Figure 7 ( B ) ACE2 , Syndecan 1 and Syndecan 4 cell surface expression on nasal epithelial cells , compared to polarized epithelial Calu - 3 was confirmed by quantitative real - time PCR . ( C , D ) Nasal epithelial cells were exposed to SARS - CoV - 2 isolate ( hCoV - 19 / Italy , 100 TCID / mL ) either directly or after pre - treatment with antibodies against ACE2 cell surface receptors ( 1 hour at 37 ° C ) or after pre - treatment with LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . Detection of viral binding after 4 hours at 4 ° C ( C ) and persistently - infected cells lysed after 24 hours at 37 ° C ( D ) was determined by quantitative real - time PCR . Data information : Data show the mean values and error bars are the SEM . Statistical analysis was performed using ( C , D ) ordinary on - way ANOVA with Tukey ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( C ) ( n = 3 ) , ( D ) ( n = 3 in duplicates ) .

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

Figure 7(B) ACE2, Syndecan 1 and Syndecan 4 cell surface expression on nasal epithelial cells, compared to polarized epithelial Calu-3 was confirmed by quantitative real-time PCR. (C, D) Nasal epithelial cells were exposed to SARS-CoV-2 isolate (hCoV-19/Italy, 100 TCID/mL) either directly or after pre-treatment with antibodies against ACE2 cell surface receptors (1 hour at 37°C )or after pre-treatment with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Detection of viral binding after 4 hours at 4°C (C) and persistently-infected cells lysed after 24 hours at 37°C (D) was determined by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (C, D) ordinary on-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3), (D) (n=3 in duplicates).


figf1 ( b ) THPN - induced cell death is prevented by the autophagic inhibitors wortmannin ( 100 nM ) and chloroquine ( 20 μM ) but not by the apoptotic inhibitor zVAD ( 20 μM ) or the necrotic inhibitors butylated hydroxyanisole ( 100 μM ) and necrostatin - 1 ( 30 μM ) . The cells were pretreated with each inhibitor for 1 h separately before the THPN treatment ( 20 μM , 48 h ) . Ctrl , control . ( c ) THPN induces autophagy in melanoma cells . Top , THPN ( 20 μM , 24 h ) induces a change in the localization of GFP - LC3 fusion proteins from a ubiquitous , diffuse pattern to cytoplasmic puncta . Serum starvation ( SS , 24 h ) was used as a positive control ( scale bars , 10 μm ) . Middle , the GFP - LC3 - positive cells with puncta were calculated from five randomly picked regions . Bottom , the conversion of LC3 - I to LC3 - II induced by THPN was determined by western blotting . Images of full blots are shown in Supplementary Figure 7 . All of the data are presented as the mean ± s . e . m . of three independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; NS , not significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(b) THPN-induced cell death is prevented by the autophagic inhibitors wortmannin (100 nM) and chloroquine (20 μM) but not by the apoptotic inhibitor zVAD (20 μM) or the necrotic inhibitors butylated hydroxyanisole (100 μM) and necrostatin-1 (30 μM). The cells were pretreated with each inhibitor for 1 h separately before the THPN treatment (20 μM, 48 h). Ctrl, control.(c) THPN induces autophagy in melanoma cells. Top, THPN (20 μM, 24 h) induces a change in the localization of GFP-LC3 fusion proteins from a ubiquitous, diffuse pattern to cytoplasmic puncta. Serum starvation (SS, 24 h) was used as a positive control (scale bars, 10 μm). Middle, the GFP-LC3-positive cells with puncta were calculated from five randomly picked regions. Bottom, the conversion of LC3-I to LC3-II induced by THPN was determined by western blotting. Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.


figf2 ( a ) THPN induces TR3 translocation from the cytoplasm to the mitochondria , detected by western blotting for the various cell fractionations ( top ) and by confocal microscopy ( bottom ) . The cells were treated with THPN at the times indicated and detected by TR3 ( red ) and Hsp60 ( green ) . The nuclei were stained with DAPI ( blue ) ( scale bars , 10 μm ) . PARP , Tubulin and Hsp60 were used as loading controls for nuclear extracts ( NE ) , cytoplasmic extracts ( CE ) and mitochondrial fractions ( Mito ) , respectively . IB , immunoblotting . ( b ) TR3 mutant Flag - ΔNLS but not Flag - NLS promotes the induction of autophagy in response to THPN treatment . The siRNA - resistant versions of Flag - ΔNLS and Flag - NLS were transfected into TR3 - knockdown A375 cells . Top , the cells cotransfected with GFP - LC3 were further treated with THPN for 24 h before microscopic analysis ( scale bars , 10 μm ) . Middle , the conversion of LC3 - I to LC3 - II was analyzed by western blotting . The rate of cell survival was determined after THPN treatment for 48 h ( bottom ) . ' - ' represents vehicle treatment only . ( c ) Nix assists the TR3 mitochondrial targeting . Cells with Nix , Pink1 and Bnip3 knockdown were treated with THPN ( 20 μM , 6 h ) . Top , mitochondrial TR3 was assayed by western blotting . Nix knockdown abolishes the THPN - induced ( 20 μM , 24 h ) formation of LC3 foci ( middle and bottom ; scale bars , 10 μm ) . Images of full blots are shown in Supplementary Figure 7 . All of the data are presented as the mean ± s . e . m . of three independent experiments . * * P 0 . 01 ; NS , not significant ; ctrl , control .

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 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

figf2(a) THPN induces TR3 translocation from the cytoplasm to the mitochondria, detected by western blotting for the various cell fractionations (top) and by confocal microscopy (bottom). The cells were treated with THPN at the times indicated and detected by TR3 (red) and Hsp60 (green). The nuclei were stained with DAPI (blue) (scale bars, 10 μm). PARP, Tubulin and Hsp60 were used as loading controls for nuclear extracts (NE), cytoplasmic extracts (CE) and mitochondrial fractions (Mito), respectively. IB, immunoblotting.(b) TR3 mutant Flag-ΔNLS but not Flag-NLS promotes the induction of autophagy in response to THPN treatment. The siRNA-resistant versions of Flag-ΔNLS and Flag-NLS were transfected into TR3-knockdown A375 cells. Top, the cells cotransfected with GFP-LC3 were further treated with THPN for 24 h before microscopic analysis (scale bars, 10 μm). Middle, the conversion of LC3-I to LC3-II was analyzed by western blotting. The rate of cell survival was determined after THPN treatment for 48 h (bottom). '-' represents vehicle treatment only.(c) Nix assists the TR3 mitochondrial targeting. Cells with Nix, Pink1 and Bnip3 knockdown were treated with THPN (20 μM, 6 h). Top, mitochondrial TR3 was assayed by western blotting. Nix knockdown abolishes the THPN-induced (20 μM, 24 h) formation of LC3 foci (middle and bottom; scale bars, 10 μm). Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01; NS, not significant; ctrl, control.


figf3 ( b ) Site C of LBD is critical for Nix binding , as determined by the in vitro pull - down assay . The purified bead - bound GST or GST - Nix proteins were incubated with different mutants as indicated and THPN ( 20 μM ) for 3 h ; the proteins were then analyzed by western blotting . IB , immunoblotting . ( e ) DPDO cannot facilitate Nix binding to TR3 . Top , the structure of DPDO . Middle , DPDO binding could not form the interacting surface for Nix , unlike THPN . The structures of Arg563 and Ser553 are the same as those in the native structure . Bottom , DPDO does not enhance the interaction between TR3 and Nix . Different plasmids were transfected into A375 cells as indicated , and cells were treated with DPDO ( 20 μM , 3 h ) . The TR3 interaction with Nix was determined by co - immunoprecipitation . Images of full blots are shown in Supplementary Figure 7 . IP , immunoprecipitation ; HA , hemagglutinin .

