A binding in RKO . PLA showed HOPS / p53 interaction with or without etoposide treatment at indicated time . Red spots showed a single interaction . counterstaining was performed with DAPI . α - Tubulin - FITC antibody was used to identify edge . Background control was performed according to manufacturer ' s instruction . Scale bars , 10µm . graph represents binding increase . Non treated binding was assumed as 1 . Data information : experiments were performed three times , representative examples are shown . In A data are presented as mean ± SD .

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A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1. Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD.


B Biochemical of - distribution . RKO were treated with etoposide , collected after 2 4 hours processed to obtain fractions . Aliquots relative to same number per fraction were immunoblotted using anti - HOPS anti - p53 antibodies . GAPDH Lamin B1 are purity controls of fraction respectively .

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B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.


C HOPS p53 binding in cytoplasm following apoptosis induction . RKO untreated or treated with etoposide as above were used to isolate fractions . WCL fractions were immunoprecipitated with anti - HOPS antibody subsequently immunoblotted using anti - p53 antibody .

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C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.


A HOPS localization at mitochondria . RKO were stained by MitoTracker ™ ( red ) , immediately fixed stained to visualize HOPS ( green ) . Nuclei were DAPI stained ( blue ) . Samples were analysed with microscope representative were shown . Scale bars , 10µm .

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A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm.


B RKO were lysed processed for mitochondria isolation . WCL , cytoplasm mitochondria were analysed using anti - HOPS antibody . GAPDH , Lamin B1 Tom20 are purity controls of mitochondrial fraction .

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B RKO cells were lysed and processed for mitochondria isolation. WCL, cytoplasm and mitochondria were analysed using anti-HOPS antibody. GAPDH, Lamin B1 and Tom20 are purity controls of mitochondrial fraction.


C HOPS p53 translocation to mitochondria . RKO were treated with etoposide as indicated processed for mitochondrial extraction subsequent immunoblotting for p53 , HOPS reported extraction purity controls .

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C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls.


D HOPS / p53 binding at mitochondria . RKO were treated with etoposide subjected to mitochondrial purification , subsequent immunoprecipitation using anti - HOPS antibody . Samples were immunoblotted to detect p53 , HOPS reported controls .

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D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls.


E HOPS p53 mitochondrial translocation in vivo . Hops + / + Hops − / − mice were treated with etoposide thymus , spleen testis were harvested . Organs were homogenized mitochondrial fraction were isolated analysed by immunoblotting versus p53 . GAPDH Tom20 are purity controls . p53 mitochondrial were semi - quantified using Tom20 as loading control p53 at time 0 were assumed as 1 . Data information : assays involving were performed three times in three different mice per condition . In E data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two Student ' s t - .

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E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1. Data information: assays involving animals were performed three times in three different mice per condition. In E data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


F p53 - dependent mitochondrial potential ( Δψm ) induction in MEFs . Hops + / + Hops − / − MEFs were treated with etoposide then incubated with JC - 1 vital dye . were collected by microscopy representative are shown ( left panel ) . Scale bars , 10µm . was analysed by TECAN plate reader plotted in graph ( right panel ) . Data information : assays involving were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times . In F data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two Student ' s t - .

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F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel). Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


G p53 MOMP induction in HOPS deficiency . Mitochondria from Hops + / + Hops − / − MEFs , were purified incubated with p53 recombinant . Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting . Samples were tested to detect p53 as control Tom20 as mitochondria structural marker . Data information : assays involving were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times .

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G p53 MOMP induction in HOPS deficiency. Mitochondria from Hops+/+ and Hops−/− MEFs, were purified and incubated with p53 recombinant protein. Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting. Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker. Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times.


A HOPS expression p53 accumulation . HOPS was overexpressed in RKO were fixed stained with antibody against p53 ( green ) . Nuclei were stained with DAPI ( blue ) . Samples were analysed with microscope merged are shown ( right panel ) . Scale bars , 10µm . were scored for p53 localization . results are shown as of versus p53 localization in untransfected HOPS transfected ( left panel ) . Data information : experiments detailed above were performed three times , representative panels are shown . In A data are presented as mean ± SD .

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A HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel). Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD.


