Figure 6H : CRISPR / Cas9 control ( CTR _ 1 and CTR _ 2 ) or PRMT1 - deleted ( KO _ 1 and KO _ 2 ) HeLa cell lines were irradiated at 150 J / cm2 UVC or not irradiated . After 4 hours , immunoprecipitations ( IP ) of endogenous p14ARF were performed from cell lysates using α - p14ARF antibody or IgG as negative control . IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies . Short and long exposure times are specified .

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Figure 6H: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC or not irradiated. After 4 hours, immunoprecipitations (IP) of endogenous p14ARF were performed from cell lysates using α-p14ARF antibody or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Short and long exposure times are specified.


Figure 7A : Kaplan - Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short - term ( < 500 days , left plot ) and long - term survival ( > 500 days , right plot ) . B : Representative immunohistochemistry ( IHC ) stainings of PRMT1 ( brown ) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients . Based on the immunostaining intensity and the percentage of stained tumor cells , all specimens were divided into three PRMT1 expression scores : not elevated , moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas . Right images are magnifications of the left images , as indicated by the black rectangles . Scale bars in the left images : 100 μm . Scale bars in the right images : 50 μm . E : Immunofluorescence ( IF ) stainings of the 75 PDAC specimens were performed using α - p14ARF ( red ) , α - CK8 / 18 ( green , staining ductal cells in normal pancreas and neoplastic cells in PDAC ) antibodies and DAPI ( blue , nuclei / DNA ) . Representative IF images are shown for normal human pancreatic tissue , two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells . Left and corresponding right images show the same tissue section . Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC . The last image pair shows a magnification of the image pair above ( indicated by the rectangle ) . Scale bars : 35 μm .

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Figure 7A: Kaplan-Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short-term (<500 days, left plot) and long-term survival (>500 days, right plot).B: Representative immunohistochemistry (IHC) stainings of PRMT1 (brown) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients. Based on the immunostaining intensity and the percentage of stained tumor cells, all specimens were divided into three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas. Right images are magnifications of the left images, as indicated by the black rectangles. Scale bars in the left images: 100 μm. Scale bars in the right images: 50 μm.E: Immunofluorescence (IF) stainings of the 75 PDAC specimens were performed using α-p14ARF (red), α-CK8/18 (green, staining ductal cells in normal pancreas and neoplastic cells in PDAC) antibodies and DAPI (blue, nuclei/DNA). Representative IF images are shown for normal human pancreatic tissue, two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells. Left and corresponding right images show the same tissue section. Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC. The last image pair shows a magnification of the image pair above (indicated by the rectangle). Scale bars: 35 μm.


Figure 8B : PaTu8988t cells were treated with 0 , 1 , 3 , and 10 μM gemcitabine ( GEM ) . After 48 hours , cell lysates were analyzed by immunoblotting using the indicated antibodies . The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal , as specified by the numbers below the blots , with the untreated condition ( - GEM ) set to 1 . The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot . C : PaTu8988t cells were treated with 3 μM gemcitabine ( GEM ) or left untreated ( 0 hour ) . After 0 , 6 , 12 , 24 and 48 hours , methylation of endogenous p14ARF was analyzed by immunoblotting using α - me - p14ARF , α - p14ARF and α - GAPDH ( loading control ) antibodies . The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal , as specified by the numbers below the blots , with the untreated condition ( 0 hour ) set to 1 . D , PaTu8988t and MiaPaCa - 2 cells were treated ( + ) with 20 μM MS023 or left untreated ( - ) for 3 days . For the last 2 days , the cells were additionally exposed to 0 , 1 , or 3 μM gemcitabine ( GEM ) . Subsequently , cell death was quantified by flow cytometry ( in ( D ) ) using FITC - labeled AnnexinV and propidium iodide ( PI ) for four independent experiments ( mean ± SD , * : p - value ≤ 0 . 05 using Welch ´ s t - test ) . G : Apoptosis was quantified by flow cytometry ( G ) using FITC - labeled AnnexinV and propidium iodide ( PI ) for six independent experiments ( mean ± SD , * * : p - value ≤ 0 . 005 , * : p - value ≤ 0 . 05 , ns : not significant using Welch ´ s t - test ) .

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Figure 8B: PaTu8988t cells were treated with 0, 1, 3, and 10 μM gemcitabine (GEM). After 48 hours, cell lysates were analyzed by immunoblotting using the indicated antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (- GEM) set to 1. The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot. C: PaTu8988t cells were treated with 3 μM gemcitabine (GEM) or left untreated (0 hour). After 0, 6, 12, 24 and 48 hours, methylation of endogenous p14ARF was analyzed by immunoblotting using α-me-p14ARF, α-p14ARF and α-GAPDH (loading control) antibodies. The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (0 hour) set to 1.D, PaTu8988t and MiaPaCa-2 cells were treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed to 0, 1, or 3 μM gemcitabine (GEM). Subsequently, cell death was quantified by flow cytometry (in (D)) using FITC-labeled AnnexinV and propidium iodide (PI) for four independent experiments (mean ± SD, *: p-value ≤ 0.05 using Welch´s t-test).G: Apoptosis was quantified by flow cytometry (G) using FITC-labeled AnnexinV and propidium iodide (PI) for six independent experiments (mean ± SD, **: p-value ≤ 0.005, *: p-value ≤ 0.05, ns: not significant using Welch´s t-test).


Figure 1B . Subcellular fractionation and Western blots of 293T cells with ectopically expressed GASZ , NRF1 , MVH , or MitoPLD . C . Localization of GFP fused with wild - type or mutant MLS of GASZ in HeLa cells . Mitochondrial localization was monitored by MitoTracker ( Red ) staining . Scale bars : 15 m .

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Figure 1B. Subcellular fractionation and Western blots of 293T cells with ectopically expressed GASZ, NRF1, MVH, or MitoPLD.C. Localization of GFP fused with wild-type or mutant MLS of GASZ in HeLa cells. Mitochondrial localization was monitored by MitoTracker (Red) staining. Scale bars: 15 m.


Figure 2A . IHF of GASZ and Cytochrome C ( Cyt C ) of testical sections from mice at day 7 postnatal , or MILI and MVH of gonad sections from wild - type embryos or embryos with MLS deletion at 16 . 5 dpc . Scale bars : 5 m . B . Western blots of GASZ expression of testes at day 5 postnatal . C . Gross morphology of testes from adult mice at 3 months postnatal with mutated MLS compared to their wild - type littermates . D . Histological studies of testis sections from wild - type mice or mice with MLS deletion at different time points after birth . Scale bars : 50 m . E . EM of testis sections from wild - type neonatal mice or mice with MLS mutation . Red arrows point to IMC among mitochondria . F . Expression of transposable elements analyzed by real - time RT - PCR on testes from wild - type mice or mice with mutated MLS at various time points after birth . Data were shown as mean ( of expression from three biological replicates ) ± one s . e . m . n = 3 for each genotype . * * : p < 0 . 01 . * : p < 0 . 05 .

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Figure 2A. IHF of GASZ and Cytochrome C (Cyt C) of testical sections from mice at day 7 postnatal, or MILI and MVH of gonad sections from wild-type embryos or embryos with MLS deletion at 16.5 dpc. Scale bars: 5 m.B. Western blots of GASZ expression of testes at day 5 postnatal.C. Gross morphology of testes from adult mice at 3 months postnatal with mutated MLS compared to their wild-type littermates.D. Histological studies of testis sections from wild-type mice or mice with MLS deletion at different time points after birth. Scale bars: 50 m.E. EM of testis sections from wild-type neonatal mice or mice with MLS mutation. Red arrows point to IMC among mitochondria.F. Expression of transposable elements analyzed by real-time RT-PCR on testes from wild-type mice or mice with mutated MLS at various time points after birth. Data were shown as mean (of expression from three biological replicates) ± one s.e.m. n=3 for each genotype. **: p<0.01. *: p<0.05.