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

figf3(b) Site C of LBD is critical for Nix binding, as determined by the in vitro pull-down assay. The purified bead-bound GST or GST-Nix proteins were incubated with different mutants as indicated and THPN (20 μM) for 3 h; the proteins were then analyzed by western blotting. IB, immunoblotting.(e) DPDO cannot facilitate Nix binding to TR3. Top, the structure of DPDO. Middle, DPDO binding could not form the interacting surface for Nix, unlike THPN. The structures of Arg563 and Ser553 are the same as those in the native structure. Bottom, DPDO does not enhance the interaction between TR3 and Nix. Different plasmids were transfected into A375 cells as indicated, and cells were treated with DPDO (20 μM, 3 h). The TR3 interaction with Nix was determined by co-immunoprecipitation. Images of full blots are shown in Supplementary Figure 7. IP, immunoprecipitation; HA, hemagglutinin.


figf4 ( a ) THPN induces the dissipation of ΔΨm in both time - and dose - dependent manners . The compromised ΔΨm is shifted to the right upon THPN treatment in flow cytometry . ' - ' represents vehicle treatment only . ( b ) Effects of different plasmids on the THPN - induced dissipation of ΔΨm . Top , endogenous TR3 was knocked down by shRNA , and the siRNA - resistant version of TR3 , TR3 - NLS , TR3 - ΔNLS or TAD ( Supplementary Fig . 2c ) were separately transfected into the cells as indicated . Bottom , similarly , the siRNA - resistant version of Nix or ΔTM were separately transfected into Nix knockdown cells . Transfected cells were treated with THPN ( 20 μM , 12 h ) , and their ΔΨm was assessed by flow cytometry . Ctrl , control . ( c ) ANT1 and VDAC1 are required for the THPN - induced autophagy . The ANT1 and VDAC1 shRNA - expressing plasmids , which also encoded EGFP for evaluating the transfection efficiency , were separately transfected into cells ; mCherry - LC3 was cotransfected to display autophagy . Green cells are those transfected with ANT1 or VDAC1 shRNA , and the cells with red cytoplasmic puncta are autophagic cells ( top ; scale bars , 10 μm ) . ( d ) ANT1 and VDAC1 are required for the THPH - induced dissipation of ΔΨm and cell death . Endogenous ANT1 or VDAC1 was separately knocked down by shRNA , and the dissipation of ΔΨm was determined after THPN ( 20 μM , 12 h ) treatment ( left ) . ANT1 and VDAC1 knockdown abolishes the THPN - induced ( 20 μM , 48 h ) cell death ( right ) . All of the data are presented as the mean ± s . e . m . of three independent experiments . * * P 0 . 01 .

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 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

figf4(a) THPN induces the dissipation of ΔΨm in both time- and dose-dependent manners. The compromised ΔΨm is shifted to the right upon THPN treatment in flow cytometry. '-' represents vehicle treatment only.(b) Effects of different plasmids on the THPN-induced dissipation of ΔΨm. Top, endogenous TR3 was knocked down by shRNA, and the siRNA-resistant version of TR3, TR3-NLS, TR3-ΔNLS or TAD (Supplementary Fig. 2c) were separately transfected into the cells as indicated. Bottom, similarly, the siRNA-resistant version of Nix or ΔTM were separately transfected into Nix knockdown cells. Transfected cells were treated with THPN (20 μM, 12 h), and their ΔΨm was assessed by flow cytometry. Ctrl, control.(c) ANT1 and VDAC1 are required for the THPN-induced autophagy. The ANT1 and VDAC1 shRNA-expressing plasmids, which also encoded EGFP for evaluating the transfection efficiency, were separately transfected into cells; mCherry-LC3 was cotransfected to display autophagy. Green cells are those transfected with ANT1 or VDAC1 shRNA, and the cells with red cytoplasmic puncta are autophagic cells (top; scale bars, 10 μm).(d) ANT1 and VDAC1 are required for the THPH-induced dissipation of ΔΨm and cell death. Endogenous ANT1 or VDAC1 was separately knocked down by shRNA, and the dissipation of ΔΨm was determined after THPN (20 μM, 12 h) treatment (left). ANT1 and VDAC1 knockdown abolishes the THPN-induced (20 μM, 48 h) cell death (right). All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01.


figf5 ( a ) Endogenous TR3 is localized in the MIM in response to THPN stimulation . Left , isolated mitochondria from cells treated with THPN ( 20 μM , 6 h ) were incubated with proteinase K ( 20 μM and 40 μM ) for 30 min . TR3 expression was determined . Tom20 in MOM and Tim23 in MIM were used as controls . Right , the mitochondria were further subjected to submitochondrial fractionation . VDAC1 , Tim23 and Hsp60 were used to represent MOM , MIM and mitochondrial matrix protein , respectively . ' - ' represents vehicle treatment only . ( b ) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM . Endogenous Toms were separately knocked down by their corresponding shRNAs , and the TR3 expression of mitochondrial fractions was detected as described above . Ctrl , control . ( c ) Tom40 and Tom70 are required for the THPN - associated dissipation of ΔΨm ( left ) , conversion of LC3 - I to LC3 - II ( right and top ) and cell death ( right and bottom ) . Endogenous Toms were separately knocked down by the corresponding shRNAs . ΔΨm , LC3 - II and cell death were detected as described above . Images of full blots are shown in Supplementary Figure 7 . ( d ) The mitochondria were markedly eliminated after treatment with THPN for 24 h . Right , endogenous Tom40 and Tom70 were knocked down , and the amount of mitochondria was measured by the fluorescence intensity of Tom20 ( green ) . Left , the volume of mitochondria from five randomly picked regions was quantified by ImageJ software . All of the data are presented as the mean ± s . e . m . of three independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; NS , not significant .

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

figf5(a) Endogenous TR3 is localized in the MIM in response to THPN stimulation. Left, isolated mitochondria from cells treated with THPN (20 μM, 6 h) were incubated with proteinase K (20 μM and 40 μM) for 30 min. TR3 expression was determined. Tom20 in MOM and Tim23 in MIM were used as controls. Right, the mitochondria were further subjected to submitochondrial fractionation. VDAC1, Tim23 and Hsp60 were used to represent MOM, MIM and mitochondrial matrix protein, respectively. '-' represents vehicle treatment only.(b) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM. Endogenous Toms were separately knocked down by their corresponding shRNAs, and the TR3 expression of mitochondrial fractions was detected as described above. Ctrl, control.(c) Tom40 and Tom70 are required for the THPN-associated dissipation of ΔΨm (left), conversion of LC3-I to LC3-II (right and top) and cell death (right and bottom). Endogenous Toms were separately knocked down by the corresponding shRNAs. ΔΨm, LC3-II and cell death were detected as described above. Images of full blots are shown in Supplementary Figure 7.(d) The mitochondria were markedly eliminated after treatment with THPN for 24 h. Right, endogenous Tom40 and Tom70 were knocked down, and the amount of mitochondria was measured by the fluorescence intensity of Tom20 (green). Left, the volume of mitochondria from five randomly picked regions was quantified by ImageJ software. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.


figf6 ( a ) THPN inhibits the formation of tumors in the liver and lung in a TR3 - dependent manner . B16 cells transfected with either control shRNA , TR3 shRNA or ATG7 shRNA ( 5 × 105 ) were separately injected into the lateral tail vein of 6 - week - old C57BL / 6J mice ( n = 7 ) . THPN were administered 3 d after inoculation . The tumors formed in the lung and liver are indicated ( top ; scale bars , 5 mm ) and were counted ( bottom ) . Ctrl , control . ( b ) Crossed Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 + / + and Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 − / − mice ( n = 12 ) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms , and mice were treated with THPN as described in Online Methods . The representative images of dorsal melanomas ( top ) and the tumor numbers ( bottom ) are presented . ( c ) The survival curves of crossed Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 + / + and Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 − / − mice are shown ( n > 16 ) .