B Biochemical of HOPS intracellular localization . RKO were transfected or not with HOPS fractionated into component . Aliquots relative to same number per fraction were immunoblotted using anti - p53 anti - HOPS antibodies . GAPDH Lamin B1 are purity controls of fraction respectively . Data information : experiments detailed above were performed three times , representative panels are shown .

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B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


HOPS p53 binding to importin α . Untransfected or HOPS transfected RKO were treated with etoposide each sample was evaluated by immunoprecipitation with anti - p53 analysed by immunoblotting using anti - importin α antibody . Arrowhead indicates importin α specific signal . Data information : experiments detailed above were performed three times , representative panels are shown .

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HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


D PCAF - mediated p53 acetylation . p53 PCAF Flag - tagged were co - expressed in H1299 with or without HOPS treated with trichostatin A nicotinamide . p53 acetylation were evaluated by immunoprecipitation with anti - p53 antibody followed by immunoblotting with anti - acetylated - Lysine ( AcK ) , anti - p53 , anti - Flag , anti - HOPS anti - α - tubulin antibodies . Data information : experiments detailed above were performed three times , representative panels are shown .

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D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


( A ) Wild type ( left middle panels ) p53 - / - ( right panel ) HCT116 were transfected with control ( mixture of control DS scrambledNeg , siLuci , siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 ) siRNAs for 36 h , treated with 5 nM actinomycin D , harvested at indicated times . Whole extracts were analyzed by immunoblotting with indicated antibodies . Coomassie Brilliant Blue ( CBB ) serves as a loading control .

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(A) Wild type (left and middle panels) and p53-/- (right panel) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 36 h, treated with 5 nM actinomycin D, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. Coomassie Brilliant Blue (CBB) staining serves as a loading control.


( B ) Control U2OS infected with control retroviruses with empty vector U2OS overexpressing siRNA - resistant HA - GRWD1 were treated analyzed as above .

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(B) Control U2OS cells infected with control retroviruses with the empty vector and U2OS cells overexpressing the siRNA-resistant HA-GRWD1 were treated and analyzed as above.


( C ) HCT116 were transfected with control ( mixture of control DS scrambledNeg , siLuci , siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 ) siRNAs for 24 h , treated with 50 µg / ml cycloheximide , harvested at indicated times . Whole extracts were analyzed by immunoblotting with indicated antibodies .

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(C) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 24 h, treated with 50 µg/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies.


( D ) HFF2 / T stably overexpressing HA - GRWD1 were established by retroviral infection . were cultured in presence or absence of actinomycin D ( 5 nM ) for 12h then treated with or without MG132 ( 20uM ) for 6 h as indicated . Whole extracts were analyzed as above .

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(D) HFF2/T cells stably overexpressing HA-GRWD1 were established by retroviral infection. Cells were cultured in the presence or absence of actinomycin D (5 nM) for 12h and then treated with or without MG132 (20uM) for 6 h as indicated. Whole cell extracts were analyzed as above.


( E ) Control HA - GRWD1 - overexpressing HFF2 / T were first treated with actinomycin D ( 5nM ) for 12 h , then further treated with 50 ug / ml cycloheximide , harvested at indicated times . Whole extracts were analyzed by immunoblotting with indicated antibodies . means SDs from two independent experiments are shown .

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(E) Control and HA-GRWD1-overexpressing HFF2/T cells were first treated with actinomycin D (5nM) for 12 h, then further treated with 50 ug/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. The means and SDs from two independent experiments are shown.


( A ) U2OS were transfected with control ( mixture of control DS scrambledNeg , siLuci , siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 or siGRWD1 - 1 ) siRNAs for 24 h . Whole extracts were then analyzed by immunoblotting with indicated antibodies .

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(A) U2OS cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h. Whole cell extracts were then analyzed by immunoblotting with the indicated antibodies.


B ) U2OS treated as above were stained with propidium iodide subjected to flow cytometry .

B) U2OS cells treated as above were stained with propidium iodide and subjected to flow cytometry.


( C ) HCT116 treated with actinomycinD ( 5nM ) or vehicle ( DMSO ) for 12h were first extracted with Triton X - 100 to remove nucleoplasmic , double - immunostained with anti - GRWD1 ( green ) anti - fibrillarin ( red ) antibodies as a marker for nucleoli , counterstained with DAPI . Scale bar , 20 um .