Figure 3B . 293T cells with ectopically expressed proteins were extracted for soluble and membrane bound fraction and examined by Western blots . C . Mitochondrial fraction from 293T cells ectopically expressing GASZ , MitoPLD , AIFM1 , or SIRT3 were treated in the presence or absence of Triton X - 100 and / or Proteinase K , and examined by Western blots . D . GST pull - down assay of GST - tagged GASZ with FLAG - GASZ produced from 293T cells . E . Co - IP assays of FLAG or GFP - tagged GASZ in 293T cells . Antibodies for Co - IP ( IP ) or Western blots ( WB ) were listed on the left . F . BiFC assays in which YFP was detected by flow cytometry in control 293T cells , or 293T cells co - expressed with GASZ fused with N - or C - terminal half of YFP . Ctrl : empty vector control . G . Co - IP assays of GFP - GASZ with various FLAG - tagged wild - type GASZ or its deletion mutants in 293T

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Figure 3B. 293T cells with ectopically expressed proteins were extracted for soluble and membrane bound fraction and examined by Western blots.C. Mitochondrial fraction from 293T cells ectopically expressing GASZ, MitoPLD, AIFM1, or SIRT3 were treated in the presence or absence of Triton X-100 and/or Proteinase K, and examined by Western blots.D. GST pull-down assay of GST-tagged GASZ with FLAG-GASZ produced from 293T cells.E. Co-IP assays of FLAG or GFP-tagged GASZ in 293T cells. Antibodies for Co-IP (IP) or Western blots (WB) were listed on the left.F. BiFC assays in which YFP was detected by flow cytometry in control 293T cells, or 293T cells co-expressed with GASZ fused with N- or C-terminal half of YFP. Ctrl: empty vector control.G. Co-IP assays of GFP-GASZ with various FLAG-tagged wild-type GASZ or its deletion mutants in 293T cells


Figure 4A . Mitochondria visualized by MitoTracker ( Red ) when wild - type GASZ or GASZ with MLS mutation was expressed in HeLa cells . Scale bar : 15 m . B . EM on 293T cells with empty vector control ( Ctrl ) , wild - type GASZ , or GASZ with F to P mutation in MLS . Scale bar : 1 m . N : nucleus . C . Averaged percentage of HeLa cells ( determined from 100 cells in each experiment ) with different types of mitochondria determined by MitoTracker ( Red ) staining when transfected with GASZ , its SAM / ZIP deletion mutant , or GASZ with MLS mutation . D . HeLa cells transfected with COX4 - DsRed , mito - PAGFP , and empty vector control , GASZ or GASZ with MLS mutation , followed by photoactivation of PAGFP . Representative images of PAGFP and DsRed were taken at 0 , 10 , and 20 min post photoactivation . Scale Bar : 10 m . Insets show images with higher magnification . The averaged exponential decay constant ( ± s . e . m . ) of normalized PAGFP fluorescence within the designated region ( white circle ) at photoactivation area over a period of 10 min was shown as a bar graph below . n > 20 cells for each group . E . Averaged percentage of C18 - 4 cells ( determined from 100 cells in experiment ) with different types of mitochondria determined by MitoTracker ( Red ) staining when transfected with Su9 - GFP or GFP - GASZ . F . Real - time PCR analyses of mtDNA copy numbers on testes at different time points postnatal , relative to a single copy gene , PECAM , encoded by nuclear DNA . n = 5 per genotype . G . Histochemistry of respiratory enzymes , COX , NADH , and SDH on testical sections from mice 10 days postnatal . Scale bar : 100 m .

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Figure 4A. Mitochondria visualized by MitoTracker (Red) when wild-type GASZ or GASZ with MLS mutation was expressed in HeLa cells. Scale bar: 15 m.B. EM on 293T cells with empty vector control (Ctrl), wild-type GASZ, or GASZ with F to P mutation in MLS. Scale bar: 1 m. N: nucleus.C. Averaged percentage of HeLa cells (determined from 100 cells in each experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with GASZ, its SAM/ZIP deletion mutant, or GASZ with MLS mutation.D. HeLa cells transfected with COX4-DsRed, mito-PAGFP, and empty vector control, GASZ or GASZ with MLS mutation, followed by photoactivation of PAGFP. Representative images of PAGFP and DsRed were taken at 0, 10, and 20 min post photoactivation. Scale Bar: 10 m. Insets show images with higher magnification. The averaged exponential decay constant (±s.e.m.) of normalized PAGFP fluorescence within the designated region (white circle) at photoactivation area over a period of 10 min was shown as a bar graph below. n>20 cells for each group.E. Averaged percentage of C18-4 cells (determined from 100 cells in experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with Su9-GFP or GFP-GASZ.F. Real-time PCR analyses of mtDNA copy numbers on testes at different time points postnatal, relative to a single copy gene, PECAM, encoded by nuclear DNA. n=5 per genotype.G. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.


Figure 5A - B . Co - IP assays of ectopically expressed FLAG - GASZ and MFN - HA in 293T cells ( A ) A - B . Co - IP assays of endogenous proteins of GASZ and MFNs from adult testes ( B ) . C . Co - IP assays of HA - tagged MFN1 with various FLAG - tagged wild - type GASZ , its domain deletion mutants , or GASZ with MLS mutations in 293T cells . D . Averaged percentage of MEFs with different types of mitochondria were determined according to the mitochondrial morphology by MitoTracker ( Red ) staining in wild - type MEFs , or MEFs containing MFN null mutations transfected with empty vector control ( Ctrl ) or a GASZ expressing plasmid . E . PEG fusion assays of Su9 - GFP and COX4 - DsRed labeled wild - type MEFs , or MEFs containing MFN null mutations transfected with empty vector control ( Ctrl ) or a GASZ expressing plasmid . Bar graph represents averaged percentage of areas with different levels of mitochondrial fusion .

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Figure 5A-B. Co-IP assays of ectopically expressed FLAG-GASZ and MFN-HA in 293T cells (A)A-B. Co-IP assays of endogenous proteins of GASZ and MFNs from adult testes (B).C. Co-IP assays of HA-tagged MFN1 with various FLAG-tagged wild-type GASZ, its domain deletion mutants, or GASZ with MLS mutations in 293T cells.D. Averaged percentage of MEFs with different types of mitochondria were determined according to the mitochondrial morphology by MitoTracker (Red) staining in wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid.E. PEG fusion assays of Su9-GFP and COX4-DsRed labeled wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid. Bar graph represents averaged percentage of areas with different levels of mitochondrial fusion.


Figure 6A . Gross morphology of testes from MFN1f / f - Cre compared to their wild - type littermates . B . Histological studies of testis sections from wild - type mice or MFN1f / f - Cre littermates at different time points postnatal . Red stars : tubules with spermatocytes . C . IHF of PLZF ( day 14 postnatal ) , MILI , MVH , and GASZ / CytC ( day 7 ) of testes sections . Scale bars for PLZF are 50 m and 10 m for others . D . EM of testical sections from wild - type neonatal mice or MFN1f / f - Cre littermates . N : nucleus . E . Histochemistry of respiratory enzymes , COX , NADH , and SDH on testical sections from mice 10 days postnatal . Scale bar : 100 m .

