis. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines. One-way ANONA, ****p＜0.0001 for CTRL versus sMDD; ***p=0.0002 for sMDD versus sMDD+Trzd. The representative trajectory of average intensity changes over time from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs in response to 67 mM KCL. CTRL, n=20 neurons; CTRL+Trzd, n=20 neurons; sMDD, n=20 neurons; sMDD+Trzd, n=21 neurons. Tracs are from a representative experiment (the whole quantification result is shown in Fig EV5E).Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per group, CTRL, CTRL+Trzd, sMDD and sMDD+Trzd all from 3 cell lines. T-test, *p=0.0495 for CTRL versus sMDD, **p=0.0075 for sMDD versus sMDD+Trzd.Representative images of calcium imaging in sMDD and sMDD+Trzd ventral forebrain organoids at different states.Quantification of the effect of Trzd on peak [Ca2+] (Fmax-F0)/F0 in ventral forebrain organoids (CTRL from IMR90-4, n=40 cells; CTRL+Trzd from IMR90-4, n=58 cells; sMDD from SA007, n=49 cells; sMDD+Trzd from SA007, n=64 cells). Two-tailed test, ****p＜0.0001 for CTRL versus sMDD; ***p=0.0007 for sMDD versus sMDD+Trzd.Representative electrophysiological traces of AP at a holding potential of -70 mV from GINs in sMDD and sMDD+Trzd groups. Average number of APs evoked during 500ms stepwise depolarization (CTRL, n=35 neurons from 3 lines; CTRL+Trzd, n=30 neurons from 3 lines; sMDD, n=55 neurons from 3 lines; sMDD+Trzd, n=44 neurons from 3 lines).Traces of Na+/K+ currents were recorded from GINs in sMDD and sMDD+Trzd groups. Average peak values of Na+ currents (CTRL, n=46 neurons from 3 lines; CTRL+Trzd, n=32 neurons; sMDD, n=38 neurons from 3 lines; sMDD+Trzd, n=36 neurons from 3 lines).


Figure 1A , Volcano plot of the differentially modulated proteins in 22rv1 ( CRPC ) versus CWR22res ( HN ) tumours . Red and blue dots represent the proteins that are significantly up - and down - regulated , respectively ( p - value < 0 . 05 , FC = 1 . 5 ) . Data information : Data reproducibility : n = 3 tumours per group . B , Western blot analysis of THEM6 expression in CRPC ( 22rv1 and LNCaP AI ) and HN ( CWR22res and LNCaP ) prostate orthografts . VCL was used as a sample loading control . Data information : Data reproducibility : : n = 1 gel loaded with three prostate orthografts per condition . G , Tumour volume ( measured by ultrasound ) of CTL and THEM6 KO 22rv1 - derived orthografts developed in surgically castrated mice . H , Tumour volume of CTL and THEM6 KO LNCaP AI - derived xenografts developed in surgically castrated mice . I , Tumour volume ( measured by ultrasound ) of CTL and THEM6 KO CWR22res - derived orthografts . Orchidectomy was performed 3 - weeks after cell implantation . Data information : Data are presented as mean values + / - SD . Statistical analysis : G , H : two - tailed Mann - Whitney U test . I : Kruskal - Wallis test . Data reproducibility : G , I : n = 5 mice per group . Panel H : n = 7 mice per group .

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 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 O O O O O O O O O O O O O O O O O O O O O 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 1A, Volcano plot of the differentially modulated proteins in 22rv1 (CRPC) versus CWR22res (HN) tumours. Red and blue dots represent the proteins that are significantly up- and down-regulated, respectively (p-value < 0.05, FC = 1.5). Data information: Data reproducibility: n = 3 tumours per group.B, Western blot analysis of THEM6 expression in CRPC (22rv1 and LNCaP AI) and HN (CWR22res and LNCaP) prostate orthografts. VCL was used as a sample loading control. Data information: Data reproducibility: : n = 1 gel loaded with three prostate orthografts per condition.G, Tumour volume (measured by ultrasound) of CTL and THEM6 KO 22rv1-derived orthografts developed in surgically castrated mice. H, Tumour volume of CTL and THEM6 KO LNCaP AI-derived xenografts developed in surgically castrated mice. I, Tumour volume (measured by ultrasound) of CTL and THEM6 KO CWR22res-derived orthografts. Orchidectomy was performed 3-weeks after cell implantation. Data information: Data are presented as mean values +/- SD. Statistical analysis: G, H: two-tailed Mann-Whitney U test. I: Kruskal-Wallis test. Data reproducibility: G, I: n = 5 mice per group. Panel H: n = 7 mice per group.


Figure 2B - C , Changes in lipid content ( total amount ) observed in THEM6 KO CRPC cells when compared to CTL . Data information : Data are presented as mean values + / - SD . Statistical analysis : * p - value < 0 . 05 using a 1 - way ANOVA with a Dunnett ' s multiple comparisons test . Data reproducibility : n = 3 independent biological experiments . DAG : diacylglycerol ; TG : triglyceride ; PC : phosphatidylcholine ; PE : phosphatidylethanolamine ; PI : phosphatidylinositol ; LysoPC : lysophosphatidylcholine ; LysoPE : lysophosphatidylethanolamine ; Cer : Ceramide ; SM : sphingomyelin ; CE : Cholesteryl ester .

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Figure 2B-C, Changes in lipid content (total amount) observed in THEM6 KO CRPC cells when compared to CTL. Data information: Data are presented as mean values +/- SD. Statistical analysis : *p-value < 0.05 using a 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent biological experiments. DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester.