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(C) HCT116 cells treated with actinomycinD (5nM) or vehicle (DMSO) for 12h were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 (green) and anti-fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. Scale bar, 20 um.


( D ) HCT116 were transfected with control ( mixture of control siLuci siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 or siGRWD1 - 1 ) siRNAs for 24 h then immunostained as above . Scale bar , 20 um .

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(D) HCT116 cells were transfected with control (mixture of control siLuci and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h and then immunostained as above. Scale bar, 20 um.


( A ) 293T were co - transfected with HA - GRWD1 ( 1 . 6 ug ) RPL5 - FLAG ( 2 . 4 ug ) or RPL11 - FLAG ( 2 . 4 ug ) as indicated for 42h subjected to immunoprecipitation with anti - FLAG antibody . Immunoprecipitates ( IPs ) 3 . 125 % of inputs were immunoblotted with indicated antibodies . * , immunoglobulin chains .

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(A) 293T cells were co-transfected with HA-GRWD1 (1.6 ug) and RPL5-FLAG (2.4 ug) or RPL11-FLAG (2.4 ug) as indicated for 42h and subjected to immunoprecipitation with anti-FLAG antibody. Immunoprecipitates (IPs) and 3.125% of inputs were immunoblotted with the indicated antibodies. *, immunoglobulin light chains.


( B ) Lysates from untransfected 293T were immunoprecipitated with anti - GRWD1 antibody or control IgG . Immunoprecipitates ( IPs ) 2 . 7 % of inputs were immunoblotted with indicated antibodies .

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(B) Lysates from untransfected 293T cells were immunoprecipitated with anti-GRWD1 antibody or control IgG. Immunoprecipitates (IPs) and 2.7% of inputs were immunoblotted with the indicated antibodies.


( C ) HCT116 were treated with 5nM actinomycin D for 12 h or left untreated subjected to immunoprecipitation with anti - GRWD1 antibody or control IgG . Immunoprecipitates ( IPs ) 3 . 5 % of inputs were immunoblotted with indicated antibodies .

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(C) HCT116 cells were treated with 5nM actinomycin D for 12 h or left untreated and subjected to immunoprecipitation with anti-GRWD1 antibody or control IgG. Immunoprecipitates (IPs) and 3.5% of inputs were immunoblotted with the indicated antibodies.


( D ) GST - RPL11 or GST alone was incubated with purified GRWD1 bound analyzed by immunoblotting with indicated antibodies . I , input . P , pull - down .

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(D) GST-RPL11 or GST alone was incubated with purified GRWD1 and bound proteins analyzed by immunoblotting with the indicated antibodies. I, input. P, pull-down.


( E ) lysates were prepared from 293T transfected with wild - type ( WT ) HA - GRWD1 - FLAG ( 12 µg ) or various N - terminal truncated mutants of HA - GRWD1 - FLAG ( 12 µg ) for 42h , then subjected to GST pull - down with purified GST - RPL11 or GST . Bound were analyzed by CBB ( GST GSR - RPL11 ) immunoblotting with indicated antibodies . * , non - specific bands .

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(E) Cell lysates were prepared from 293T cells transfected with wild-type (WT) HA-GRWD1-FLAG (12 µg) or various N-terminal truncated mutants of HA-GRWD1-FLAG (12 µg) for 42h, and then subjected to GST pull-down assay with purified GST-RPL11 or GST. Bound proteins were analyzed by CBB staining (GST and GSR-RPL11) and immunoblotting with the indicated antibodies. *, non-specific bands.


( G ) HCT116 were transfected with RPL11 - FLAG expression vector ( 0 . 56 ug ) or empty vector ( 0 . 56 ug ) for 24 h then treated with 5 nM actinomycinD or vehicle ( PBS ) for 12 h . were first extracted with Triton X - 100 to remove nucleoplasmic , double - immunostained with anti - GRWD1 antibody ( green ) anti - FLAG M2 antibody ( red ) , counterstained with DAPI . Scale bar , 20 um .

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(G) HCT116 cells were transfected with RPL11-FLAG expression vector (0.56 ug) or empty vector (0.56 ug) for 24 h and then treated with 5 nM actinomycinD or vehicle (PBS) for 12 h. The cells were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 antibody (green) and anti-FLAG M2 antibody (red), and counterstained with DAPI. Scale bar, 20 um.