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

figf6(a) THPN inhibits the formation of tumors in the liver and lung in a TR3-dependent manner. B16 cells transfected with either control shRNA, TR3 shRNA or ATG7 shRNA (5 × 105) were separately injected into the lateral tail vein of 6-week-old C57BL/6J mice (n = 7). THPN were administered 3 d after inoculation. The tumors formed in the lung and liver are indicated (top; scale bars, 5 mm) and were counted (bottom). Ctrl, control.(b) Crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice (n = 12) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms, and mice were treated with THPN as described in Online Methods. The representative images of dorsal melanomas (top) and the tumor numbers (bottom) are presented.(c) The survival curves of crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice are shown (n > 16).


Figure 1B . Comparisons of MTAP mRNA and protein expression between CL1 - 0 and CL1 - 5 cells detected by RT - qPCR ( top , mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) and Western blot assays using anti - MTAP antibody ( bottom , representative of three independent experiments ) . C . Cell invasion abilities of MTAP - overexpressing CL1 - 5 or MTAP - knockout CL1 - 0 and H1650 were determined by Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) . sgCTRL indicates cells transduced with a non - targeting control sgRNA , and sgMTAP indicates cells transduced with a sgRNA targeting MTAP . Bottom : the protein expression levels of V5 - MTAP and endogenous MTAP were detected by Western blots . E . Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays . Top : representative photographs of lungs and H & E staining of the lung sections . The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows . Bottom : quantification of averaged primary tumor sizes , metastatic incidence and nodule number . F . Kaplan - Meier analyses of overall survival ( top ) and progression - free survival ( bottom ) for 101 patients with lung adenocarcinoma grouped into high - or low - MTAP mRNA expression measured by RT - qPCR . p values were obtained by log - rank test .

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

Figure 1B. Comparisons of MTAP mRNA and protein expression between CL1-0 and CL1-5 cells detected by RT-qPCR (top, mean ± SD, Student t test, n=3, biological replicates, *p < 0.05) and Western blot assays using anti-MTAP antibody (bottom, representative of three independent experiments).C. Cell invasion abilities of MTAP-overexpressing CL1-5 or MTAP-knockout CL1-0 and H1650 were determined by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). sgCTRL indicates cells transduced with a non-targeting control sgRNA, and sgMTAP indicates cells transduced with a sgRNA targeting MTAP. Bottom: the protein expression levels of V5-MTAP and endogenous MTAP were detected by Western blots.E. Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays. Top: representative photographs of lungs and H&E staining of the lung sections. The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows. Bottom: quantification of averaged primary tumor sizes, metastatic incidence and nodule number.F. Kaplan-Meier analyses of overall survival (top) and progression-free survival (bottom) for 101 patients with lung adenocarcinoma grouped into high- or low-MTAP mRNA expression measured by RT-qPCR. p values were obtained by log-rank test.


Figure 2A . The necessity of MTAP activity in cell invasion . CL1 - 5 and A549 cells were introduced with indicated plasmids and subjected to Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 compared to the vector control group ) . The protein expression levels of exogenous wild - type and mutant V5 - MTAP were detected by Western blots . B . Western blot analysis of the symmetric dimethylarginine ( sDMA ) , monomethylarginine ( mMA ) and asymmetric dimethylarginine ( aDMA ) levels in MTAP - overexpressing and MTAP - knockout cells . V5 - MTAP wild - type and D220A mutant were restored in H1650 MTAP - knockout cells . Data shown are representative of three independent experiments . C . In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti - sDMA antibody . Arrow marks dimethyl - vimentin . D . Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells . Arrow marks dimethyl - vimentin . Data shown are representative of three independent experiments . E - F . Immunoprecipitation analysis for vimentin dimethylation in control and MTAP - knockout cells . Arrows mark vimentin , asterisks mark heavy chains , and arrowheads mark nonspecific bands . Data shown are representative of three independent experiments . G . Vimentin - knockdown counteracts MTAP - loss - induced effect on cell invasion . MTAP - knockout and control H1650 cells were transfected with vimentin - specific siRNAs for 72 hours and analyzed by Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) . The silence efficiency of vimentin was examined by Western blots .

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Figure 2A. The necessity of MTAP activity in cell invasion. CL1-5 and A549 cells were introduced with indicated plasmids and subjected to Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05 compared to the vector control group). The protein expression levels of exogenous wild-type and mutant V5-MTAP were detected by Western blots.B. Western blot analysis of the symmetric dimethylarginine (sDMA), monomethylarginine (mMA) and asymmetric dimethylarginine (aDMA) levels in MTAP-overexpressing and MTAP-knockout cells. V5-MTAP wild-type and D220A mutant were restored in H1650 MTAP-knockout cells. Data shown are representative of three independent experiments.C. In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti-sDMA antibody. Arrow marks dimethyl-vimentin.D. Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells. Arrow marks dimethyl-vimentin. Data shown are representative of three independent experiments.E-F. Immunoprecipitation analysis for vimentin dimethylation in control and MTAP-knockout cells. Arrows mark vimentin, asterisks mark heavy chains, and arrowheads mark nonspecific bands. Data shown are representative of three independent experiments.G. Vimentin-knockdown counteracts MTAP-loss-induced effect on cell invasion. MTAP-knockout and control H1650 cells were transfected with vimentin-specific siRNAs for 72 hours and analyzed by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). The silence efficiency of vimentin was examined by Western blots.


Figure 3F . Validation of the PRMT5 - catalyzed methylation sites on V5 - vimentin . HEK293T cells were transfected with indicated constructs and subjected to immunoprecipitation analysis . The intensity of sDMA signals was quantified by ImageJ software , normalized to the intensity of corresponding immunoprecipitated V5 - vimentin , then relative to the V5 - vimentin wild - type group and represented as percentage . Data were the averaged percentage from three independent experiments .

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Figure 3F. Validation of the PRMT5-catalyzed methylation sites on V5-vimentin. HEK293T cells were transfected with indicated constructs and subjected to immunoprecipitation analysis. The intensity of sDMA signals was quantified by ImageJ software, normalized to the intensity of corresponding immunoprecipitated V5-vimentin, then relative to the V5-vimentin wild-type group and represented as percentage. Data were the averaged percentage from three independent experiments.