Figure 3C , Representative electron microscopy ( EM ) pictures of CTL and THEM6 KO 22rv1 cells taken at low ( left ) and high ( right ) magnification . Red arrows point towards abnormal ER structure . Scale bar represents 500 nm . I , Western blot analysis of THEM6 , AMFR , SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells . Data information : Data reproducibility : representative image from 2 independent biological experiments . J , Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing . Data information : Data reproducibility : representative image from 3 independent biological experiments .

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Figure 3C, Representative electron microscopy (EM) pictures of CTL and THEM6 KO 22rv1 cells taken at low (left) and high (right) magnification. Red arrows point towards abnormal ER structure. Scale bar represents 500 nm.I, Western blot analysis of THEM6, AMFR, SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells. Data information: Data reproducibility: representative image from 2 independent biological experiments.J, Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing. Data information: Data reproducibility: representative image from 3 independent biological experiments.


Figure 4B , RT - qPCR analysis of MVD , FDPS and DHCR7 expression in CTL and THEM6 KO 22rv1 cells . CASC3 was used as a normalising control . Data information : Data are presented as mean values + / - SD . Statistical analysis : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons test . Data reproducibility : : n = 3 independent biological experiments . F , Labelled cholesterol fraction derived from 13C - glucose and 13C - glutamine in CTL and THEM6 KO CWR22res cells after 72 hours of incubation . G , Labelled cholesterol fraction derived from 13C - glucose and 13C - glutamine in CTL and THEM6 KO MCF - 7 cells after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical analysis : , F , : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons test . G : * p - value < 0 . 05 using a two - tailed Student t - test . Data reproducibility : : n = 3 independent wells from the same cell culture . J , Labelled palmitic , oleic and stearic acid fractions derived from 13C - glucose and 13C - glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical analysis : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons test . Data reproducibility : n = 3 independent wells from the same cell culture .

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 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 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

Figure 4B, RT-qPCR analysis of MVD, FDPS and DHCR7 expression in CTL and THEM6 KO 22rv1 cells. CASC3 was used as a normalising control. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: : n = 3 independent biological experiments.F, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO CWR22res cells after 72 hours of incubation. G, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO MCF-7 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: , F, : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. G: *p-value < 0.05 using a two-tailed Student t-test. Data reproducibility: : n = 3 independent wells from the same cell culture.J, Labelled palmitic, oleic and stearic acid fractions derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent wells from the same cell culture.


Figure 5B , Proteomic analysis highlighting proteins associated with the ER stress response significantly down - regulated in THEM6 KO 22rv1 cells when compared to CTL ( p - value ≤ 0 . 05 , FC = 1 . 3 ) . Data information : Data are presented as mean values + / - SD . Data reproducibility : n = 3 independent biological experiments . E , Proteomic analysis highlighting ATF4 targets significantly down - regulated in THEM6 KO LNCaP AI cells when compared to CTL ( p - value ≤ 0 . 05 , FC = 1 . 3 ) . Data information : Data are presented as mean values + / - SD . Data reproducibility : n = 3 independent biological experiments . J , Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid ( 200 µM ) or hexadecylglycerol ( 50 µM ) for 48 hours . Data information : HSC70 was used as a sample loading control . Data reproducibility : representative image from 3 independent biological experiments .

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Figure 5B, Proteomic analysis highlighting proteins associated with the ER stress response significantly down-regulated in THEM6 KO 22rv1 cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.E, Proteomic analysis highlighting ATF4 targets significantly down-regulated in THEM6 KO LNCaP AI cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.J, Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid (200 µM) or hexadecylglycerol (50 µM) for 48 hours. Data information: HSC70 was used as a sample loading control. Data reproducibility: representative image from 3 independent biological experiments.


Figure 6C , Kaplan - Meier progression - free survival analysis of PCa patients stratified according high and low THEM6 expression using the PRAD TCGA dataset . D , Kaplan - Meier recurrence - free survival analysis of PCa patients stratified according to median THEM6 expression using the GSE21034 dataset ( n = 179 ) . Data information : Statistical analysis : logrank test . Data reproducibility : C : n = 489 tumours . D : n = 179 . E , IHC staining of THEM6 expression in treatment naïve , NHT - treated , CRPC and NEPC tumours . Scale bar represents 100 µm . Data information : Data reproducibility : : n = 132 ; 90 ; 66 ; 137 ; 44 ; 30 tumours for Untreated ; NHT 1 - 3 ; NHT4 - 6 ; NHT > 7 ; CRPC ; NEPC respectively . I - J , Kaplan - Meier recurrence - free ( I ) and overall ( J ) survival analysis of PCa patients stratified according to median THEM6 expression . Data information : Statistical analysis : logrank test . Data reproducibility : n = 35 tumours for THEM6 low and n = 34 tumours for THEM6 high . K , Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in F . L , Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in I - J . Data information : Centre line corresponds to median of data , top and bottom of box correspond to 90th and 10th percentile , respectively . Whiskers extend to adjacent values ( minimum and maximum data points not considered outliers ) . Statistical analysis : two - tailed Mann - Whitney U test . Data reproducibility : K : n = 269 tumours for Ki67 low and n = 270 tumours for Ki67 high . L : n = 35 tumours for Ki67 low and n = 29 tumours for Ki67 high . M , Volcano plot of the differentially modulated genes in PCa patients from the PRAD TCGA stratified according to THEM6 expression . Red dots represent the UPR - related genes extracted from the UPR - gene signature Data information : Data reproducibility : n = 489 tumours .