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Figure 6A. Gross morphology of testes from MFN1f/f-Cre compared to their wild-type littermates.B. Histological studies of testis sections from wild-type mice or MFN1f/f-Cre littermates at different time points postnatal. Red stars: tubules with spermatocytes.C. IHF of PLZF (day 14 postnatal), MILI, MVH, and GASZ/CytC (day 7) of testes sections. Scale bars for PLZF are 50 m and 10 m for others.D. EM of testical sections from wild-type neonatal mice or MFN1f/f-Cre littermates. N: nucleus.E. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.


Figure 1Heatmap of overlapped expressed genes in response to sorafenib - resistance . D The mRNA expression level of METTL3 in sorafenib - sensitive ( n = 3 ) and sorafenib - resistant ( n = 3 ) human liver tumors . Data information : In all relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . E Expression of METTL3 was significantly down - regulated in advanced stage of liver cancer from TCGA . F METTL3 RNA levels between naïve HepG - 2 cells ( n = 3 ) and sorafenib - resistant HepG - 2 cells ( n = 10 ) . Data information : In all relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . G The IC50 of sorafenib - resistant HepG - 2 cells treated with sorafenib under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . H The protein level of METTL3 in sorafenib - resistant HepG - 2 cells treated with sorafenib under hypoxia condition ( 1 % O2 ) . I The global RNA m6A level in naïve HepG - 2 and sorafenib - resistant HepG - 2 determined by dot - blotting assay under hypoxia condition ( 1 % O2 ) .

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Figure 1Heatmap of overlapped expressed genes in response to sorafenib-resistance.D The mRNA expression level of METTL3 in sorafenib-sensitive (n=3) and sorafenib-resistant (n=3) human liver tumors. Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.E Expression of METTL3 was significantly down-regulated in advanced stage of liver cancer from TCGA.F METTL3 RNA levels between naïve HepG-2 cells (n=3) and sorafenib-resistant HepG-2 cells (n=10). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.G The IC50 of sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.H The protein level of METTL3 in sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2).I The global RNA m6A level in naïve HepG-2 and sorafenib-resistant HepG-2 determined by dot-blotting assay under hypoxia condition (1%O2).


Figure 2A - B Clonogenic survival of METTL3 - knockdown ( A ) and METTL3 - overexpression ( B ) in normal liver cell line WRL68 cells for 7 days in normoxia condition ( 21 % O2 ) and quantification of clusters in A - 1 and B - 1 , respectively . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . C - D The IC50 of METTL3 - knockdown SMMC - 7721 cells ( C ) and Bel - 7402 cells ( D ) after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . E Overexpression of wild type METTL3 or catalytic mutant METTL3 in sorafenib - resistant HepG - 2 cells . F The global RNA m6A level in sorafenib - resistant HepG - 2 cells with wild type METTL3 - overexpression or catalytic mutant METTL3 - overexpression by dot - blotting assay . G - I Rescue of shRNA - resistant wild type METTL3 but not catalytic mutant METTL3 sensitized METTL3 - knockdown SMMC - 7721 cells ( G ) , Bel - 7402 cells ( H ) and sorafenib - resistant HepG - 2 cells ( I ) to sorafenib treatment . The IC50 of the cells was measured after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . J Cell survival assay of sorafenib - resistant HepG - 2 cells with wild type METTL3 overexpression or catalytic mutant METTL3 overexpression after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Scale bar , 1mm . K Capillary - like structures in HUVECs treated with the tumor - conditioned medium ( TCM ) from control HCCs and METTL3 - knockdown HCCs cultured under hypoxic conditions for 48 h . Quantification of the number of tubes with image J in K - 1 . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . L - M The relative mRNA expression levels of angiogenesis genes were detected in SMMC - 7721 cells ( L ) and Bel - 7402 cells ( M ) with METTL3 - knockdown by RT - PCR assay . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . N The protein levels of VEGF - A in METTL3 - knockdown HCCs . O The protein levels of PDGF - B in METTL3 - knockdown HCCs .

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 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 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 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 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 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 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

Figure 2A-B Clonogenic survival of METTL3-knockdown (A) and METTL3-overexpression (B) in normal liver cell line WRL68 cells for 7 days in normoxia condition (21% O2) and quantification of clusters in A-1 and B-1, respectively. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.C-D The IC50 of METTL3-knockdown SMMC-7721 cells (C) and Bel-7402 cells (D) after treated with sorafenib for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.E Overexpression of wild type METTL3 or catalytic mutant METTL3 in sorafenib-resistant HepG-2 cells.F The global RNA m6A level in sorafenib-resistant HepG-2 cells with wild type METTL3-overexpression or catalytic mutant METTL3-overexpression by dot-blotting assay.G-I Rescue of shRNA-resistant wild type METTL3 but not catalytic mutant METTL3 sensitized METTL3-knockdown SMMC-7721 cells (G), Bel-7402 cells (H) and sorafenib-resistant HepG-2 cells (I) to sorafenib treatment. The IC50 of the cells was measured after treated with sorafenib for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.J Cell survival assay of sorafenib-resistant HepG-2 cells with wild type METTL3 overexpression or catalytic mutant METTL3 overexpression after treated with sorafenib for 24h under hypoxia condition (1%O2). Scale bar, 1mm.K Capillary-like structures in HUVECs treated with the tumor-conditioned medium (TCM) from control HCCs and METTL3-knockdown HCCs cultured under hypoxic conditions for 48 h. Quantification of the number of tubes with image J in K-1. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.L-M The relative mRNA expression levels of angiogenesis genes were detected in SMMC-7721 cells (L) and Bel-7402 cells (M) with METTL3-knockdown by RT-PCR assay. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.N The protein levels of VEGF-A in METTL3-knockdown HCCs. O The protein levels of PDGF-B in METTL3-knockdown HCCs.


Figure 3A Electron micrographs of METTL3 - knockdown SMMC - 7721 cells under hypoxia for 48 h . Protein levels of LC3 I / II in METTL3 - knockdown SMMC - 7721 ( B ) , Bel - 7402 ( C ) cells under hypoxia condition for 48h . Protein levels of LC3 I / II in HepG - 2 ( D ) cells under hypoxia condition for 48h . E Protein level of LC3 I / II in HepG - 2 and sorafenib - resistant HepG - 2 cells under hypoxia condition for 48h . F - H Overexpression of shRNA - resistant wild type METTL3 but not catalytic mutant METTL3 rescue autophagy phenotype in METTL3 - knockdown SMMC - 7721 cells ( F ) , Bel - 7402 cells ( G ) and sorafenib - resistant HepG2 cells ( H ) by representative immunostaining images of LC3 under hypoxia condition for 48h . Scale bar , 200μm . Quantification of the numbers of GFP - LC3 puncta / cell by Imaris in F - 1 , G - 1 and H - 1 , respectively . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . I - K Protein levels of LC3 I / II in METTL3 - knockdown SMMC - 7721 cells ( I ) , Bel - 7402 cells ( J ) and HepG - 2 cells ( K ) treated with or without 3 - MA under hypoxia condition for 24h . L - N The IC50 of sorafenib in SMMC - 7721 cells ( L ) , Bel - 7402 cells ( M ) and HepG - 2 cells ( N ) treated with or without 3 - MA for 24h under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