Figure 4B . Reconstitution of vimentin - knockout cells with wild - type , 4RK - and 4RF - mutated V5 - vimentin . Both protein and mRNA levels of vimentin were determined by Western blots and RT - qPCR . Data shown are representative of three independent experiments . C . Vimentin protein and mRNA levels in indicated paired cells were measured by Western blots and RT - qPCR . Data shown are representative of three independent experiments . D . Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT - qPCR . Data shown are representative of three independent experiments . E . Western blot analysis of vimentin protein levels in MTAP - knockout and control cells transfected with PRMT5 siRNAs ( left ) or Flag - PRMT5 ( right ) . Arrows mark the site of Flag - PRMT5 and endogenous PRMT5 . Data shown are representative of three independent experiments . F . Effect of PRMT5 on the levels of ectopically expressed wild - type , 4RK - and 4RF - mutated V5 - vimentin in HEK293T cells assessed by Western blots . Data shown are representative of three independent experiments . G . Effects of wild - type and catalytically dead R368A mutant PRMT5 on the level of V5 - vimentin in HEK293T cells detected by Western blot assays . Data shown are representative of three independent experiments .

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Figure 4B. Reconstitution of vimentin-knockout cells with wild-type, 4RK- and 4RF-mutated V5-vimentin. Both protein and mRNA levels of vimentin were determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.C. Vimentin protein and mRNA levels in indicated paired cells were measured by Western blots and RT-qPCR. Data shown are representative of three independent experiments.D. Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.E. Western blot analysis of vimentin protein levels in MTAP-knockout and control cells transfected with PRMT5 siRNAs (left) or Flag-PRMT5 (right). Arrows mark the site of Flag-PRMT5 and endogenous PRMT5. Data shown are representative of three independent experiments.F. Effect of PRMT5 on the levels of ectopically expressed wild-type, 4RK- and 4RF-mutated V5-vimentin in HEK293T cells assessed by Western blots. Data shown are representative of three independent experiments. G. Effects of wild-type and catalytically dead R368A mutant PRMT5 on the level of V5-vimentin in HEK293T cells detected by Western blot assays. Data shown are representative of three independent experiments.


Figure 5A . Measurement of V5 - vimentin wild - type , 4RK and 4RF mutant protein levels in CL1 - 5 vimentin - knockout cells treated with 10 μM MG132 by Western blots . Data shown are representative of three independent experiments . B . Western blot analysis of vimentin protein levels in control and MTAP - knockout cells treated with 10 μM MG132 . Data shown are representative of three independent experiments . Immunoprecipitation analysis of ubiquitination ( C ) of V5 - vimentin in HEK293T cells transfected with wild - type , 4RK - or 4RF - mutated vimentin in the presence of HA - tagged ubiquitin . Arrows mark the sites of V5 - vimentin . Data shown are representative of three independent experiments . K48 - linked polyubiquitination ( D ) of V5 - vimentin in HEK293T cells transfected with wild - type , 4RK - or 4RF - mutated vimentin in the presence of HA - tagged ubiquitin . Arrows mark the sites of V5 - vimentin . Data shown are representative of three independent experiments . E . Immunoprecipitation analysis for K48 - linked polyubiquitination of vimentin in CL1 - 0 control and MTAP - knockout cells . Arrow marks the site of vimentin . Data shown are representative of three independent experiments . F - G . Western blot analysis of V5 - vimentin wild - type , 4RK and 4RF mutant levels in CL1 - 0 cells ( F ) or endogenous vimentin levels in control and MTAP - knockout cells ( G ) treated with 50 μg / ml cycloheximide ( CHX ) . Bottom : the intensity of V5 - vimentin and vimentin signals was quantified by ImageJ software , normalized to the internal control β - actin , then normalized to zero time point , and plotted against time points ( mean ± SE , n = 3 , biological replicates ) .

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Figure 5A. Measurement of V5-vimentin wild-type, 4RK and 4RF mutant protein levels in CL1-5 vimentin-knockout cells treated with 10 μM MG132 by Western blots. Data shown are representative of three independent experiments.B. Western blot analysis of vimentin protein levels in control and MTAP-knockout cells treated with 10 μM MG132. Data shown are representative of three independent experiments.Immunoprecipitation analysis of ubiquitination (C) of V5-vimentin in HEK293T cells transfected with wild-type, 4RK- or 4RF-mutated vimentin in the presence of HA-tagged ubiquitin. Arrows mark the sites of V5-vimentin. Data shown are representative of three independent experiments.K48-linked polyubiquitination (D) of V5-vimentin in HEK293T cells transfected with wild-type, 4RK- or 4RF-mutated vimentin in the presence of HA-tagged ubiquitin. Arrows mark the sites of V5-vimentin. Data shown are representative of three independent experiments.E. Immunoprecipitation analysis for K48-linked polyubiquitination of vimentin in CL1-0 control and MTAP-knockout cells. Arrow marks the site of vimentin. Data shown are representative of three independent experiments.F-G. Western blot analysis of V5-vimentin wild-type, 4RK and 4RF mutant levels in CL1-0 cells (F) or endogenous vimentin levels in control and MTAP-knockout cells (G) treated with 50 μg/ml cycloheximide (CHX). Bottom: the intensity of V5-vimentin and vimentin signals was quantified by ImageJ software, normalized to the internal control β-actin, then normalized to zero time point, and plotted against time points (mean ± SE, n=3, biological replicates).


Figure 6A - B . The correlation of MTAP and vimentin or PRMT5 and vimentin at protein ( A ) and mRNA ( B ) levels from 89 lung adenocarcinoma patients . r is the Spearman ' s rank correlation coefficient . C . Representative images of IHC staining . D . An association between MTAP and vimentin levels in 124 lung cancer patients was examined by Spearman ' s rank correlation . E . Kaplan - Meier analyses of overall survival for lung cancer patients grouped by MTAP and vimentin levels from IHC staining . p value was obtained by log - rank test .

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Figure 6A-B. The correlation of MTAP and vimentin or PRMT5 and vimentin at protein (A) and mRNA (B) levels from 89 lung adenocarcinoma patients. r is the Spearman's rank correlation coefficient.C. Representative images of IHC staining.D. An association between MTAP and vimentin levels in 124 lung cancer patients was examined by Spearman's rank correlation.E. Kaplan-Meier analyses of overall survival for lung cancer patients grouped by MTAP and vimentin levels from IHC staining. p value was obtained by log-rank test.


Figure 1 ( A ) ATG - 5 staining in non - cancerous gastric tissues scored 285 ( × 200 ) ; ( B ) ATG - 5 staining in GC tumor tissues scored 50 ( × 400 ) ; ( C ) ATG - 5 staining in GC tumor tissues scored 270 ( × 400 ) ; ( D ) ATG - 5 staining in GC tumor tissues scored 400 ( × 200 ) .

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Figure 1  (A) ATG-5 staining in non-cancerous gastric tissues scored 285(×200); (B) ATG-5 staining in GC tumor tissues scored 50(×400); (C) ATG-5 staining in GC tumor tissues scored 270(×400); (D) ATG-5 staining in GC tumor tissues scored 400(×200).


Figure 2 ( A and B ) Patients were divided into four following subgroups based on ATG - 5 immunostaining : scored 0 - 99 ( curve a ) , scored 100 - 199 ( curve b ) , scored 200 - 299 ( curve c ) and scored 300 - 400 ( curve d ) . The difference among different subgroups was statistically significant as evaluated by overall Log - rank comparisons ( OS : P < 0 . 001 , DFS : P = 0 . 003 ) . Pairwise Log - rank comparisons showed that the subgroup D exhibited the poorest survival rates as compared to other subgroups ( OS : P < 0 . 05 , DFS : P < 0 . 01 ) . ( C and D ) Patients were divided into four following subgroups based on MRP - 1 immunostaining : scored 0 - 99 ( curve a ) , scored 100 - 199 ( curve b ) , scored 200 - 299 ( curve c ) and scored 300 - 400 ( curve d ) . The difference among various subgroups was statistically significant by overall Log - rank comparisons ( OS : P = 0 . 001 , DFS : P = 0 . 018 ) . Pairwise Log - rank comparisons showed that the subgroup A had the most favorable prognosis among the four subgroups ( OS : P < 0 . 05 , DFS : P < 0 . 001 ) . ( E and F ) Patients were divided into four subgroups according to different TNM stages . The difference among different subgroups was statistically significant by overall Log - rank comparisons ( OS : P < 0 . 001 , DFS : P < 0 . 001 ) . Pairwise Log - rank comparisons showed that subgroups III or IV exhibited poorer survival rates than that of subgroups IB and II ( OS : P < 0 . 01 , DFS : P < 0 . 001 ) .