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Figure 6C, Kaplan-Meier progression-free survival analysis of PCa patients stratified according high and low THEM6 expression using the PRAD TCGA dataset. D, Kaplan-Meier recurrence-free survival analysis of PCa patients stratified according to median THEM6 expression using the GSE21034 dataset (n = 179). Data information: Statistical analysis: logrank test. Data reproducibility: C: n = 489 tumours. D: n = 179.E, IHC staining of THEM6 expression in treatment naïve, NHT-treated, CRPC and NEPC tumours. Scale bar represents 100 µm. Data information: Data reproducibility: : n = 132; 90; 66; 137; 44; 30 tumours for Untreated; NHT 1-3; NHT4-6; NHT>7; CRPC; NEPC respectively.I-J, Kaplan-Meier recurrence-free (I) and overall (J) survival analysis of PCa patients stratified according to median THEM6 expression. Data information: Statistical analysis: logrank test. Data reproducibility: n = 35 tumours for THEM6 low and n = 34 tumours for THEM6 high.K, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in F. L, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in I-J. Data information: Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: two-tailed Mann-Whitney U test. Data reproducibility: K: n = 269 tumours for Ki67 low and n = 270 tumours for Ki67 high. L: n = 35 tumours for Ki67 low and n = 29 tumours for Ki67 high.M, Volcano plot of the differentially modulated genes in PCa patients from the PRAD TCGA stratified according to THEM6 expression. Red dots represent the UPR-related genes extracted from the UPR-gene signature Data information: Data reproducibility: n = 489 tumours.


Figure 1 ( B ) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and cell density recorded ( x105 / mL ) . 3 independent experiments are shown with mean and SEM . The dotted line shows seeding density ( 10 , 000 cells / 100µL ) . Data information : For statistical analysis , an ordinary one - way ANOVA with Dunnett ' s correction for multiple comparisons was performed ( C ) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and viability measured . 3 independent experiments are shown with mean and SEM . The live cell fractions ( Annexin V - , 7 - AAD - ) were used for statistical analysis . Data information : For statistical analysis , an ordinary one - way ANOVA with Dunnett ' s correction for multiple comparisons was performed ( D ) Western blotting was used to visualise ASS1 protein levels in untreated or imatinib treated ( 2µM , 48 hours ) CML CD34 + samples , normal CD34 + samples and indicated cell lines following 16 hours arginine starvation . ( E ) ASS1 expression from the MILE study . The dotted line revers for average of Healthy Bone Marrow . Data information : For statistical analysis , an ordinary one - way ANOVA with Dunnett ' s correction for multiple comparisons was performed a Kruskal - Wallis test was performed on E with * * * * referring to P < . 0001 in E .

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Figure 1(B) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and cell density recorded (x105/mL). 3 independent experiments are shown with mean and SEM. The dotted line shows seeding density (10,000 cells/100µL). Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed(C) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and viability measured. 3 independent experiments are shown with mean and SEM. The live cell fractions (Annexin V-, 7-AAD-) were used for statistical analysis. Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed(D) Western blotting was used to visualise ASS1 protein levels in untreated or imatinib treated (2µM, 48 hours) CML CD34+ samples, normal CD34+ samples and indicated cell lines following 16 hours arginine starvation.(E) ASS1 expression from the MILE study. The dotted line revers for average of Healthy Bone Marrow. Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed a Kruskal-Wallis test was performed on E with **** referring to P<.0001 in E.


Figure 2 ( A ) CTV analysis of CML CD34 + patient samples after 72 hours arginine starvation ( n = 3 ) . Data are shown with mean and SEM . Data information : For statistical analysis , multiple unpaired tests with Benjamini , Krieger , and Yekutieli Benjamini two - stage step up correction was performed out on log - transformed data in A ( B ) Viability data from CML CD34 + patient samples recorded after 72 hours starvation of indicated amino acid . Data are shown are from 3 biological replicates with mean and SEM plotted . Mean and SEM are shown . Data information for B - E Mann - Whitney tests were performed . ( D ) CFCs from CML CD34 + patient samples generated after 72 hours starvation of indicated amino acid . The upper panel shows representative image of colonies , counts are plotted in the lower panel . Data are shown are from 4 ( - arginine ) or two ( - ornithine or - citrulline ) biological replicates . Mean and SEM are shown . ( E ) CFCs from Normal CD34 + samples generated after 72 hours starvation of indicated amino acid . Upper panel shows representative image of colonies , counts are plotted in lower panel . Data are shown are from 3 biological replicates . Mean and SEM are shown . Data information : Mann - Whitney tests were performed .

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Figure 2(A) CTV analysis of CML CD34+ patient samples after 72 hours arginine starvation (n=3). Data are shown with mean and SEM. Data information: For statistical analysis, multiple unpaired tests with Benjamini, Krieger, and Yekutieli Benjamini two-stage step up correction was performed out on log-transformed data in A(B) Viability data from CML CD34+ patient samples recorded after 72 hours starvation of indicated amino acid. Data are shown are from 3 biological replicates with mean and SEM plotted. Mean and SEM are shown. Data information for B-E Mann-Whitney tests were performed.(D) CFCs from CML CD34+ patient samples generated after 72 hours starvation of indicated amino acid. The upper panel shows representative image of colonies, counts are plotted in the lower panel. Data are shown are from 4 (-arginine) or two (-ornithine or -citrulline) biological replicates. Mean and SEM are shown. (E) CFCs from Normal CD34+ samples generated after 72 hours starvation of indicated amino acid. Upper panel shows representative image of colonies, counts are plotted in lower panel. Data are shown are from 3 biological replicates. Mean and SEM are shown. Data information: Mann-Whitney tests were performed.