Figure 3A Electron micrographs of METTL3-knockdown SMMC-7721 cells under hypoxia for 48 h.Protein levels of LC3 I/II in METTL3-knockdown SMMC-7721 (B), Bel-7402 (C) cells under hypoxia condition for 48h.Protein levels of LC3 I/II in HepG-2 (D) cells under hypoxia condition for 48h.E Protein level of LC3 I/II in HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia condition for 48h.F-H Overexpression of shRNA-resistant wild type METTL3 but not catalytic mutant METTL3 rescue autophagy phenotype in METTL3-knockdown SMMC-7721 cells (F), Bel-7402 cells (G) and sorafenib-resistant HepG2 cells (H) by representative immunostaining images of LC3 under hypoxia condition for 48h. Scale bar, 200μm. Quantification of the numbers of GFP-LC3 puncta/cell by Imaris in F-1, G-1 and H-1, respectively. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.I-K Protein levels of LC3 I/II in METTL3-knockdown SMMC-7721 cells (I), Bel-7402 cells (J) and HepG-2 cells (K) treated with or without 3-MA under hypoxia condition for 24h.L-N The IC50 of sorafenib in SMMC-7721 cells (L), Bel-7402 cells (M) and HepG-2 cells (N) treated with or without 3-MA for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


Figure 4A Gene Set Enrichment Analysis ( GSEA ) output images of cellular response to stress pathways displaying a correlation of differentially regulated genes in METTL3 - knockdown HepG2 , SMMC - 7721 and Bel - 7402 cells with the C5 . biological process symbol set . ( p < 0 . 001 ) . C Protein level of FOXO3 in METTL3 - knockdown HCCs under hypoxia for 48h . D Half - life of FOXO3 mRNA in Bel - 7402 cells and correspondent METTL3 - knockdown Bel - 7402 cells treated with actinomycin D . E Half - life of FOXO3 protein in Bel - 7402 cells and correspondent METTL3 - knockdown Bel - 7402 cells treated with cycloheximide . Quantification of the protein optical density by Image J in E - 1 . F Analysis of FOXO3 mRNA in non - ribosome portion ( < 40S ) , 40S , 60S , 80S and polysome for the METTL3 - knockdown Bel - 7402 cells compare to control cells under hypoxia for 48h . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . G Knockdown of YTHDF1 and YTHDF2 in Bel - 7402 cells by lentiviral shRNAs . H Knockdown of YTHDF1 but not YTHDF2 abolished the increase of FOXO3 protein level in METTL3 - overexpressing Bel - 7402 cells . Quantification of the protein optical density by Image J in H - 1 and H - 2 . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . I RNA level of FOXO3 in YTHDF1 - knockdown Bel - 7402 cells by RT - PCR . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . J The m6A - IP sequencing under hypoxia proved the m6A modification participated in regulation of FOXO3 . The YTHDF1 binding site locates at the 3 ' UTR of FOXO3 . L Relative luciferase activity of the wild - type and mutant form of FOXO3 3 ' UTR reporter vectors in Bel - 7402 cells with various levels of METTL3 and YTHDF1 . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . M Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the m6A MeRIP analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . N Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the YTHDF1 - RIP analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . O Correlation analysis of the RNA expression levels of METTL3 and FOXO3 in liver tumor cohort ( Kim - 90 ) by Pearson . P Correlation analysis of the RNA expression levels of YTHDF1 and FOXO3 in liver tumor cohort ( Kim - 90 ) by Pearson .

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

Figure 4A Gene Set Enrichment Analysis (GSEA) output images of cellular response to stress pathways displaying a correlation of differentially regulated genes in METTL3-knockdown HepG2, SMMC-7721 and Bel-7402 cells with the C5. biological process symbol set. (p < 0.001).C Protein level of FOXO3 in METTL3-knockdown HCCs under hypoxia for 48h.D Half-life of FOXO3 mRNA in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with actinomycin D.E Half-life of FOXO3 protein in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with cycloheximide. Quantification of the protein optical density by Image J in E-1.F Analysis of FOXO3 mRNA in non-ribosome portion (<40S), 40S, 60S, 80S and polysome for the METTL3-knockdown Bel-7402 cells compare to control cells under hypoxia for 48h. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.G Knockdown of YTHDF1 and YTHDF2 in Bel-7402 cells by lentiviral shRNAs.H Knockdown of YTHDF1 but not YTHDF2 abolished the increase of FOXO3 protein level in METTL3-overexpressing Bel-7402 cells. Quantification of the protein optical density by Image J in H-1 and H-2. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.I RNA level of FOXO3 in YTHDF1-knockdown Bel-7402 cells by RT-PCR. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.J The m6A-IP sequencing under hypoxia proved the m6A modification participated in regulation of FOXO3. The YTHDF1 binding site locates at the 3'UTR of FOXO3.L Relative luciferase activity of the wild-type and mutant form of FOXO3 3'UTR reporter vectors in Bel-7402 cells with various levels of METTL3 and YTHDF1. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.M Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the m6A MeRIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.N Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the YTHDF1-RIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.O Correlation analysis of the RNA expression levels of METTL3 and FOXO3 in liver tumor cohort (Kim-90) by Pearson.P Correlation analysis of the RNA expression levels of YTHDF1 and FOXO3 in liver tumor cohort (Kim-90) by Pearson.


Figure 5A Validation of biomarker genes related to autophagy in FOXO3 - knockdown Bel - 7402 cells by RT - PCR . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . B Protein level of FOXO3 in naïve HepG - 2 and sorafenib - resistant HepG - 2 cells under hypoxia for 48h . C Protein levels of LC3 I / II in FOXO3 - knockdown SMMC - 7721 cells . D Overexpression of FOXO3 in sorafenib - resistant HepG - 2 cells by Western blot . E Protein levels of LC3 I / II in METTL3 - knockdown & FOXO3 - overexpression SMMC - 7721 cells under hypoxia for 48 h . F Electron micrographs of METTL3 - knockdown SMMC7721 cells and METTL3 - knockdown & FOXO3 - overexpression SMMC7721 cells under hypoxia for 48 h . G Representative Immunostaining images of LC3 in METTL3 - knockdown SMMC7721 cells and METTL3 - knockdown & FOXO3 - overexpression SMMC7721 cells under hypoxia for 48 h . Scale bar , 200μm . The quantification of the numbers of GFP - LC3 puncta / cell by Imaris in G - 1 . H Representative Immunostaining images of LC3 in naïve HepG - 2 cells and sorafenib - resistant HepG - 2 cells under hypoxia for 48 h . Scale bar , 200μm . The quantification of the numbers of GFP - LC3 puncta / cell by Imaris in H - 1 . I The IC50 of METTL3 - knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . J The IC50 of naïve HepG - 2 cells and sorafenib - resistant HepG - 2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments . K Cell survival assay of METTL3 - knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition . L Cell survival assay of naïve HepG - 2 cells and sorafenib - resistant HepG - 2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition .

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Figure 5A Validation of biomarker genes related to autophagy in FOXO3-knockdown Bel-7402 cells by RT-PCR. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.B Protein level of FOXO3 in naïve HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia for 48h.C Protein levels of LC3 I/II in FOXO3-knockdown SMMC-7721 cells.D Overexpression of FOXO3 in sorafenib-resistant HepG-2 cells by Western blot.E Protein levels of LC3 I/II in METTL3-knockdown & FOXO3-overexpression SMMC-7721 cells under hypoxia for 48 h.F Electron micrographs of METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. G Representative Immunostaining images of LC3 in METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in G-1. H Representative Immunostaining images of LC3 in naïve HepG-2 cells and sorafenib-resistant HepG-2 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in H-1. I The IC50 of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.J The IC50 of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.K Cell survival assay of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.L Cell survival assay of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.