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Figure 2(A and B) Patients were divided into four following subgroups based on ATG-5 immunostaining: scored 0-99 (curve a), scored 100-199 (curve b), scored 200-299 (curve c) and scored 300-400 (curve d). The difference among different subgroups was statistically significant as evaluated by overall Log-rank comparisons (OS: P<0.001, DFS: P = 0.003). Pairwise Log-rank comparisons showed that the subgroup D exhibited the poorest survival rates as compared to other subgroups (OS: P<0.05, DFS: P<0.01).(C and D) Patients were divided into four following subgroups based on MRP-1 immunostaining: scored 0-99 (curve a), scored 100-199 (curve b), scored 200-299 (curve c) and scored 300-400 (curve d). The difference among various subgroups was statistically significant by overall Log-rank comparisons (OS: P = 0.001, DFS: P = 0.018). Pairwise Log-rank comparisons showed that the subgroup A had the most favorable prognosis among the four subgroups (OS: P<0.05, DFS: P<0.001).(E and F) Patients were divided into four subgroups according to different TNM stages. The difference among different subgroups was statistically significant by overall Log-rank comparisons (OS: P<0.001, DFS: P<0.001). Pairwise Log-rank comparisons showed that subgroups III or IV exhibited poorer survival rates than that of subgroups IB and II (OS: P<0.01, DFS: P<0.001).


Figure 3 ( A ) The level of ATG5 was detected in cell lines using western blot analysis . β - actin was used as internal controls . ( B ) The IC 25 , IC50 and IC75 of SGC - 7901 and SGC - 7901 / DDP cells were tested using the MTT assays after DPP treatment .

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Figure 3(A) The level of ATG5 was detected in cell lines using western blot analysis. β -actin was used as internal controls.(B) The IC 25, IC50 and IC75 of SGC-7901 and SGC-7901/DDP cells were tested using the MTT assays after DPP treatment.


Figure 4 ( A ) The mRNA level of ATG - 5 was detected by real time PCR after treatment with siRNAs . GAPDH was used as internal controls . ( B ) The protein level of ATG - 5 was detected by western blot after treatment with siRNAs . β - actin was used as internal controls . ( C ) The proliferation ability was tested using MTT assay 48 hours or 72 hours after different treatment .

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Figure 4(A) The mRNA level of ATG-5 was detected by real time PCR after treatment with siRNAs. GAPDH was used as internal controls.(B) The protein level of ATG-5 was detected by western blot after treatment with siRNAs. β -actin was used as internal controls.(C) The proliferation ability was tested using MTT assay 48 hours or 72 hours after different treatment.


Figure 5 ( A ) The autophagy was detected by immunofluorescence assay of LC3B in the cells 48 hours after different treatment . ( B ) The protein levels of LC3A and LC3B were tested by western blot . β - actin was used as internal controls .

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Figure 5(A) The autophagy was detected by immunofluorescence assay of LC3B in the cells 48 hours after different treatment.(B) The protein levels of LC3A and LC3B were tested by western blot. β-actin was used as internal controls.


Figure 1A Effects of myeloid - specific Lamtor1 deficiency on peritoneal IL - 1β production , CD11b + Ly6C + monocyte recruitment , and CD11b + Ly6G + neutrophil recruitment after an intraperitoneal injection of 600 μL alum solution ( 20 mg / mL , 6 h ) , Data are shown as means ± SEM . * indicates P < 0 . 05 and * * * indicates P < 0 . 001 by Student ' s t - test , n = 6 mice ( peritoneal IL - 1β production ) and 4 mice ( FACS analysis ) . B Effects of myeloid - specific Lamtor1 deficiency in an acute gouty arthritis model . MSU crystals ( 0 . 5 mg ) or the PBS control were injected intra - articularly into the tibia - tarsal joint of Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre mice . Mice were assessed for joint swelling using electronic calipers . Images show photomicrographs ( upper panel ) and hematoxylin and eosin - stained sections ( middle and lower panels ) of ankle joints obtained at 24 h . Data are shown as the means ± SEM . * * * indicates P < 0 . 001 by Student ' s t - test , n = 7 mice each . C Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation . ELISA assay shows IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with LPS ( 200 ng / mL , 4 h ) , followed by nigericin ( 15 μM , 1 h ) , ATP ( 10 mM , 1 h ) , or GPN ( 100 nM , 6 h ) . D Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO different single clones , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) . E , F Effects of Lamtor1 deficiency on IL - 1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous ROS ( hydrogen peroxide ) . ELISA assay shows IL - 1β secretion in supernatants after treatment with LPS ( 200 ng / mL ) , Rotenone ( 10 μM ) , or Antimycin ( 10 μg / mL ) for 6 h or followed by hydrogen peroxide ( 3 . 86 mM ) . G Effects of Lamtor1 reconstitution in Lamtor1 - deficient macrophages on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in BMDM supernatants from Lamtor1flox / flox cells , Lamtor1flox / flox LysM - Cre cells , and Lamtor1flox / flox LysM - Cre cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with LPS ( 200 ng / mL , 4 h ) . H Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) . I Effects of Lamtor1 deficiency on IL - 18 secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 18 secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) . J Effects of Lamtor1 deficiency on IL - 1β secretion after Pyrin inflammasome activation . ELISA assay showing IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with TcdB ( 0 . 5 μg / mL , 3 h ) after LPS ( 200 ng / mL , 4 h ) stimulation . K Effects of Lamtor1 deficiency on IL - 1β secretion after AIM2 inflammasome activation . ELISA assay showing IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with Poly ( dA : dT ) ( 5 μg / mL , overnight ) and Lipofectamine 3000 after LPS ( 200 ng / mL , 4 h ) stimulation .

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Figure 1A Effects of myeloid-specific Lamtor1 deficiency on peritoneal IL-1β production, CD11b+ Ly6C+ monocyte recruitment, and CD11b+ Ly6G+ neutrophil recruitment after an intraperitoneal injection of 600 μL alum solution (20 mg/mL, 6 h), Data are shown as means ± SEM. * indicates P < 0.05 and *** indicates P < 0.001 by Student's t-test, n = 6 mice (peritoneal IL-1β production) and 4 mice (FACS analysis).B Effects of myeloid-specific Lamtor1 deficiency in an acute gouty arthritis model. MSU crystals (0.5 mg) or the PBS control were injected intra-articularly into the tibia-tarsal joint of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre mice. Mice were assessed for joint swelling using electronic calipers. Images show photomicrographs (upper panel) and hematoxylin and eosin-stained sections (middle and lower panels) of ankle joints obtained at 24 h. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 7 mice each.C Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation. ELISA assay shows IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h), followed by nigericin (15 μM, 1 h), ATP (10 mM, 1 h), or GPN (100 nM, 6 h).D Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO different single clones, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).E, F Effects of Lamtor1 deficiency on IL-1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous ROS (hydrogen peroxide). ELISA assay shows IL-1β secretion in supernatants after treatment with LPS (200 ng/mL), Rotenone (10 μM), or Antimycin (10 μg/mL) for 6 h or followed by hydrogen peroxide (3.86 mM).G Effects of Lamtor1 reconstitution in Lamtor1-deficient macrophages on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in BMDM supernatants from Lamtor1flox/flox cells, Lamtor1flox/flox LysM-Cre cells, and Lamtor1flox/flox LysM-Cre cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with LPS (200 ng/mL, 4 h).H Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).I Effects of Lamtor1 deficiency on IL-18 secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-18 secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).J Effects of Lamtor1 deficiency on IL-1β secretion after Pyrin inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with TcdB (0.5 μg/mL, 3 h) after LPS (200 ng/mL, 4 h) stimulation.K Effects of Lamtor1 deficiency on IL-1β secretion after AIM2 inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with Poly(dA:dT) (5 μg/mL, overnight) and Lipofectamine 3000 after LPS (200 ng/mL, 4 h) stimulation.