( A ) H1299 were co - transfected with indicated expression vectors ( p53 , 7 . 5 ng ; HA - Ub , 0 . 5 ug ; His - Xpress - MDM2 , 2 ug ; RPL11 - FLAG , 1 ug ; HA - GRWD1 - FLAG , 1 . 5 ug ; GFP , 0 . 04 ug ) for 48 h analyzed by immunoblotting with indicated antibodies . GFP serves as a control to show equal transfection efficiencies .

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(A) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug; GFP, 0.04 ug) for 48 h and analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies.


( B ) Lysates were prepared from 293T co - transfected with His - Xpress - MDM2 ( 1 . 2 ug ) , FLAG - RPL11 ( 1 . 8 ug ) , HA - GRWD1 ( 1 ug ) as indicated for 42 h then immunoprecipitated with anti - FLAG antibody . Immunoprecipitates ( IPs ) 1 . 5 % of inputs were immunoblotted with indicated antibodies .

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(B) Lysates were prepared from 293T cells co-transfected with His-Xpress-MDM2 (1.2 ug), FLAG-RPL11 (1.8 ug), and HA-GRWD1 (1 ug) as indicated for 42 h and then immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (IPs) and 1.5% of inputs were immunoblotted with the indicated antibodies.


( C ) In vivo ubiquitination to detect MDM2 autoubiquitination in H1299 . Lysates were prepared from H1299 transfected with indicated expression vectors ( His - Xpress - MDM2 , 2 ug ; HA - Ub , 0 . 5 ug ; RPL11 - FLAG , 1 ug ; HA - GRWD1 - FLAG , 1 . 5 ug ) for 48 h , treated with proteasome inhibitors for 6 h before harvest , then immunoprecipitated with anti - MDM2 antibody . Immunoprecipitates ( IPs ) inputs were immunoblotted with indicated antibodies .

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(C) In vivo ubiquitination assay to detect MDM2 autoubiquitination in H1299 cells. Lysates were prepared from H1299 cells transfected with the indicated expression vectors (His-Xpress-MDM2, 2 ug; HA-Ub, 0.5 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug) for 48 h, treated with proteasome inhibitors for 6 h before harvest, and then immunoprecipitated with anti-MDM2 antibody. Immunoprecipitates (IPs) and inputs were immunoblotted with the indicated antibodies.


( D ) In vitro ubiquitination of p53 by immunopurified MDM2 . His - Xpress - MDM2 was immunopurified from transfected 293T with anti - Omni probe antibody . Recombinant p53 was incubated with E1 , E2 ( UbcH5a ) , His - ubiquitin , ATP , GST - RPL11 , GRWD1 - His , immunopurified His - Xpress - MDM2 or control immunoprecipitates at 30 ℃ for 120 min as indicated . samples were resolved by SDS - PAGE followed by immunoblotting with indicated antibodies .

O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) In vitro ubiquitination of p53 by immunopurified MDM2. His-Xpress-MDM2 was immunopurified from transfected 293T cells with anti-Omni probe antibody. Recombinant p53 was incubated with E1, E2 (UbcH5a), His-ubiquitin, ATP, GST-RPL11, GRWD1-His, and immunopurified His-Xpress-MDM2 or control immunoprecipitates at 30℃ for 120 min as indicated. The samples were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies.


( A ) Expression of introduced effect on p53 . HFF2 / T stably expressing HA - GRWD1 , E7 , KRAS G12V were established with retroviral vectors analyzed by immunoblotting with indicated antibodies . * , endogenous RAS .

O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O

(A) Expression of the introduced proteins and the effect on p53. HFF2/T cells stably expressing HA-GRWD1, E7, and KRAS G12V were established with retroviral vectors and analyzed by immunoblotting with the indicated antibodies. *, endogenous RAS proteins.