Figure 6A Depletion of METTL3 in Hepa1 - 6 cells by CRISPR - Cas9 knockout enhanced sorafenib - resistance in vivo . Growth curve ( B ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days ( n = 5 ) . tumor weight ( C ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days ( n = 5 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD . D Knockdown of METTL3 slightly increased liver tumor growth treated with sorafenib in orthotopic xenograft mouse model . Stable METTL3 - knockdown Bel - 7402 cells and control cells were injected into the liver of each NOD / SCID mouse . 3 weeks after injection , livers were separated for pathological analysis . Red circle indicates tumors in the livers . E Liver tumors in orthotopic xenograft mouse model were confirmed by hematoxylin and eosin staining . F Overexpression of FOXO3 rescued sorafenib - resistance mediated by METTL3 - knockdown in xenograft liver tumors . Representative xenograft tumors at endpoint , containing 6 groups . Growth curve ( G ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days ( n = 5 ) . tumor weight ( H ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days ( n = 5 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD . I H & E and immunohistochemical images of Ki67 , LC3 - B , VEGF - A in Hepa1 - 6 cells derived xenografts in 6 treatment groups . J - L Quantification of Ki67 ( K ) , LC3 - B ( K ) , and VEGF - A ( L ) expression in immunohistochemical images by Image Pro Plus ( IPP ) analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD .

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

Figure 6A Depletion of METTL3 in Hepa1-6 cells by CRISPR-Cas9 knockout enhanced sorafenib-resistance in vivo.Growth curve (B) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days (n=5).tumor weight (C) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days (n=5). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.D Knockdown of METTL3 slightly increased liver tumor growth treated with sorafenib in orthotopic xenograft mouse model. Stable METTL3-knockdown Bel-7402 cells and control cells were injected into the liver of each NOD/SCID mouse. 3 weeks after injection, livers were separated for pathological analysis. Red circle indicates tumors in the livers.E Liver tumors in orthotopic xenograft mouse model were confirmed by hematoxylin and eosin staining.F Overexpression of FOXO3 rescued sorafenib-resistance mediated by METTL3-knockdown in xenograft liver tumors. Representative xenograft tumors at endpoint, containing 6 groups.Growth curve (G) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days (n=5).tumor weight (H) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days (n=5). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.I H&E and immunohistochemical images of Ki67, LC3-B, VEGF-A in Hepa1-6 cells derived xenografts in 6 treatment groups.J-L Quantification of Ki67 (K), LC3-B (K), and VEGF-A (L) expression in immunohistochemical images by Image Pro Plus (IPP) analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.


Figure 7In vivo analyses of tumor ( B ) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups . In vivo analyses of tumor growth ( C ) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups . In vivo analyses of tumor weight ( D ) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD . E H & E and immunohistochemical images of Ki67 , LC3 - B , VEGF - A in randomly selected PDX # 1 and PDX # 2 tumors . Quantification of Ki67 ( F ) expression in immunohistochemical images from PDX tumors by Image Pro Plus ( IPP ) analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD . Quantification of LC3 - B ( G ) , and VEGF - A ( H ) expression in immunohistochemical images from PDX tumors by Image Pro Plus ( IPP ) analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD . I Knockdown of METTL3 by siRNA in PDX # 1 and PDX # 2 tissues were confirmed by Western blot .

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Figure 7In vivo analyses of tumor (B) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups.In vivo analyses of tumor growth (C) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups.In vivo analyses of tumor weight (D) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.E H&E and immunohistochemical images of Ki67, LC3-B, VEGF-A in randomly selected PDX#1 and PDX#2 tumors.Quantification of Ki67 (F) expression in immunohistochemical images from PDX tumors by Image Pro Plus (IPP) analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.Quantification of LC3-B (G), and VEGF-A (H) expression in immunohistochemical images from PDX tumors by Image Pro Plus (IPP) analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.I Knockdown of METTL3 by siRNA in PDX#1 and PDX#2 tissues were confirmed by Western blot.


Figure 1A . Cartoon representation of the Dln1 dimer ( PDB 4ZNO ) . Subunit A is colored in gray . The lectin module of subunit B is colored in yellow , whereas the middle and C - terminal moieties of the aerolysin module are colored in hot pink and green , respectively . The pre - stem hairpin is colored in blue . The bound sucrose molecules are shown as light pink sticks . B . The dimeric interface . Residues involved in dimeric interactions are shown as sticks .

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Figure 1A. Cartoon representation of the Dln1 dimer (PDB 4ZNO). Subunit A is colored in gray. The lectin module of subunit B is colored in yellow, whereas the middle and C-terminal moieties of the aerolysin module are colored in hot pink and green, respectively. The pre-stem hairpin is colored in blue. The bound sucrose molecules are shown as light pink sticks.B. The dimeric interface. Residues involved in dimeric interactions are shown as sticks.


Figure 2A . A close - up view of the sucrose - binding pocket . Residues hydrogen - bonding with sucrose are shown as yellow sticks , and the sucrose molecule is shown as light pink stick . B . The glycan binding profile of Dln1 . This profile represents FITC - labeled Dln1 at 200 μg / ml screened against 609 printed glycans on Mammalian Printed Array version 5 . 2 in replicates of six . A list of top 8 hits reacted with a concentration of 200 μg / ml is shown in Appendix Table S1 . The two kinds of Dln1 - binding glycan structures ( No . 214 and 316 ) are illustrated in schematic . C & amp ; amp ; D . Close - up views of Manα1 - 2Man and Manα1 - 3Man binding sites . The mannobiose molecules are shown as cyan and gray sticks , respectively . The Fo - Fc electron density maps of disaccharides and the disaccharide - binding sites are shown as gray mesh and countered at 3 . 0 . The hydrogen - bonding residues are shown as yellow sticks . Polar interactions with the mannobiose are shown as dashed lines .

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Figure 2A. A close-up view of the sucrose-binding pocket. Residues hydrogen-bonding with sucrose are shown as yellow sticks, and the sucrose molecule is shown as light pink stick.B. The glycan binding profile of Dln1. This profile represents FITC-labeled Dln1 at 200 μg/ml screened against 609 printed glycans on Mammalian Printed Array version 5.2 in replicates of six. A list of top 8 hits reacted with a concentration of 200 μg/ml is shown in Appendix Table S1. The two kinds of Dln1-binding glycan structures (No. 214 and 316) are illustrated in schematic.C&amp;D. Close-up views of Manα1-2Man and Manα1-3Man binding sites. The mannobiose molecules are shown as cyan and gray sticks, respectively. The Fo-Fc electron density maps of disaccharides and the disaccharide-binding sites are shown as gray mesh and countered at 3.0 . The hydrogen-bonding residues are shown as yellow sticks. Polar interactions with the mannobiose are shown as dashed lines.


Figure 3A & amp ; amp ; B . ELISA of Dln1 towards glycosylated gp120 or yeast mannan . The curve represents the average of triplicate detections , using BSA as the control . Points are averages ( ± standard error of the mean ) of triplicate determinations . C . Binding of FITC - labeled Dln1 and D135A mutant to saccharomyces cerevisiae cells . The scale bar represents 5 μm . D . Immunoblot analysis of Dln1 and D135A mutant after incubating with S . cerevisiae cells . The seeing bands represent the cell - bound protein after extensive washing the yeast cells . The monomer and oligomer bands are highlighted by the black arrow .