Figure 3 ( B ) Indicated cell lines ( Control or knock - down ( sg - 2 or sg - 3 ) ) grown for 24 hours in absence or presence of BCT - 100 ( 1000ng / mL ) and ASS1 protein levels measured . Data information : For statistical analysis , ordinary one - way ANOVA with Tukey ' s correction for multiple comparisons was performed ( C ) Indicated cell lines ( Control or knock - down ( sg - 2 or sg - 3 ) ) grown for 72 hours in absence or presence of BCT - 100 ( 1000ng / mL ) and viability measured . 3 independent experiments are shown with mean and SD . Live cells ( Annexin V - , 7 - AAD - ) were analysed . Data information : For statistical analysis , ordinary one - way ANOVA with Tukey ' s correction for multiple comparisons was performed ( E ) Viability data from CML CD34 + patient samples ( 5 biological replicates ) recorded after 72 hours of treatment as in D , Mean and SEM are shown . Data information : For statistical analysis , ordinary one - way ANOVA with Tukey ' s correction for multiple comparisons was performed ( G ) CFCs from CML CD34 + samples are shown . Cells were treated as in D before seeding for CFCs . The left panel shows representative image of colonies , counts are plotted in the right panel . Data are shown are from 7 biological replicates . Mean and SEM are shown . Data information : For statistical analysis , ordinary one - way ANOVA with Tukey ' s correction for multiple comparisons was

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Figure 3(B) Indicated cell lines (Control or knock-down (sg-2 or sg-3)) grown for 24 hours in absence or presence of BCT-100 (1000ng/mL) and ASS1 protein levels measured. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed(C) Indicated cell lines (Control or knock-down (sg-2 or sg-3)) grown for 72 hours in absence or presence of BCT-100 (1000ng/mL) and viability measured. 3 independent experiments are shown with mean and SD. Live cells (Annexin V-, 7-AAD-) were analysed. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed(E) Viability data from CML CD34+ patient samples (5 biological replicates) recorded after 72 hours of treatment as in D, Mean and SEM are shown. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed(G) CFCs from CML CD34+ samples are shown. Cells were treated as in D before seeding for CFCs. The left panel shows representative image of colonies, counts are plotted in the right panel. Data are shown are from 7 biological replicates. Mean and SEM are shown. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed


Figure 4 ( B ) Volcano plot showing differentially expressed ( DE ) genes between CML CD34 + vehicle and BCT - 100 treated cells , red denotes q - value < 0 . 1 . Data information : , DESEQ2 and GSEA were used as described in methods .

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Figure 4(B) Volcano plot showing differentially expressed (DE) genes between CML CD34+ vehicle and BCT-100 treated cells, red denotes q-value <0.1. Data information: , DESEQ2 and GSEA were used as described in methods.


Figure 5Absolute concentration of indicated amino acid in serum is shown . Dashed line denotes lowest linear point from standard curve . Data information : Metabonalyst was used to conduct both two - way ANOVA and Multivariate Empirical Bayes Analysis ( MEBA ) on R - Log transformed data in A and B . ( F ) Representative data showing CD34 + CD38 - population from each treatment group . ( G ) The percentage of CD34 + CD38 - cells ( from CD34 + ) is shown . Biological replicate data from all mice , average and SD are plotted . Vehicle : n = 8 mice , Imatinib : n = 8 mice , BCT - 100 : n = 6 mice , Combo : n = 7 mice . Data information A Kruskal - Wallis test was used to analyse data in G ( H ) The absolute number of CD34 + CD38 - cells is shown . Biological replicate data from all mice , average and SD are plotted . Vehicle : n = 8 mice , Imatinib : n = 8 mice , BCT - 100 : n = 6 mice , Combo : n = 7 mice . Data information A Kruskal - Wallis test was used to analyse data in H .

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Figure 5Absolute concentration of indicated amino acid in serum is shown. Dashed line denotes lowest linear point from standard curve. Data information: Metabonalyst was used to conduct both two-way ANOVA and Multivariate Empirical Bayes Analysis (MEBA) on R-Log transformed data in A and B.(F) Representative data showing CD34+CD38- population from each treatment group.(G) The percentage of CD34+CD38- cells (from CD34+) is shown. Biological replicate data from all mice, average and SD are plotted. Vehicle: n=8 mice, Imatinib: n=8 mice, BCT-100: n=6 mice, Combo: n=7 mice. Data information A Kruskal-Wallis test was used to analyse data in G(H) The absolute number of CD34+CD38- cells is shown. Biological replicate data from all mice, average and SD are plotted. Vehicle: n=8 mice, Imatinib: n=8 mice, BCT-100: n=6 mice, Combo: n=7 mice. Data information A Kruskal-Wallis test was used to analyse data in H.


Figure 2A . Representative western blot of hTERT - RPE1 cells transfected for 72 hours with siRNA against indicated targets or non - targeting ( NT1 ) prior to harvesting and cell lysis . B . Quantification of BNIP3 , NIX and HIF1α protein levels for data represented in A . Individual datapoint from n = 5 ( BNIP3 , NIX ) or 4 ( HIF1α ) independent colour coded experiments are shown . Shown is the fold change normalised to NT1 control . Error bars show standard deviation . One way ANOVA and Dunnett ' s multiple comparison test * * P < 0 . 01 , * * * * P < 0 . 0001 .

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Figure 2A. Representative western blot of hTERT-RPE1 cells transfected for 72 hours with siRNA against indicated targets or non-targeting (NT1) prior to harvesting and cell lysis.B. Quantification of BNIP3, NIX and HIF1α protein levels for data represented in A. Individual datapoint from n=5 (BNIP3, NIX) or 4 (HIF1α) independent colour coded experiments are shown. Shown is the fold change normalised to NT1 control. Error bars show standard deviation. One way ANOVA and Dunnett's multiple comparison test **P<0.01, ****P<0.0001.