( B C ) Anchorage - independent growth of HFF2 / T overexpressing E7 with or without HA - GRWD1 . Three after seeding , were acquired colonies > 100 µm in diameter were counted . Representative are shown in ( B ) . Scale bars , 500 um . Average numbers of colonies per field , with SDs , are shown in ( C ) . Six randomly selected areas were counted per sample .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) Anchorage-independent growth of HFF2/T cells overexpressing E7 with or without HA-GRWD1. Three weeks after seeding, images were acquired and colonies >100 µm in diameter were counted. Representative images are shown in (B). Scale bars, 500 um. Average numbers of colonies per field, with SDs, are shown in (C). Six randomly selected areas were counted per sample.


( D E ) Anchorage - independent growth of HFF2 / T overexpressing E7 activated KRAS G12V with or without HA - GRWD1 . At 9 days after seeding , colonies were analyzed as above . Representative are shown in ( D ) . Scale bars , 500 um . average numbers of colonies ( > 200 um in diameter ) per field , with SDs , are shown in ( E ) . Twenty randomly selected areas were counted .

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(D and E) Anchorage-independent growth of HFF2/T cells overexpressing E7 and activated KRAS G12V with or without HA-GRWD1. At 9 days after seeding, the colonies were analyzed as above. Representative images are shown in (D). Scale bars, 500 um. The average numbers of colonies (>200 um in diameter) per field, with SDs, are shown in (E). Twenty randomly selected areas were counted.


( F ) Tumorigenesis of HFF2 / T expressing E7 KRAS G12V with or without HA - GRWD1 . were subcutaneously injected into nude mice ( 1 x 106 / mouse ) , was measured . Average volumes , with SDs , are shown ( n = 5 ) .

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(F) Tumorigenesis of HFF2/T cells expressing E7 and KRAS G12V with or without HA-GRWD1. Cells were subcutaneously injected into nude mice (1 x 106 cells/mouse), and tumor size was measured. Average tumor volumes, with SDs, are shown (n=5).


( G ) High expression of GRWD1 is associated with poor prognosis of brain lower grade glioma with wild type p53 but not with mutated p53 . data were obtained from TCGA databases ( cBioPortal for Cancer Genomics ; http : / / www . cbioportal . org ) . For detail , see Materials Methods .

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

(G) High expression of GRWD1 is associated with poor prognosis of brain lower grade glioma patients with wild type p53 but not with mutated p53. The data were obtained from the TCGA databases (the cBioPortal for Cancer Genomics; http://www.cbioportal.org). For detail, see Materials and Methods.


( A ) 293T were co - transfected with HA - GRWD1 wild - type ( WT ) ( 0 . 5 ug ) or HA - GRWD1 Δ1 - 136 ( 3 . 5 ug ) RPL11 - FLAG ( 1 . 5 ug ) as indicated for 42h , then immunoprecipitated with an anti - FLAG antibody . Immunoprecipitates ( IPs ) 1 . 6 % of inputs were immunoblotted with indicated antibodies .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 293T cells were co-transfected with HA-GRWD1 wild-type (WT) (0.5 ug) or HA-GRWD1 Δ1-136 (3.5 ug) and RPL11-FLAG (1.5 ug) as indicated for 42h, and then immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies.


( B ) 293T were co - transfected with His - Xpress - MDM2 ( 0 . 6 ug ) , RPL11 - FLAG ( 0 . 9 ug ) , HA - GRWD1 WT ( 0 . 5 ug ) or HA - GRWD1 Δ1 - 136 ( 4 . 5 ug ) as indicated for 42h subjected to immunoprecipitation with an anti - FLAG antibody . Immunoprecipitates ( IPs ) 1 . 6 % of inputs were immunoblotted with indicated antibodies .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with His-Xpress-MDM2 (0.6 ug), RPL11-FLAG (0.9 ug), and HA-GRWD1 WT (0.5 ug) or HA-GRWD1 Δ1-136 (4.5 ug) as indicated for 42h and subjected to immunoprecipitation with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies.


( C ) H1299 were co - transfected with indicated expression vectors ( p53 , 7 . 5 ng ; HA - Ub , 0 . 5 ug ; His - Xpress - MDM2 , 2 ug ; RPL11 - FLAG , 1 ug ; HA - GRWD1 - FLAG WT , 1 . 5 ug ; HA - GRWD1 - FLAG Δ1 - 136 , 1 . 5 ug ; GFP , 0 . 04 ug ) for 48 h , then analyzed by immunoblotting with indicated antibodies . GFP serves as a control to show equal transfection efficiencies .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG WT, 1.5 ug; HA-GRWD1-FLAG Δ1-136, 1.5 ug; GFP, 0.04 ug) for 48 h, and then analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies.