Figure 2Effects of Lamtor1 deficiency on Caspase - 1 processing after NLRP3 inflammasome activation . Western blot shows caspase - 1 / p10 , pro - caspase - 1 , Lamtor1 , and β - actin . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs ( A ) ( B ) were treated with nigericin ( 15 μM , 1 h ) after LPS [ 200 ng / mL , for 4 h ( BMDM ) stimulation . Effects of Lamtor1 deficiency on Caspase - 1 processing after NLRP3 inflammasome activation . Western blot shows caspase - 1 / p10 , pro - caspase - 1 , Lamtor1 , and β - actin . WT and Lamtor1 KO THP - 1 cells ( B ) were treated with nigericin ( 15 μM , 1 h ) after LPS [ 200 ng / mL 2 h ( THP - 1 ) ] stimulation . C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation . Western blot shows GSDMD , Lamtor1 , and β - actin . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with nigericin ( 15 μM , 1 h ) after LPS ( 200 ng / mL , 4 h ) stimulation . D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with nigericin ( 15 μM , 1 h ) after LPS ( 200 ng / mL , 4 h ) stimulation and the percentage of propidium iodide positive cells were counted by flow - cytometry . E Effects of Lamtor1 deficiency on IL - 1β dynamics . Western blot shows IL - 1β and β - actin . WT cells and Lamtor1 KO cells were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) stimulation . F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP - 1 cells . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with active form of GSDMD , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) . G Confirmation of the reconstitution of the active form Caspase - 1 ( p10 and p20 ) or GSDMD ( GSDMD - NT ) . H Effects of Lamtor1 deficiency on the priming phase . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with treated with LPS ( 200 ng / mL , 4 h ) . I Effects of IL - 1β and active form of Caspase - 1 reconstitution on Lamtor1 KO THP - 1 cells . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with IL - 1β , active form of Caspase - 1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) .

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 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 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 O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 2Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs (A) (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL, for 4 h (BMDM) stimulation.Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. WT and Lamtor1 KO THP-1 cells (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL 2 h (THP-1)] stimulation.C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation. Western blot shows GSDMD, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation.D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation and the percentage of propidium iodide positive cells were counted by flow-cytometry.E Effects of Lamtor1 deficiency on IL-1β dynamics. Western blot shows IL-1β and β-actin. WT cells and Lamtor1 KO cells were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) stimulation.F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with active form of GSDMD, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).G Confirmation of the reconstitution of the active form Caspase-1 (p10 and p20) or GSDMD (GSDMD-NT).H Effects of Lamtor1 deficiency on the priming phase. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with treated with LPS (200 ng/mL, 4 h).I Effects of IL-1β and active form of Caspase-1 reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with IL-1β, active form of Caspase-1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).


Figure 3A Representative images of mutant Lamtor1 . PMA - primed Lamtor1 KO THP - 1 macrophages reconstituted with full - length or truncated Lamtor1 . B Effects of the Lamtor1 point mutation on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion in THP - 1 cell supernatants from WT cells , Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full - length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine ( G2A ) ; all cell types were then treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) . C Effects of Lamtor1 deficiency on mTORC1 activity . Immunoblot analysis of Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with LPS ( 200 ng / mL , 4 h ) . D Effect of the rapamycin treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from PMA ( 50 nM , overnight ) primed WT THP - 1 cells treated with rapamycin at indicated concentration for 2 h , then treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) . E Effect of rapamycin treatment on Caspase - 1 processing . Western blot shows caspase - 1 / p20 . LPS ( 200 ng / mL , 4 h ) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin ( 15 μM , 1 h ) . F Effect of Torin - 1 treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from WT BMDMs pretreated with Torin - 1 ( 0 , 1 , 10 , 100 , or 250 nM ) for 2 h before LPS ( 200 ng / ml , 4 h ) and nigericin ( 15 μM , 1 h ) treatment ( left panel ) . Western blot shows phospho - p70 - S6K from BMDMs pretreated with Torin - 1 at the indicated concentrations for 2 h before LPS and nigericin treatment ( right panel ) .

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

Figure 3A Representative images of mutant Lamtor1. PMA-primed Lamtor1 KO THP-1 macrophages reconstituted with full-length or truncated Lamtor1.B Effects of the Lamtor1 point mutation on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion in THP-1 cell supernatants from WT cells, Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full-length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine (G2A); all cell types were then treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).C Effects of Lamtor1 deficiency on mTORC1 activity. Immunoblot analysis of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h).D Effect of the rapamycin treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from PMA (50 nM, overnight) primed WT THP-1 cells treated with rapamycin at indicated concentration for 2 h, then treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h).E Effect of rapamycin treatment on Caspase-1 processing. Western blot shows caspase-1/p20. LPS (200 ng/mL, 4 h) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin (15 μM, 1 h).F Effect of Torin-1 treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from WT BMDMs pretreated with Torin-1 (0, 1, 10, 100, or 250 nM) for 2 h before LPS (200 ng/ml, 4 h) and nigericin (15 μM, 1 h) treatment (left panel). Western blot shows phospho-p70-S6K from BMDMs pretreated with Torin-1 at the indicated concentrations for 2 h before LPS and nigericin treatment (right panel).


Figure 4A Effects of the HDAC6 inhibitor treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion and caspase - 1 processing ( p10 ) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin ( left to right : 10 and 20 mM ) or rocilinostat ( left to right : 30 and 60 mM ) . Co - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmidsCo - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmidsCo - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Lamtor1 - Flag co - immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1 - Flag knocked - in THP - 1 cells . ( D ) Endogenous Lamtor1 co - immunoprecipitated with endogenous HDAC6 from lysates of WT THP - 1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6 . Co - immunoprecipitation assay shows interactions between Lamtor1 and HDAC6 . Endogenous Lamtor1 co - immunoprecipitated with endogenous HDAC6 from lysates of WT THP - 1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6 . Co - immunoprecipitation assay shows interactions between Lamtor1 and HDAC6 . G PLA to confirm endogenous binding between Lamtor1 and HDAC6 . Lamtor1 - HDAC6 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over nine images in each experiment . H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells . HEK293T cells were transiently transfected with NanoLuc - fused Lamtor1 and Halo - tag - fused HDAC6 using Lipofectamine 2000 . Luminescence was measured 48 h after transfection following the addition of Nano - Glo Luciferase Assay Substrate ( Promega ) . I Immunoblot analysis of the truncated form of Flag - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - 144 ; ∆ 95 - 161 , Met1 - Val94 ) co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids . Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP - 1 cells . ELISA assay showing IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , Lamtor1 KO cells reconstituted with full - length Lamtor1 , and Met1 - Val94 Lamtor1 cells ; all cell types were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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