Figure 3A . Representative western blot of two matched pairs of control ( Ctrl ) and FBXL4 KO pool RPE1 - Cas9i - mt - mKeima cells , generated with two distinct sgRNAs . B . Quantification of data represented in A . Individual datapoints from n = 3 independent colour coded experiments are shown . Error bars show standard deviation of the fold change in BNIP3 and NIX in each KO pool normalised to their matched control pools ; one - way ANOVA and Tukey ' s multiple comparison ' s test , * * P < 0 . 01 , * * * * P < 0 . 0001 . D . Representative images of control or FBXL4 KO pool RPE1 - Cas9i - mt - mKeima cells . " Magenta only " puncta ( Ex . 561 ) correspond to acidic mitolysosomes . Scale bar 20 µm . E . Graphs show the quantification of mitolysosomes ( mt - mkeima " magenta only " puncta ) per cell . A minimum of 45 cells were analysed per condition in each experiment . Individual data from two ( for control 1 ( Ctrl 1 ) and knockout 1 ( KO1 ) ) or three ( control 2 ( Ctrl 2 ) and knockout 2 ( KO2 ) ) are shown using different colours for each independent experiment . Mean and standard deviation are indicated . Bottom graph shows the individual data points . One - way ANOVA with Tukey ' s multiple comparison ' s test , * P < 0 . 05 . G . Quantitative RT - PCR reactions of BNIP3 and NIX ( normalised to Actin ) were performed with cDNA derived from control and FBXL4 KO RPE1 - Cas9i - mt - mKeima cells . Two primer pairs were used per target indicated by symbol shape . Three independent colour - coded experiments are shown . Error bars show standard deviation of the fold change of BNIP3 and NIX mRNA in each KO pool normalised to their matched control pools .

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Figure 3A. Representative western blot of two matched pairs of control (Ctrl) and FBXL4 KO pool RPE1-Cas9i-mt-mKeima cells, generated with two distinct sgRNAs. B. Quantification of data represented in A. Individual datapoints from n=3 independent colour coded experiments are shown. Error bars show standard deviation of the fold change in BNIP3 and NIX in each KO pool normalised to their matched control pools; one-way ANOVA and Tukey's multiple comparison's test, **P<0.01, ****P<0.0001.D. Representative images of control or FBXL4 KO pool RPE1-Cas9i-mt-mKeima cells. "Magenta only" puncta (Ex. 561) correspond to acidic mitolysosomes. Scale bar 20 µm. E. Graphs show the quantification of mitolysosomes (mt-mkeima "magenta only" puncta) per cell. A minimum of 45 cells were analysed per condition in each experiment. Individual data from two (for control 1 (Ctrl 1) and knockout 1 (KO1)) or three (control 2 (Ctrl 2) and knockout 2 (KO2)) are shown using different colours for each independent experiment. Mean and standard deviation are indicated. Bottom graph shows the individual data points. One-way ANOVA with Tukey's multiple comparison's test, *P<0.05.G. Quantitative RT-PCR reactions of BNIP3 and NIX (normalised to Actin) were performed with cDNA derived from control and FBXL4 KO RPE1-Cas9i-mt-mKeima cells. Two primer pairs were used per target indicated by symbol shape. Three independent colour-coded experiments are shown. Error bars show standard deviation of the fold change of BNIP3 and NIX mRNA in each KO pool normalised to their matched control pools.


Figure 4A and B . hTERT - RPE1 cells were co - transfected with Flag ( V ) or FBXL4 - Flag ( FL4 ) and either GFP ( Vect ) , GFP - BNIP3 or GFP - NIX . Lysates were subjected to immunoprecipitation ( IP ) with either GFP - nanobody coupled beads ( A ) or Flag - antibody coupled agarose beads ( B ) . IPs were probed alongside the input samples for Flag and GFP . D . hTERT - RPE1 cells were transfected with empty vector ( V ) , FBXL4 - Flag ( WT ) or FBXL4 - R482W - Flag ( RW ) . Lysates were subjected to IP with Flag - antibody coupled agarose beads and probed for SKP1 and Flag . Note that FBXL4 - Flag presents with an as yet uncharacterized higher molecular weight band ( indicated by a blue arrowhead ) E . Quantitation of SKP1 protein co - immunoprecipitated with FBXL4 - R482W - Flag relative to FBXL4 - Flag ( WT ) . Data from two independent experiments are shown , normalised to the amount of SKP1 in the input and the FBXL4 - Flag in the IP . F and G . Representative images of Control ( Ctrl ) and FBXL4 KO RPE1 - Cas9i - mt - mKeima cells transfected with FBXL4 - Flag wild - type ( WT ) or FBXL4 - Flag [ R482W ] for 24 hours , then fixed and stained for either BNIP3 or NIX . Scale bar 20 µm . Quantitation of the frequency of high BNIP3 and high NIX phenotypes observed in F and G . A minimum of 32 Flag - positive ( Transfected , indicated by an asterisk in the images ) and Flag - negative cells were analysed per condition in each experiment . Shown are the mean and individual datapoints of two colour - coded experiments .