( D ) HFF2 / T stably expressing HA - GRWD1 wild - type ( WT ) or Δ1 - 136 , E7 , KRAS G12V were established using retroviral vectors analyzed by immunoblotting with indicated antibodies . * , endogenous RAS .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O

(D) HFF2/T cells stably expressing HA-GRWD1 wild-type (WT) or Δ1-136, E7, and KRAS G12V were established using retroviral vectors and analyzed by immunoblotting with the indicated antibodies. *, endogenous RAS proteins.


( E F ) Anchorage - independent growth of HFF2 / T overexpressing E7 activated KRAS G12V with HA - GRWD1 WT or Δ1 - 136 ( or without HA - GRWD1 ) . At 9 days after seeding , colonies were analyzed as above . Representative are shown in ( E ) . Scale bars , 500 um . Average numbers of colonies ( > 200 um in diameter ) per photographed with SDs are shown in ( F ) . Seventy randomly selected areas were counted .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E and F) Anchorage-independent growth of HFF2/T cells overexpressing E7 and activated KRAS G12V with HA-GRWD1 WT or Δ1-136 (or without HA-GRWD1). At 9 days after seeding, colonies were analyzed as above. Representative images are shown in (E). Scale bars, 500 um. Average numbers of colonies (>200 um in diameter) per photographed area with SDs are shown in (F). Seventy randomly selected areas were counted.


collected in GFP channel for each truncation ( columns ) of each studied MRP ( rows ) grouped by N - terminus targeting properties on theoretical expectation summarized in ( B ) with MRPs possessing MTS - like N - termini in panel ( C )

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

Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) with the MRPs possessing MTS-like N-termini in panel (C)


collected in GFP channel for each truncation ( columns ) of each studied MRP ( rows ) grouped by N - terminus targeting properties on theoretical expectation summarized in ( B ) MRPs with intermediate phenotype in panel ( D ) MRPs without N - terminal signal in panel ( E ) , for each MRP a yeast gene name new nomenclature name is shown on left . Scale bar for is 5 µm .

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

Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) MRPs with intermediate phenotype in panel (D) and MRPs without N-terminal signal in panel (E), for each MRP a yeast gene name and new nomenclature protein name is shown on the left. Scale bar for all micrographs is 5 µm.


( A ) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by microscopy with MitoTracker Orange straining . Scale bar for is 10 µm .

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(A) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by fluorescent microscopy with MitoTracker Orange straining. Scale bar for all micrographs is 10 µm.


( B ) Characterization of Mrp17 translocation signal using an in vitro import : shown are autoradiographs of full - Mrp17 or its truncations fused to DHFRmut , translated in vitro with radiolabeled - , incubated with isolated yeast mitochondria for 2 , 5 , or 10 min , treated with proteinase K ( PK ) to remove nonimported visualized by 16 % SDS - PAGE / autoradiography . As a negative control , mitochondria were treated with valinomycin , antimycin oligomycin ( VAO ) that eliminate potential . For comparison , 20 % of used per import reaction was loaded on first lane .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(B) Characterization of Mrp17 translocation signal using an in vitro import assay: shown are autoradiographs of full-length Mrp17 or its truncations fused to DHFRmut, translated in vitro with radiolabeled amino-acids, incubated with isolated yeast mitochondria for 2, 5, or 10 min, treated with proteinase K (PK) to remove nonimported proteins and visualized by 16% SDS-PAGE/autoradiography. As a negative control, mitochondria were treated with valinomycin, antimycin and oligomycin (VAO) that eliminate membrane potential. For comparison, 20% of the protein used per import reaction was loaded on the first lane.


( B ) In vitro mitochondria translocation capacity of WT Mrp17 , Mrp17K - R with lysines ( K ) substituted with arginines ( R ) , Mrp17K - A with lysines ( K ) substituted with alanines ( A ) fused to DHFRmut . Import was performed as described in Fig . 2 legend .

O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O 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) In vitro mitochondria translocation capacity of WT Mrp17, Mrp17K-R with all lysines (K) substituted with arginines (R), and Mrp17K-A with all lysines (K) substituted with alanines (A) fused to DHFRmut. Import was performed as described in Fig. 2 legend.