Figure 4A Effects of the HDAC6 inhibitor treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion and caspase-1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM).Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmidsCo-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmidsCo-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Lamtor1-Flag co-immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1-Flag knocked-in THP-1 cells. (D)Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.G PLA to confirm endogenous binding between Lamtor1 and HDAC6. Lamtor1-HDAC6 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over nine images in each experiment.H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc-fused Lamtor1 and Halo-tag-fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano-Glo Luciferase Assay Substrate (Promega).I Immunoblot analysis of the truncated form of Flag-tagged Lamtor1 mutant (∆145-161, Met1-144; ∆95-161, Met1-Val94) co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids.Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP-1 cells. ELISA assay showing IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full-length Lamtor1, and Met1-Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


Figure 5A Yeast two hybrid ( Y2H ) assays for Lamtor1 interactions with NLRP3 . Positive Y2H interaction between Lamtor1 as prey , and NLRP3 as bait . Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control . B PLA to confirm endogenous binding between Lamtor1 and NLRP3 . Lamtor1 - NLRP3 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over 10 images in each experiment . C , D Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids . Immunoblot analysis of the truncated form of V5 - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - Ser144 ) co - immunoprecipitated with NLRP3 - flag from lysates of HEK293T cells transfected with the indicated plasmids . Immunoblot analysis of the truncated form of V5 - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - Ser144 ) co - immunoprecipitated with NLRP3 - flag from lysates of HEK293T cells transfected with the indicated plasmids . G Effect of the HDAC6 knockdown on NLRP3 - Lamtor1 interactions . Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids .

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

Figure 5A Yeast two hybrid (Y2H) assays for Lamtor1 interactions with NLRP3. Positive Y2H interaction between Lamtor1 as prey, and NLRP3 as bait. Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control.B PLA to confirm endogenous binding between Lamtor1 and NLRP3. Lamtor1-NLRP3 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over 10 images in each experiment.C, D Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.G Effect of the HDAC6 knockdown on NLRP3-Lamtor1 interactions. Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.


Figure 6A , B Representative images of Lamtor1 and ASC . PMA - treated THP - 1 cells stably expressing GFP - tagged ASC and td - Tomato - tagged Lamtor1 were primed with LPS ( 200 ng / mL , 2 h ) and then stimulated with nigericin for the indicated duration . Time shows after nigericin stimulation ( min : s ) . C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation . Representative images of PMA - primed THP - 1 macrophages stably expressing GFP - tagged ASC treated with or without LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) . Scale bars = 10 μm .

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Figure 6A, B Representative images of Lamtor1 and ASC. PMA-treated THP-1 cells stably expressing GFP-tagged ASC and td-Tomato-tagged Lamtor1 were primed with LPS (200 ng/mL, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s).C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA-primed THP-1 macrophages stably expressing GFP-tagged ASC treated with or without LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm.


Figure 7Effects of DL - all - rac - α - tocopherol on the interactions between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL - all - rac - α - tocopherol ( B ) IL - 1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL - all - rac - α - tocopherol ( C ) at the indicated concentrations . ELISA assay showing IL - 1β secretion in supernatants from WT THP - 1 cells treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) . D ) IL - 6 secretion after overnight pretreatment with DL - all - rac - α - tocopherol . ELISA assay showing IL - 6 secretion in supernatants from WT BMDMs treated with LPS ( 200 ng / mL , overnight ) and DL - all - rac - α - tocopherol at the indicated concentrations ( overnight ) . Effects of α - tocopherol on the interactions between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D - α - tocopherol ( E ) IL - 1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D - α - tocopherol ( F ) at the indicated concentrations . ELISA assay showing IL - 1β secretion in supernatants from WT THP - 1 cells treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) . G Effects of DL - all - rac - α - tocopherol in an acute gouty arthritis model . MSU crystals ( 0 . 5 mg ) suspended in 20 μL endotoxin - free PBS or PBS control were injected intra - articularly into the tibia - tarsal joint ( ankle ) of C57BL / 6 WT mice with or without DL - all - rac - α - tocopherol . Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin - stained sections of ankle joints . H Effects of DL - all - rac - α - tocopherol in an MSU - induced peritonitis model . Twelve hours after DL - all - rac - α - tocopherol pretreatment , 100 μL MSU solution ( 10 mg / mL ) was intraperitoneally injected for 4 h . Peritoneal IL - 1β production was then measured by ELISA , and CD11b + Ly6G + neutrophil recruitment was measured by FACS .

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Figure 7Effects of DL-all-rac-α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL-all-rac-α-tocopherol (B)IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL-all-rac-α-tocopherol (C) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).D) IL-6 secretion after overnight pretreatment with DL-all-rac-α-tocopherol. ELISA assay showing IL-6 secretion in supernatants from WT BMDMs treated with LPS (200 ng/mL, overnight) and DL-all-rac-α-tocopherol at the indicated concentrations (overnight).Effects of α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D -α-tocopherol (E)IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D -α-tocopherol (F) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).G Effects of DL-all-rac-α-tocopherol in an acute gouty arthritis model. MSU crystals (0.5 mg) suspended in 20 μL endotoxin-free PBS or PBS control were injected intra-articularly into the tibia-tarsal joint (ankle) of C57BL/6 WT mice with or without DL-all-rac-α-tocopherol. Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin-stained sections of ankle joints.H Effects of DL-all-rac-α-tocopherol in an MSU-induced peritonitis model. Twelve hours after DL-all-rac-α-tocopherol pretreatment, 100 μL MSU solution (10 mg/mL) was intraperitoneally injected for 4 h. Peritoneal IL-1β production was then measured by ELISA, and CD11b+ Ly6G+ neutrophil recruitment was measured by FACS.


Figure 1B . Western blot analysis of purified autophagic vesicles . Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms , respectively , and are represented with total lysate ( TL ) . Depicted are representative blots of 14 independent approaches .

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Figure 1B. Western blot analysis of purified autophagic vesicles. Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms, respectively, and are represented with total lysate (TL). Depicted are representative blots of 14 independent approaches.


Figure 2A . Differential interference contrast microscopy images of purified autophagic vesicles at high ( I ) or low ( II ) dilution . Images are representative of 3 independent approaches . B . Negative stain electron microscopy images of isolated vesicles . C . Size evaluation of isolated vesicles . The diameters of approx . 60 individual vesicles were determined using EM images . D . Western blot analysis of isolated autophagic vesicles upon proteinase K digestion . Mechanically opened vesicles served as positive control . For negative control , isolates were incubated with BSA instead of proteinase K . Depicted are two different blots that are representative for 5 independent

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Figure 2A. Differential interference contrast microscopy images of purified autophagic vesicles at high (I) or low (II) dilution. Images are representative of 3 independent approaches. B. Negative stain electron microscopy images of isolated vesicles. C. Size evaluation of isolated vesicles. The diameters of approx. 60 individual vesicles were determined using EM images.D. Western blot analysis of isolated autophagic vesicles upon proteinase K digestion. Mechanically opened vesicles served as positive control. For negative control, isolates were incubated with BSA instead of proteinase K. Depicted are two different blots that are representative for 5 independent experiments


Figure 3E . Phospholipids identified in autophagic vesicles isolated upon different conditions . Relative amounts were calculated based on total levels of detected phospholipids . Abbreviations are depicted in A .