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Figure 4A and B. hTERT-RPE1 cells were co-transfected with Flag (V) or FBXL4-Flag (FL4) and either GFP (Vect), GFP-BNIP3 or GFP-NIX. Lysates were subjected to immunoprecipitation (IP) with either GFP-nanobody coupled beads (A) or Flag-antibody coupled agarose beads (B). IPs were probed alongside the input samples for Flag and GFP.D. hTERT-RPE1 cells were transfected with empty vector (V), FBXL4-Flag (WT) or FBXL4- R482W-Flag (RW). Lysates were subjected to IP with Flag-antibody coupled agarose beads and probed for SKP1 and Flag. Note that FBXL4-Flag presents with an as yet uncharacterized higher molecular weight band (indicated by a blue arrowhead) E. Quantitation of SKP1 protein co-immunoprecipitated with FBXL4-R482W-Flag relative to FBXL4-Flag (WT). Data from two independent experiments are shown, normalised to the amount of SKP1 in the input and the FBXL4-Flag in the IP.F and G. Representative images of Control (Ctrl) and FBXL4 KO RPE1-Cas9i-mt-mKeima cells transfected with FBXL4-Flag wild-type (WT) or FBXL4-Flag [R482W] for 24 hours, then fixed and stained for either BNIP3 or NIX. Scale bar 20 µm. Quantitation of the frequency of high BNIP3 and high NIX phenotypes observed in F and G. A minimum of 32 Flag-positive (Transfected, indicated by an asterisk in the images) and Flag-negative cells were analysed per condition in each experiment. Shown are the mean and individual datapoints of two colour-coded experiments.


Figure 5Control ( Ctrl ) or FBXL4 KO RPE1 - Cas9i - mt - mkeima cells were treated for 72 hours with either non - targeting ( NT1 ) , BNIP3 ( B3 ) , NIX or HIF1α ( HIF ) siRNA . A . Representative images of the mt - mKeima reporter in Control ( Ctrl2 ) or FBXL4 KO2 RPE1 - Cas9i - mt - mkeima . " Magenta only " puncta ( Ex . 561 ) correspond to acidic mitolysosomes . Scale bar : 20 µm . C . Representative western blot verifying the knockdown efficiency for samples shown in B .

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Figure 5Control (Ctrl) or FBXL4 KO RPE1-Cas9i-mt-mkeima cells were treated for 72 hours with either non-targeting (NT1), BNIP3 (B3), NIX or HIF1α (HIF) siRNA. A. Representative images of the mt-mKeima reporter in Control (Ctrl2) or FBXL4 KO2 RPE1-Cas9i-mt-mkeima. "Magenta only" puncta (Ex. 561) correspond to acidic mitolysosomes. Scale bar: 20 µm.C. Representative western blot verifying the knockdown efficiency for samples shown in B.


Figure 6C . Representative western blot of lysates prepared from hTERT - RPE1 cells transfected with siRNA targeting BNIP3 ( B3 ) , NIX or both for 72 hours . Cells were treated with either DMSO or MLN4924 ( 1 µM ) for the last 24 hours of transfection . Untransfected cells were treated alongside transfected ones with DMSO , antimycin and oligomycin ( AO ; 1 and 10 µM respectively ) , or MLN4924 . D . Quantification of fold change in protein levels calculated from data represented in C for three colour - coded independent experiments ; One way ANOVA and Dunnett ' s multiple comparison test * P < 0 . 05 , * * * * P < 0 . 0001 . E . Representative western blot of hTERT - RPE1 cells treated with either DMSO or MLN4924 ( 1 µM ) for the indicated time points . G . Quantification of fold change in protein levels calculated from data represented in F for six colour - coded independent experiments . Error bars show standard deviation ; One way ANOVA and Tukey ' s multiple comparison test * * P < 0 . 01 , * * * * P < 0 . 0001 .

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Figure 6C. Representative western blot of lysates prepared from hTERT-RPE1 cells transfected with siRNA targeting BNIP3 (B3), NIX or both for 72 hours. Cells were treated with either DMSO or MLN4924 (1 µM) for the last 24 hours of transfection. Untransfected cells were treated alongside transfected ones with DMSO, antimycin and oligomycin (AO; 1 and 10 µM respectively), or MLN4924. D. Quantification of fold change in protein levels calculated from data represented in C for three colour-coded independent experiments; One way ANOVA and Dunnett's multiple comparison test *P<0.05, ****P<0.0001.E. Representative western blot of hTERT-RPE1 cells treated with either DMSO or MLN4924 (1 µM) for the indicated time points.G. Quantification of fold change in protein levels calculated from data represented in F for six colour-coded independent experiments. Error bars show standard deviation; One way ANOVA and Tukey's multiple comparison test **P<0.01, ****P<0.0001.


Figure 7A . Representative western blot of lysates from control or FBXL4 KO RPE1 - Cas9i - mt - mKeima cells treated with MLN4924 ( 1 µM ) for 6 hours . B . Representative western blot of lysates from control or FBXL4 KO RPE1 - Cas9i - mt - mKeima cells pre - treated without or with MLN4924 ( 1 µM ) for 6 hours followed by a cycloheximide ( CHX ) chase ( 100 µg / ml ) . C and D . Quantification of protein levels calculated from data represented in B for three colour - coded independent experiments . Error bars show standard deviation . F . TUBES - pulldown of ubiquitylated NIX . FBXL4 KO RPE1 - Cas9i - mt - mkeima cells and matched control cells were treated as in E and lysates subjected to a TUBES pulldown .

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Figure 7A. Representative western blot of lysates from control or FBXL4 KO RPE1-Cas9i-mt-mKeima cells treated with MLN4924 (1 µM) for 6 hours. B. Representative western blot of lysates from control or FBXL4 KO RPE1-Cas9i-mt-mKeima cells pre-treated without or with MLN4924 (1 µM) for 6 hours followed by a cycloheximide (CHX) chase (100 µg/ml).C and D. Quantification of protein levels calculated from data represented in B for three colour-coded independent experiments. Error bars show standard deviation.F. TUBES-pulldown of ubiquitylated NIX. FBXL4 KO RPE1-Cas9i-mt-mkeima cells and matched control cells were treated as in E and lysates subjected to a TUBES pulldown.