( E ) Mrp17 variants rescue ∆ mrp17 growth defect on respiratory media : indicated variants or empty vector ( EV ) were introduced in WT yeast then genomic MRP17 was disrupted by knock out , resulting mutants were serially diluted 10 × spotted on media containing glucose or glycerol as a sole carbon source .

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Mrp17 variants rescue ∆mrp17 strain growth defect on respiratory media: the indicated variants or empty vector (EV) were introduced in WT yeast and then the genomic MRP17 was disrupted by knock out, the resulting mutants were serially diluted 10× and spotted on media containing glucose or glycerol as a sole carbon source.


( A ) in vitro import of Mrp17 - DHFRmut is sensitive to elimination of outer mitochondrial by trypsinization ( left ) , more so than model import substrate Atp1 ( right ) . Mitochondria were incubated with indicated concentrations of trypsin import was performed as described in legend for Fig . 2 . Proteinase K ( PK ) was not added to sample without mitochondria . ( B ) In vitro translocation of Mrp17 - DHFRmut into mitochondria isolated from WT ∆ tom20 yeast showing reduced translocation in ∆ tom20 background .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O

(A) The in vitro import of Mrp17-DHFRmut is sensitive to the elimination of outer mitochondrial membrane proteins by trypsinization (left), more so than the model import substrate Atp1 (right). Mitochondria were incubated with the indicated concentrations of trypsin and the import assay was performed as described in the legend for Fig. 2. Proteinase K (PK) was not added to the sample without mitochondria. (B) In vitro translocation of Mrp17-DHFRmut into mitochondria isolated from WT and ∆tom20 yeast showing reduced translocation in ∆tom20 background.


( G ) , ( H ) Import of Mrp17 - DHFRmut control Atp1 Aac1 into WT , tim17 - ts ( G ) tim22 - ts ( H ) mitochondria . Import was performed at indicated temperatures , proteinase K was added to samples except loading control in first lane of each autoradiograph ( 20 % of amount used for each import reaction ) . full autoradiograph for Aac1 with molecular markers is shown in Appendix Figure S6D .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G),(H) Import of Mrp17-DHFRmut and control proteins Atp1 and Aac1 into WT, tim17-ts (G) and tim22-ts (H) mitochondria. Import was performed at indicated temperatures, proteinase K was added to all samples except the loading control in the first lane of each autoradiograph (20% of protein amount used for each import reaction). The full autoradiograph for Aac1 with molecular weight markers is shown in Appendix Figure S6D.


( C ) Expression of structurally conserved RPs fused to GFP in yeast ( Full in al channels are shown on Fig . EV5 ) . Scale bar is 10 µm .

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

(C) Expression of structurally conserved bacterial RPs fused to GFP in yeast cells (Full micrographs in al channels are shown on Fig. EV5). Scale bar is 10 µm.


( E ) Chimeric variants of S6 depicted in panel D imported into isolated yeast mitochondria . Lanes are labeled as described in Fig . 2 legend .

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(E) Chimeric variants of S6 depicted in panel D imported into isolated yeast mitochondria. Lanes are labeled as described in Fig. 2 legend.


( B ) On right : MRP N - termini structural environment ( solvent exposure ) : average number of atoms belonging to same , other chains or in 13Å radius from first structured acid Cα atom , plotted separately for with their most N - terminus appearing in structure vs with precisely MTS , other which have a longer unstructured extension ( see classification legend on bottom ) calculated on PDB : 5MRC ; on right : same as on left but only coordination number ( number of Cα atoms in 13Å radius ( Hamelryck , 2005 ) ) is calculated , * * p - value < 0 . 01 ( Mann - Whitney ) , absolute values were measured without replicates ; on bottom : MRPs were divided in three groups depending on relative position of MTS structured residues : " No MTS ( str ) " - structure starts at very N - terminus without a cleavable MTS , " Precise MTS " - MTS is off 1 - 2 before structure starts , " Other " - unstructured uncleaved present before structure 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) On the right: MRP N-termini structural environment (solvent exposure): average number of atoms belonging to the same protein, other protein chains or RNA in the 13Å radius from the first structured amino acid Cα atom, plotted separately for proteins with their most N-terminus appearing in the structure vs proteins with precisely cleaved MTS, and other proteins which have a longer unstructured extension (see the classification legend on the bottom) calculated based on PDB:5MRC; on the right: same as on the left but only coordination number (number of protein Cα atoms in the 13Å radius (Hamelryck, 2005)) is calculated, ** p-value<0.01 (Mann-Whitney test), absolute values were measured without replicates; on the bottom: MRPs were divided in three groups depending on relative position of the MTS and structured residues: "No MTS (str)" - structure starts at the very N-terminus without a cleavable MTS, "Precise MTS" - MTS is cleaved off 1-2 amino acids before the structure starts, "Other" - unstructured uncleaved amino acids present before the structure start.