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Figure 3A&amp;B. ELISA of Dln1 towards glycosylated gp120 or yeast mannan. The curve represents the average of triplicate detections, using BSA as the control. Points are averages (±standard error of the mean) of triplicate determinations.C. Binding of FITC-labeled Dln1 and D135A mutant to saccharomyces cerevisiae cells. The scale bar represents 5 μm.D. Immunoblot analysis of Dln1 and D135A mutant after incubating with S. cerevisiae cells. The seeing bands represent the cell-bound protein after extensive washing the yeast cells. The monomer and oligomer bands are highlighted by the black arrow.


Figure 4A . Oligomerization of Dln1 in the presence of S . cerevisiae mannan or liposomes at different pH values . Histograms are averages ( ± standard error of the mean ) of triplicate determinationsB & amp ; amp ; C . Dye release velocity from the liposomes in the presence of Dln1 at different concentrations , and various pH values . Liposomes were incubated with Dln1 of different concentrations at pH 5 . 5 , and 1 μM Dln1 at various pH values , respectively . D . Oligomerization of Dln1M5 with or without liposomes at different pH values . All samples for electrophoresis were prepared with boiling at 95 ° C for 5 min , and the comparative gels were processed in parallel . The oligomer band was quantified by Image J software with the average of triplicate detections , using the marker band as a sample processing control . The SDS - PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4 . Histograms are averages ( ± standard error of the mean ) of triplicate determinations . E . Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5 . 5 . F . The inhibition of dye release in presence of 0 . 5 M Dln1M5 and 25 mM polyethylene glycols ( PEG ) of different molecular weights at a fixed pH value of 5 . 5 . Dye efflux was monitored in a period of 1400 sec . Triton X - 100 at 0 . 1 % was added to completely disrupt liposomes . Dye efflux was measured as a percentage to the maximal release upon addition of Triton X - 100 . Carboxyfluorescein was loaded in the liposomes for the detection of dye efflux .

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Figure 4A. Oligomerization of Dln1 in the presence of S. cerevisiae mannan or liposomes at different pH values. Histograms are averages (±standard error of the mean) of triplicate determinationsB&amp;C. Dye release velocity from the liposomes in the presence of Dln1 at different concentrations, and various pH values. Liposomes were incubated with Dln1 of different concentrations at pH 5.5, and 1 μM Dln1 at various pH values, respectively.D. Oligomerization of Dln1M5 with or without liposomes at different pH values. All samples for electrophoresis were prepared with boiling at 95°C for 5 min, and the comparative gels were processed in parallel. The oligomer band was quantified by Image J software with the average of triplicate detections, using the marker band as a sample processing control. The SDS-PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4. Histograms are averages (±standard error of the mean) of triplicate determinations.E. Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5.5.F. The inhibition of dye release in presence of 0.5 M Dln1M5 and 25 mM polyethylene glycols (PEG) of different molecular weights at a fixed pH value of 5.5. Dye efflux was monitored in a period of 1400 sec. Triton X-100 at 0.1% was added to completely disrupt liposomes. Dye efflux was measured as a percentage to the maximal release upon addition of Triton X-100. Carboxyfluorescein was loaded in the liposomes for the detection of dye efflux.


Figure 5A . Negative - stain electron microscopy image of Dln1M5 in presence of liposomes . B . Side views of Dln1M5 oligomers attached to the membrane of liposomes . The inset represents the respective class averages . C & amp ; amp ; D . Transmission electron microscope micrograph of dispersed ring - like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer . The inset represents a few 2D class averages showing the octamer pattern from some individual rings . C & amp ; amp ; D . Transmission electron microscope micrograph of dispersed ring - like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer . The inset represents a few 2D class averages showing the octamer pattern from some individual rings . E . EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer . The inset represents the computed diffraction pattern Fourier transform of the image . F . Projection map with a P4 symmetry . G . The top , side and cut - open views of the 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation . The surface of membrane is indicated by dashed lines . The scale bar is 10 nm .

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 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 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 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

Figure 5A. Negative-stain electron microscopy image of Dln1M5 in presence of liposomes.B. Side views of Dln1M5 oligomers attached to the membrane of liposomes. The inset represents the respective class averages.C&amp;D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings.C&amp;D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings.E. EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer. The inset represents the computed diffraction pattern Fourier transform of the image.F. Projection map with a P4 symmetry.G. The top, side and cut-open views of the 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation. The surface of membrane is indicated by dashed lines. The scale bar is 10 nm.


Figure 6A . Top and side views of eight lectin modules docked to the 20 Å EM map . Due to the moderate resolution of the EM map , the flexible aerolysin module cannot be fit in the map . It is important to notice that this model is only a hypothetical fit . Each pair of the lectin modules are colored in yellow and orange , respectively . The β - barrel of γ - hemolysin ( PDB 3B07 ) was used to mimic the β - barrel backbone of Dln1 . The position of cell membrane is indicated by dashed lines . B . B - factor of Dln1 subunit B . The mobility is represented by coil thickness from thin ( low ) to thick ( high ) , and color from blue ( low ) to red ( high ) . Notably , the pre - stem hairpin and distal moiety of aerolysin module are particularly mobile . C . Position of Cα atoms along MD simulations of apo - form ( red ) and the pre - stem hairpin extraction form ( blue ) are shown with the lectin modules superimposed ( 200 structures for each ) .

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 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

Figure 6A. Top and side views of eight lectin modules docked to the 20 Å EM map. Due to the moderate resolution of the EM map, the flexible aerolysin module cannot be fit in the map. It is important to notice that this model is only a hypothetical fit. Each pair of the lectin modules are colored in yellow and orange, respectively. The β-barrel of γ-hemolysin (PDB 3B07) was used to mimic the β-barrel backbone of Dln1. The position of cell membrane is indicated by dashed lines.B. B-factor of Dln1 subunit B. The mobility is represented by coil thickness from thin (low) to thick (high), and color from blue (low) to red (high). Notably, the pre-stem hairpin and distal moiety of aerolysin module are particularly mobile.C. Position of Cα atoms along MD simulations of apo-form (red) and the pre-stem hairpin extraction form (blue) are shown with the lectin modules superimposed (200 structures for each).


Figure 1A - C Volcano plots depicting significantly up ( blue ) and down ( pink ) regulated phosphorylation events for DMXAA vs DMSO groups for 5 min ( A ) , 10 min ( B ) and 30 min ( C ) timepoints . - Log10 ( p - value ) is plotted on the y - axis vs Log2 ( Fold change [ FC ] ) on the x - axis . Significantly regulated events display p - value > 0 . 01 and Log2 ( FC ) > 1 or < - 1 .

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Figure 1A-C Volcano plots depicting significantly up (blue) and down (pink) regulated phosphorylation events for DMXAA vs DMSO groups for 5 min (A), 10 min (B) and 30 min (C) timepoints. -Log10(p-value) is plotted on the y-axis vs Log2(Fold change [FC]) on the x-axis. Significantly regulated events display p-value>0.01 and Log2(FC) >1 or<-1.


Figure 2A Sting - / - iBMDMs expressing mRuby3 - STING ( magenta ) and eGFP - Golgin84 ( green ) were imaged live on the 3i marianas spinning disk microscope . Z stack images were acquired either before ( i . e . , untreated ) or after 50 μg / mL DMXAA treatment ( i . e . , image captured at 12 min post DMXAA treatment ) . Data is shown as a maximum intensity projection ( MIP ) of Z stack images . D Sting - / - iBMDMs expressing mRuby3 - STING were imaged live on the spinning disk microscope . Representative images of timelapse showing STING vesicles exiting the Golgi for a recording starting 27 min after 50 μg / mL DMXAA treatment . Data is shown as a MIP of Z stack images . Square region of interest ( ROI ) indicates zoomed insert . Scale bar = 5 μm . F Sting - / - iBMDMs expressing eGFP - STING were imaged live on the spinning disk microscope . A representative image of timelapse showing transport of STING vesicles for a recording starting 1 h 18 min after 50 μg / mL DMXAA treatment . Data is shown as a MIP of Z stack images . Square ROI indicates zoomed insert . Scale bar = 5 μm .