Figure 1C Upper panel : senescence curve of a representative est2 ∆ clone . DNA samples were prepared at the indicated time points to analyze telomere length ( middle panel ) and t - circles production ( lower panel and graph ) during senescence and cell immortalization . Telomeres were analyzed by Southern blot of the XhoI cut DNA hybridized with a TG1 - 3 probe . The presence of t - circles was monitored by RCA and analyzed by TG1 - 3 probed dot blot . The graph shows signal intensities of the dots measured with the Fiji software ( arbitrary units ) . Similar results were obtained with at least 16 other est2 ∆ clones . E ) Dot blot of the RCA product either treated or not with the Exonuclease VII that degrades linear single - stranded DNA in both the 3 ' to 5 ' and 5 ' to 3 ' directions ( upper panel ) . The membrane was hybridized with a telomeric TG1 - 3 probe . The signal intensities of the dots are shown ( lower graph ) .

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Figure 1C Upper panel: senescence curve of a representative est2∆ clone. DNA samples were prepared at the indicated time points to analyze telomere length (middle panel) and t-circles production (lower panel and graph) during senescence and cell immortalization. Telomeres were analyzed by Southern blot of the XhoI cut DNA hybridized with a TG1-3 probe. The presence of t-circles was monitored by RCA and analyzed by TG1-3 probed dot blot. The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units). Similar results were obtained with at least 16 other est2∆ clones.E) Dot blot of the RCA product either treated or not with the Exonuclease VII that degrades linear single-stranded DNA in both the 3' to 5' and 5' to 3' directions (upper panel). The membrane was hybridized with a telomeric TG1-3 probe. The signal intensities of the dots are shown (lower graph).


Figure 2A Senescence curve of a representative est2 ∆ clone and corresponding dot blot of the RCA product hybridized with the indicated subtelomeric or telomeric probes . t - circle amounts were evaluated based on signal intensity of the dots and were expressed as fold increase over the signal in the absence of Φ29 ( lower graph ) . B Senescence curve of a representative est2 ∆ clone ( upper panel ) . DNA samples were prepared at the indicated time points to analyze telomere length by Southern blot ( middle panel ) and t - circles production by RCA assay performed in the absence of dCTP and dATP . ( lower panel ) . The graph shows signal intensities of the dots measured with the Fiji software ( arbitrary units ) .

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Figure 2A Senescence curve of a representative est2∆ clone and corresponding dot blot of the RCA product hybridized with the indicated subtelomeric or telomeric probes. t-circle amounts were evaluated based on signal intensity of the dots and were expressed as fold increase over the signal in the absence of Φ29 (lower graph).B Senescence curve of a representative est2∆ clone (upper panel). DNA samples were prepared at the indicated time points to analyze telomere length by Southern blot (middle panel) and t-circles production by RCA assay performed in the absence of dCTP and dATP.(lower panel). The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units).


Figure 3C Detection of the t - circles co - immunoprecipitated with Nic96 - RFP from the extracts obtained from several type II survivors . The DNA co - immunoprecipitated with Nic96 - RFP was amplified by RCA assay , loaded on a dot blot and detected with a telomeric TG1 - 3 probe . The numbers above the blot refer to the growth curves in ( A ) and correspond to type II survivors produced in independent cultures . The lower panel shows the efficiency of Nic96 - RFP IP that was evaluated by western blot with anti - mCherry antibody . D Quantification of the RCA product after co - IP of the C - circles with Nic96 - RFP showing the effect of ADA2 deletion . Dot signal intensities were quantified using the Fiji software and were calculated as the percentage of INPUT that was immunoprecipitated normalized to est2 ∆ signals . Relative signals were calculated by setting the mean of est2 ∆ signals from each independent experiment to 1 . Means and s . e . m . of the six independent co - IPs experiments each with 2 - 6 clones per genotype are shown . Outliers were removed using ROUT robust outlier test . The p - values from one - way ANOVA were adjusted using the Bonferroni ' s correction for multiple comparisons . est2 ∆ versus est2 ∆ NIC96 - RFP ( p = 0 , 000007 ) , est2 ∆ NIC96 - RFP versus est2 ∆ ada2 ∆ NIC96 - RFP ( p = 0 , 00012 ) . * * * P < 0 , 0001 . E Quantification of the RCA product after co - IP of the C - circles with Nic96 - RFP showing the effect of SUS1 deletion . The graph shows means and s . e . m . of the three independent co - IPs with 2 - 5 clones per genotype . Outliers were removed using ROUT robust outlier test . The p - values from one - way ANOVA were were adjusted using the Bonferroni ' s correction for multiple comparisons . est2 ∆ versus est2 ∆ NIC96 - RFP ( p = 0 , 002 ) , est2 ∆ versus est2 ∆ sus1 ∆ NIC96 - RFP ( p = 0 , 0006 ) . * * P < 0 , 001 , * * * P < 0 , 0001 .

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Figure 3C Detection of the t-circles co-immunoprecipitated with Nic96-RFP from the extracts obtained from several type II survivors. The DNA co-immunoprecipitated with Nic96-RFP was amplified by RCA assay, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to the growth curves in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Nic96-RFP IP that was evaluated by western blot with anti-mCherry antibody.D Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of ADA2 deletion. Dot signal intensities were quantified using the Fiji software and were calculated as the percentage of INPUT that was immunoprecipitated normalized to est2∆ signals. Relative signals were calculated by setting the mean of est2∆ signals from each independent experiment to 1. Means and s.e.m. of the six independent co-IPs experiments each with 2-6 clones per genotype are shown. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,000007), est2∆ NIC96-RFP versus est2∆ ada2∆ NIC96-RFP (p=0,00012).***P<0,0001.E Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of SUS1 deletion. The graph shows means and s.e.m. of the three independent co-IPs with 2-5 clones per genotype. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,002), est2∆ versus est2∆ sus1∆ NIC96-RFP (p= 0,0006). **P<0,001, ***P<0,0001.