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Figure 3E. Phospholipids identified in autophagic vesicles isolated upon different conditions. Relative amounts were calculated based on total levels of detected phospholipids. Abbreviations are depicted in A.


Figure 4A . Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) . B . Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) .

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Figure 4A. Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd). B. Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd).


Figure 1 ( A ) Classical , ( B ) Rolled , and ( C ) Rotated subpopulations of the 80S * HCVIRES complex . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) . ( D ) Subpopulations of the 80S * HCVIRES complex containing P - site tRNA . The 80S is in the classical configuration and the head tilt is not present . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) . ( E ) The cryo - EM map was filtered according to resolution . 40S * HCV IRES . Left column shows an overview of the final cryo - EM reconstructions with the HCV IRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCV IRES ( pink ) , tRNA ( green ) .

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Figure 1(A) Classical, (B) Rolled, and (C) Rotated subpopulations of the 80S*HCVIRES complex. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCVIRES (pink), tRNA (green).(D) Subpopulations of the 80S*HCVIRES complex containing P-site tRNA. The 80S is in the classical configuration and the head tilt is not present. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink),40S subunit (yellow),60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts, respectively.28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray),HCVIRES (pink), tRNA (green).(E) The cryo-EM map was filtered according to resolution. 40S*HCV IRES. Left column shows an overview of the final cryo-EM reconstructions with the HCV IRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCV IRES (pink), tRNA (green).


Figure 2 ( A ) Comparison of 40S subunit positions in Rolled , Rotated , Classical / no head tilt ( orange ) and Classical 80S * HCVIRES complex ( yellow ) in common 60S alignment . The cryo - EM map of the Classical 80S * HCVIRES was filtered to 6 Å . Arrows indicate the direction of movement during transition between two different states . The distance changes in the 40S subunit positions resulting from the rigid body transformation are color - coded in Å units . ( B ) Comparison of the HCVIRES in Classical 80S * HCVIRES complex ( gray ) and upon P - tRNA binding ( Classical , no head tilt ; purple ) in a common 40S body alignment . The distance of the movement of domain II was indicated with an arrow and Å

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Figure 2(A) Comparison of 40S subunit positions in Rolled, Rotated, Classical/no head tilt (orange) and Classical 80S*HCVIRES complex (yellow) in common 60S alignment. The cryo-EM map of the Classical 80S*HCVIRES was filtered to 6 Å. Arrows indicate the direction of movement during transition between two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units.(B) Comparison of the HCVIRES in Classical 80S*HCVIRES complex (gray) and upon P-tRNA binding (Classical, no head tilt; purple) in a common 40S body alignment. The distance of the movement of domain II was indicated with an arrow and Å units


Figure 3 ( A ) Adopted secondary structure of the IRES representing the tertiary fold . ( B ) Atomic model of the HCV IRES RNA . IRES domains are presented as coloured ribbons , overlaid with experimental IRES density ( gray , semi - transparent ) . ( C ) . Molecular model of the HCV IRES ( pink ) with ribosomal interaction sites ( coloured proteins and helices ) .

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Figure 3(A) Adopted secondary structure of the IRES representing the tertiary fold.(B) Atomic model of the HCV IRES RNA. IRES domains are presented as coloured ribbons, overlaid with experimental IRES density (gray, semi-transparent).(C). Molecular model of the HCV IRES (pink) with ribosomal interaction sites (coloured proteins and helices).


Figure 4 ( A ) The secondary structure of the HCVIRES domain III ( hIII1 , hIII2 , IIIe and IIId ) and helix 26 ( h26 ) from the 18S rRNA . Colors of HCVIRES Domains are the same as in Fig 3 . Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line . ( B ) The interaction core between IIId , IIIe , hIII1 of HCVIRES ( colored as in ( A ) ) and Expansion segment 7 ( ES7 ) of h26 ( dark gray ) . ( C ) The rearrangement of h26 ( red ) upon binding of HCVIRES . The 80S * HCVIRES complex ( yellow ) is compared to the elongating POST state ( gray ; PDB 5Aj0 ) ( Behrmann et al , 2015 ) . The loop closing the helix is highlighted in red and dark gray . ( E ) Close up on the interactions between GGG266 - 268 of IIId ( cyan ) and CCC1116 - 1118 of ES7 ( gray ) . ( F ) Close up on the interactions between G295 , A296 and U297 of IIIe ( green ) , A136 and A288 of hIII1 ( yellow ) and U1114 , U1115 of ES7 ( gray ) .

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Figure 4(A) The secondary structure of the HCVIRES domain III (hIII1, hIII2, IIIe and IIId) and helix 26 (h26) from the 18S rRNA. Colors of HCVIRES Domains are the same as in Fig 3. Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line.(B) The interaction core between IIId, IIIe, hIII1 of HCVIRES (colored as in (A)) and Expansion segment 7 (ES7) of h26 (dark gray).(C) The rearrangement of h26 (red) upon binding of HCVIRES. The 80S*HCVIRES complex (yellow) is compared to the elongating POST state (gray; PDB 5Aj0) (Behrmann et al, 2015). The loop closing the helix is highlighted in red and dark gray.(E) Close up on the interactions between GGG266-268 of IIId (cyan) and CCC1116-1118 of ES7 (gray).(F) Close up on the interactions between G295, A296 and U297 of IIIe (green), A136 and A288 of hIII1 (yellow) and U1114, U1115 of ES7 (gray).


Figure 5 ( A ) Comparison of the 40S subunit from Classical 80S * HCVIRES complex ( yellow ) and canonical POST state ( gray ; EMD 2875 ) ( Behrmann et al , 2015 ) in common 40S body alignment . The arrow indicates the direction of the head tilt movement . Ribosomal proteins of the 40S head ( uS7 , eS25 and eS28 ) that interact with the HCVIRES domains II and IV ( pink ) on the 40S head are illuminated with blue , green , and purple , respectively . The positions of the proteins in the canonical POST state are illuminated in lighter color . The numbers indicate interaction site of HCVIRES and the 40S subunit . ( B ) Close up view on the interaction site of HCVIRES and the 40S head region . Numbers correspond to red labels in ( A ) . The rearrangement of the uS7 loop ( blue ) compared to the Classical 80S * HCVIRES complex ( gray ) is indicated . The arrow indicates the direction of the rearrangement of the uS7 loop . ( B - 1 and B - 4 ) inter subunit view from 40S subunit . ( B - 2 and - 3 ) inter subunit view from 60S subunit .

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Figure 5(A) Comparison of the 40S subunit from Classical 80S*HCVIRES complex (yellow) and canonical POST state (gray; EMD 2875) (Behrmann et al, 2015) in common 40S body alignment. The arrow indicates the direction of the head tilt movement. Ribosomal proteins of the 40S head (uS7, eS25 and eS28) that interact with the HCVIRES domains II and IV (pink) on the 40S head are illuminated with blue, green, and purple, respectively. The positions of the proteins in the canonical POST state are illuminated in lighter color. The numbers indicate interaction site of HCVIRES and the 40S subunit.(B) Close up view on the interaction site of HCVIRES and the 40S head region. Numbers correspond to red labels in (A). The rearrangement of the uS7 loop (blue) compared to the Classical 80S*HCVIRES complex (gray) is indicated. The arrow indicates the direction of the rearrangement of the uS7 loop. (B-1 and B-4) inter subunit view from 40S subunit. (B-2 and -3) inter subunit view from 60S subunit.