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 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

Figure 2A Sting-/- iBMDMs expressing mRuby3-STING (magenta) and eGFP-Golgin84 (green) were imaged live on the 3i marianas spinning disk microscope. Z stack images were acquired either before (i.e., untreated) or after 50 μg/mL DMXAA treatment (i.e., image captured at 12 min post DMXAA treatment). Data is shown as a maximum intensity projection (MIP) of Z stack images.D Sting-/- iBMDMs expressing mRuby3-STING were imaged live on the spinning disk microscope. Representative images of timelapse showing STING vesicles exiting the Golgi for a recording starting 27 min after 50 μg/mL DMXAA treatment. Data is shown as a MIP of Z stack images. Square region of interest (ROI) indicates zoomed insert. Scale bar = 5 μm.F Sting-/- iBMDMs expressing eGFP-STING were imaged live on the spinning disk microscope. A representative image of timelapse showing transport of STING vesicles for a recording starting 1 h 18 min after 50 μg/mL DMXAA treatment. Data is shown as a MIP of Z stack images. Square ROI indicates zoomed insert. Scale bar = 5 μm.


Figure 3A Primary BMDMs were left untreated or treated with 25 μg / mL DMXAA for 2 , 4 or 6 h . Cells were lysed for immunoblot with the indicated antibodies . Data shown is representative of 3 independent experiments . B Primary BMDMs were left untreated or treated with 10 μg / mL 2 ' 3 ' - cGAM ( PS ) 2 for 2 , 4 or 6 h . Cells were lysed for immunoblot with the indicated antibodies . Data shown is representative of 3 independent experiments . D Sting - / - , WT or TBK1 / IKKε dKO iBMDMs were left untreated or treated with 10 μg / mL 2 ' 3 ' - cGAM ( PS ) 2 for 1 , 2 , or 3 h . Cells were lysed for immunoblot with the indicated antibodies . Data shown representative of 3 independent experiments .

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Figure 3A Primary BMDMs were left untreated or treated with 25 μg/mL DMXAA for 2, 4 or 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments. B Primary BMDMs were left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 2, 4 or 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.D Sting-/-, WT or TBK1/IKKε dKO iBMDMs were left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 1, 2, or 3 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown representative of 3 independent experiments.


Figure 4B Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 25 μg / mL DMXAA for 1 , 2 or 4 h . Cells were lysed for immunoblot with the indicated antibodies . Data shown is representative of 3 independent experiments . C Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 10 μg / mL 2 ' 3 ' - cGAM ( PS ) 2 for 4 h . Cells were lysed for immunoblot with the indicated antibodies . Data shown is representative of 3 independent experiments . D Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated ( UT ) or treated with 25 μg / mL DMXAA for 1 , 2 or 4 h . Cell were fixed and stained for p - STING ( white ) and the nucleus ( DAPI ; blue ) , before Z stack images were acquired on the LSM980 confocal microscope . Images are displayed as a maximum intensity projection ( MIP ) of Z stack images . Scale bar = 5 μm . Data shown is representative of 3 independent experiments . G - I Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated ( UT ) or treated with 25 μg / mL DMXAA or 10 μg / mL 2 ' 3 ' - cGAM ( PS ) 2 for 4 h . Cells were lysed for RNA purification and the expression of Ifnb1 ( G ) , Isg15 ( H ) and Il6 ( I ) was analysed by qPCR . Data is shown as mean ± SEM combined from N = 3 independent experiments . Statistical analysis was performed using two - way ANOVA using Bonferroni ' s multiple comparisons test , where * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 25 μg/mL DMXAA for 1, 2 or 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments. C Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.D Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/mL DMXAA for 1, 2 or 4 h. Cell were fixed and stained for p-STING (white) and the nucleus (DAPI; blue), before Z stack images were acquired on the LSM980 confocal microscope. Images are displayed as a maximum intensity projection (MIP) of Z stack images. Scale bar = 5 μm. Data shown is representative of 3 independent experiments.G-I Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/mL DMXAA or 10 μg/mL 2'3'-cGAM(PS)2 for 4 h. Cells were lysed for RNA purification and the expression of Ifnb1 (G) , Isg15 (H) and Il6 (I) was analysed by qPCR. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Bonferroni's multiple comparisons test, where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


Figure 5B Sting - / - iBMDMs expressing HA - STING were left UT or treated with 50 μg / mL DMXAA for 1 or 3 h . Cells were lysed and a portion of lysate underwent immunoblot with the indicated antibodies ( i . e . , lysate ) . The remaining lysate underwent immunoprecipitation with an anti - HA antibody ( i . e . , HA pull down ) . Samples then underwent immunoblot with the indicated antibodies . Data representative of 3 independent experiments . D Square ROI indicates zoomed insert from C , displayed as both merged and single colour images . Scale bar = 5 μm .

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Figure 5B Sting-/- iBMDMs expressing HA-STING were left UT or treated with 50 μg/mL DMXAA for 1 or 3 h. Cells were lysed and a portion of lysate underwent immunoblot with the indicated antibodies (i.e., lysate). The remaining lysate underwent immunoprecipitation with an anti-HA antibody (i.e., HA pull down). Samples then underwent immunoblot with the indicated antibodies. Data representative of 3 independent experiments.D Square ROI indicates zoomed insert from C, displayed as both merged and single colour images. Scale bar = 5 μm.


Figure 6A dCas9 - KRAB expressing iBMDMs , without ( i . e . , dCas9 alone ) or with HRS - targeting sgRNAs ( i . e . , sg1 , sg2 or sg3 ) were treated for 48 h with 1 μg / mL doxycycline ( Dox ) to induce sgRNA expression . Cells were then left untreated ( UT ) , or treated with 20 μg / mL DMXAA or 10 μg / mL 2 ' 3 ' - cGAM ( PS ) 2 for 6 h . Cells were lysed for immunoblot with the indicated antibodies . Data shown is representative of 3 independent experiments . B - C dCas9 - KRAB expressing iBMDMs , without ( i . e . , dCas9 alone ) or with HRS - targeting sgRNAs ( i . e . , sg1 , sg2 or sg3 ) were treated for 48 h with 1 μg / mL Dox to induce sgRNA expression . Cells were then left UT , or treated with 20 μg / mL DMXAA or 5 μg / mL 2 ' 3 ' - cGAM ( PS ) 2 for 6 h . Cell supernatant was collected and assayed for secreted IFNβ ( B ) and IL6 ( C ) by ELISA . Data is shown as mean ± SEM combined from N = 3 independent experiments . Statistical analysis was performed using two - way ANOVA using Bonferroni ' s test , where * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n . d . = not detectedD - F dCas9 - KRAB expressing iBMDMs , without ( i . e . , dCas9 alone ) or with HRS - targeting sgRNAs ( i . e . , sg1 , sg2 or sg3 ) were treated for 24 h with 1 μg / mL Dox to induce sgRNA expression . iBMDMs were further treated with 1 μg / mL ( i . e . 1000 ng / mL ) or 100 ng / mL H - 151 as indicated , and incubated for a further 24 h . Cells were lysed for RNA purification and the expression of Ifnb1 ( D ) , Isg15 ( E ) and Irf7 ( F ) was analysed by qPCR . Data is shown as mean ± SEM combined from N = 3 independent experiments . Statistical analysis was performed using two - way ANOVA using Tukey ' s multiple comparisons test , where * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Bars display significant differences between dCas9 and sgRNA knockdowns in the absence of H - 151 . Additional asterisks represent significant differences between the absence or presence of H - 151 across the same cell line .