Figure 4A Senescence curves of two est2 ∆ and two est2 ∆ ADA2 - Myc clones used to generate type II survivors for Co - IP - RCA . This experiment is representative out of the 4 independent experiments performed during this study . C Detection of the C - circles co - immunoprecipitated with Ada2 - Myc in type II survivors . The DNA co - immunoprecipitated with Ada2 - Myc was amplified by RCA , loaded on a dot blot and detected with a telomeric TG1 - 3 probe . The numbers above the blot refer to clones in ( A ) and correspond to type II survivors produced in independent cultures . The lower panel shows the efficiency of Ada2 - Myc IP analyzed by western blot with anti - Myc antibody .

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Figure 4A Senescence curves of two est2∆ and two est2∆ ADA2-Myc clones used to generate type II survivors for Co-IP-RCA. This experiment is representative out of the 4 independent experiments performed during this study.C Detection of the C-circles co-immunoprecipitated with Ada2-Myc in type II survivors. The DNA co-immunoprecipitated with Ada2-Myc was amplified by RCA, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to clones in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Ada2-Myc IP analyzed by western blot with anti-Myc antibody.


Figure 5A Mean senescence profiles of the est2 ∆ ( n = 7 ) and est2 ∆ sus1 ∆ ( n = 8 ) clones isolated from the same heterozygous diploid . The error bars are SDs . B Mean senescence profiles of the est2 ∆ ( n = 10 ) and est2 ∆ sac3 ∆ ( n = 10 ) clones isolated from one heterozygous diploid . The error bars are SDs .

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Figure 5A Mean senescence profiles of the est2∆ (n=7) and est2∆ sus1∆ (n=8) clones isolated from the same heterozygous diploid. The error bars are SDs. B Mean senescence profiles of the est2∆ (n=10) and est2∆ sac3∆ (n=10) clones isolated from one heterozygous diploid. The error bars are SDs.


Figure 6A Mean senescence profiles of the est2 ∆ ( n = 10 ) and est2 ∆ shs1 ∆ ( n = 16 ) clones . The error bars are SDs . B Mean senescence profiles of the est1 ∆ ( n = 6 ) and est1 ∆ bud6 ∆ ( n = 7 ) clones . Est1 , similarly to Est2 , is essential for telomere maintenance . est1 ∆ has been used in this experiment instead of est2 ∆ because EST2 and BUD6 genes are linked on chromosome XII . The error bars are SDs .

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Figure 6A Mean senescence profiles of the est2∆ (n=10) and est2∆ shs1∆ (n=16) clones. The error bars are SDs. B Mean senescence profiles of the est1∆ (n=6) and est1∆ bud6∆ (n=7) clones. Est1, similarly to Est2, is essential for telomere maintenance. est1∆ has been used in this experiment instead of est2∆ because EST2 and BUD6 genes are linked on chromosome XII. The error bars are SDs.


Figure 1 ( A ) RPE1 cells were transfected with either scramble or TMEM135 siRNAs and immunostained for LAMP1 as a lysosome marker ( red ) and PMP70 as a peroxisome marker ( green ) . Scale bar , 10 μm . ( B ) Quantification of overlap signal between lysosomes and peroxisomes shown in ( A ) . Data represent mean ± SD ( n = 3 experiments ) , 50 cells were scored per condition per experiment . * P < 0 . 05 , Student ' s t - test . ( C ) Subcellular fractions of a discontinuous sucrose gradient collected from top to bottom , separated by SDS - PAGE , and immunoblotted for LAMP1 , EEA1 , Rab11 , Rab8 , PMP70 , GM130 , and Na + / K + ATPase . ( D ) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - test . ( E ) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2 . ( F ) Cells transfected with TMEM135 siRNA were subjected to qPCR . Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - test .

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Figure 1(A) RPE1 cells were transfected with either scramble or TMEM135 siRNAs and immunostained for LAMP1 as a lysosome marker (red) and PMP70 as a peroxisome marker (green). Scale bar, 10 μm. (B) Quantification of overlap signal between lysosomes and peroxisomes shown in (A). Data represent mean ± SD (n=3 experiments), 50 cells were scored per condition per experiment. *P < 0.05, Student's t-test. (C) Subcellular fractions of a discontinuous sucrose gradient collected from top to bottom, separated by SDS-PAGE, and immunoblotted for LAMP1, EEA1, Rab11, Rab8, PMP70, GM130, and Na+/K+ ATPase.(D) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.(E) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2.(F) Cells transfected with TMEM135 siRNA were subjected to qPCR. Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.


Figure 2 ( A ) RPE1 cells were transfected with siRNAs as indicated , followed by serum starvation for 24 h , and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( B ) Quantification of the percentage of ciliated cells shown in ( A ) . Data represent mean ± SD ( n = 3 experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( C ) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum - starvation media followed by filipin staining . Scale bar , 20 μm . ( D ) Cells were transfected with siRNAs shown in ( C ) and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) Quantification of the percentage of ciliated cells shown in ( D ) . Data represent mean ± SD ( n = 3 experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( F ) Cells were transfected with siRNAs as indicated , incubated with cholesterol - MβCD complex in serum - starvation media , and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( G ) Quantification of the percentage of ciliated cells shown in ( F ) . Data represent mean ± SD ( n = 3experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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Figure 2(A) RPE1 cells were transfected with siRNAs as indicated, followed by serum starvation for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (B) Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (C) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm.(D) Cells were transfected with siRNAs shown in (C) and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of ciliated cells shown in (D). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (F) Cells were transfected with siRNAs as indicated, incubated with cholesterol-MβCD complex in serum-starvation media, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of ciliated cells shown in (F). Data represent mean ± SD (n=3experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.