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

Figure 6A dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/mL doxycycline (Dox) to induce sgRNA expression. Cells were then left untreated (UT), or treated with 20 μg/mL DMXAA or 10 μg/mL 2'3'-cGAM(PS)2 for 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.B-C dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/mL Dox to induce sgRNA expression. Cells were then left UT, or treated with 20 μg/mL DMXAA or 5 μg/mL 2'3'-cGAM(PS)2 for 6 h. Cell supernatant was collected and assayed for secreted IFNβ (B) and IL6 (C) by ELISA. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Bonferroni's test, where * p<0.05, *** p<0.001, **** p<0.0001. n.d. = not detectedD-F dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 24 h with 1 μg/mL Dox to induce sgRNA expression. iBMDMs were further treated with 1 μg/mL (i.e. 1000 ng/mL) or 100 ng/mL H-151 as indicated, and incubated for a further 24 h. Cells were lysed for RNA purification and the expression of Ifnb1 (D), Isg15 (E) and Irf7 (F) was analysed by qPCR. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Tukey's multiple comparisons test, where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Bars display significant differences between dCas9 and sgRNA knockdowns in the absence of H-151. Additional asterisks represent significant differences between the absence or presence of H-151 across the same cell line.


Figure 7A - B VPS4aWT or VPS4aE228Q dominant negative BMDMs were left untreated ( UT ) or treated with 1 μg / mL doxycycline ( Dox ) for 4 h . BMDMs were then further left UT or treated with 25 μg / mL DMXAA ( A ) or 10 μg / mL 2 ' 3 ' - cGAM ( PS ) 2 ( B ) for 4 h . Cells were lysed for immunoblot with the indicated antibodies . Data shown is representative of 2 independent experiments for A and 3 independent experiments for B . C - D VPS4aWT or VPS4aE228Q dominant negative BMDMs were left UT or treated with 1 μg / mL Dox for 4 h . BMDMs were then further left UT or treated with 25 μg / mL DMXAA for 4 h . Cell supernatant was collected and secreted IFNβ ( C ) or IL - 6 ( D ) was measured by ELISA . Data is shown as mean ± SEM combined from N = 3 independent experiments . Statistical analysis was performed using one - way ANOVA . using Bonferroni ' s test , where * p < 0 . 01 . n . d . = not

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

Figure 7A-B VPS4aWT or VPS4aE228Q dominant negative BMDMs were left untreated (UT) or treated with 1 μg/mL doxycycline (Dox) for 4 h. BMDMs were then further left UT or treated with 25 μg/mL DMXAA (A) or 10 μg/mL 2'3'-cGAM(PS)2 (B) for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 2 independent experiments for A and 3 independent experiments for B.C-D VPS4aWT or VPS4aE228Q dominant negative BMDMs were left UT or treated with 1 μg/mL Dox for 4 h. BMDMs were then further left UT or treated with 25 μg/mL DMXAA for 4 h. Cell supernatant was collected and secreted IFNβ (C) or IL-6 (D) was measured by ELISA. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using one-way ANOVA. using Bonferroni's test, where * p<0.01. n.d. = not detected


figf1 ( a ) p35 interacts with EndoB1 in a GST pulldown assay . ( b , c ) EndoB1 co - immunoprecipitates ( IP ) with p35 in COS - 7 cell lysates expressing p35 and full - length EndoB1 ( b ) and adult rat brain lysate ( c ) . ( e ) Dose - dependent phosphorylation of EndoB1 by Cdk5 / p35 in an in vitro kinase assay . ( f ) Cdk5 / p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay . ( g ) Cdk5 / p35 phosphorylates EndoB1 at Thr 145 in COS - 7 cells . ( h ) Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5 + / + , but not Cdk5 - / - mouse brains . Quantification of p - Thr145 - EndoB1 level is shown in the right panel . Data are means ± s . e . m . ; n = 3 . Uncropped images of blots are shown in Supplementary Fig .

O B-PANEL_START 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 B-PANEL_START 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 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

figf1(a) p35 interacts with EndoB1 in a GST pulldown assay.(b,c) EndoB1 co-immunoprecipitates (IP) with p35 in COS-7 cell lysates expressing p35 and full-length EndoB1 (b) and adult rat brain lysate (c).(e) Dose-dependent phosphorylation of EndoB1 by Cdk5/p35 in an in vitro kinase assay.(f) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay.(g) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in COS-7 cells.(h) Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5+/+, but not Cdk5-/- mouse brains. Quantification of p-Thr145-EndoB1 level is shown in the right panel. Data are means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8


figf2 ( a ) Left : starvation increases the level of RFP - LC3 puncta ( arrows ) in rat cortical neurons . Middle : quantification of the percentage of cells with RFP - LC3 puncta . Right : pretreatment with 10 mM 3 - MA attenuates the starvation - induced increase in LC3 - II level in neurons . Data are means ± s . e . m . ; n = 3 . Samples were run on the same gel and spliced together to create the image shown . ( b ) Knockdown of EndoB1 abolishes the starvation - induced increase in LC3 - II levels ( left ) and the percentage of cells with RFP - LC3 puncta ( right ) in neurons . Data are means ± s . e . m . ; n = 3 . ( c ) Starvation enhances Cdk5 activity ( top ) and increases the level of Thr 145 - phosphorylated EndoB1 in neurons ( top and bottom ) . Histone H1 was used as a Cdk5 substrate . Data are means ± s . e . m . ; n = 3 . ( d , e ) Pretreatment with Ros ( d ) or knockdown of Cdk5 ( e ) attenuates starvation - induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons . Data are means ± s . e . m . ; n = 3 for d and n = 8 for e . DMSO , dimethylsulphoxide . ( f ) Overexpression of EndoB1T145A significantly reduces the starvation - induced increase in LC3 - II levels ( left ) and the percentage of cells with RFP - LC3 puncta ( right ) in neurons . Data are means ± s . e . m . ; n = 6 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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 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 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 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

figf2(a) Left: starvation increases the level of RFP-LC3 puncta (arrows) in rat cortical neurons. Middle: quantification of the percentage of cells with RFP-LC3 puncta. Right: pretreatment with 10 mM 3-MA attenuates the starvation-induced increase in LC3-II level in neurons. Data are means ± s.e.m.; n=3. Samples were run on the same gel and spliced together to create the image shown.(b) Knockdown of EndoB1 abolishes the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP-LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=3.(c) Starvation enhances Cdk5 activity (top) and increases the level of Thr 145-phosphorylated EndoB1 in neurons (top and bottom). Histone H1 was used as a Cdk5 substrate. Data are means ± s.e.m.; n=3.(d,e) Pretreatment with Ros (d) or knockdown of Cdk5 (e) attenuates starvation-induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons. Data are means ± s.e.m.; n=3 for d and n=8 for e. DMSO, dimethylsulphoxide.(f) Overexpression of EndoB1T145A significantly reduces the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP-LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=6. Uncropped images of blots are shown in Supplementary Fig. S8.