mRNA expression of CiTLR5a - HA ( D ) , CiTLR5b - HA ( D ) , CiTLR5a - GFP ( E ) , CiTLR5b - GFP ( E ) plasmids in FHM were examined by qRT - PCR ( upper panel ) with EF1α gene as an internal control by immunoblotting ( IB ) ( lower panel ) with β - tubulin as a reference ( n = 4 ) . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used for ( D ) ( E ) . n represents biological replicates . * * * P < 0 . 001 .

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

mRNA and protein expression of CiTLR5a-HA (D), CiTLR5b-HA (D), CiTLR5a-GFP (E), and CiTLR5b-GFP (E) plasmids in FHM cells were examined by qRT-PCR (upper panel) with EF1α gene as an internal control and by immunoblotting (IB) (lower panel) with β-tubulin as a reference (n = 4). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (D) and (E). n represents biological replicates. ***P < 0.001.


FHM in 10 cm2 dishes were transfected with indicated plasmids for 24 h . Co - IP was performed with anti - HA monoclonal antibody . Mouse IgG was used as control . Immunoblotting was performed with anti - HA anti - GFP antibodies , respectively .

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

FHM cells in 10 cm2 dishes were transfected with the indicated plasmids for 24 h. Co-IP was performed with anti-HA monoclonal antibody. Mouse IgG was used as control. Immunoblotting was performed with anti-HA and anti-GFP antibodies, respectively.


Lysates of FHM overexpressing CiTLR5a were respectively incubated with FlaA , FlaB , FlaC , at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads or streptavidin agarose beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA or anti - His antibodies .

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

Lysates of FHM cells overexpressing CiTLR5a were respectively incubated with FlaA, FlaB, FlaC, at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads or streptavidin agarose beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA or anti-His antibodies.


Lysates of FHM overexpressing CiTLR5b were respectively incubated with FlaA , FlaB , FlaC , at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads or streptavidin agarose beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA or anti - His antibodies .

Lysates of FHM cells overexpressing CiTLR5b were respectively incubated with FlaA, FlaB, FlaC, at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads or streptavidin agarose beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA or anti-His antibodies.


Lysates of FHM overexpressing CiTLR5a CiTLR5b were respectively incubated with biotin - poly ( I : C ) , biotin - ssRNA ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA antibodies .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b were respectively incubated with biotin-poly(I:C), biotin-ssRNA (1 μg/mL) at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA antibodies.


N , O CiUNC93B1 - Myc CiTLR5a - HA ( M ) or CiTLR5b - HA ( N ) were transfected into FHM . Co - IP immunoblotting were conducted to interactions between CiUNC93B1 - Myc CiTLR5a - HA or CiTLR5b - HA .

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

N, O CiUNC93B1-Myc and CiTLR5a-HA (M) or CiTLR5b-HA (N) were transfected into FHM cells. Co-IP and immunoblotting were conducted to test the interactions between CiUNC93B1-Myc and CiTLR5a-HA or CiTLR5b-HA.


O , P expression of IFN1 ( O ) NF - κB1 ( P ) in spleen tissue were investigated by immunoblotting ( IB ) after RNAi in vivo for 24 h , A . hydrophila or GCRV challenge for 24 h ( n = 3 ) . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O, P Protein expression of IFN1 (O) and NF-κB1 (P) in spleen tissue were investigated by immunoblotting (IB) after RNAi in vivo for 24 h, and A. hydrophila or GCRV challenge for 24 h (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Lysates of FHM overexpressing mutants were incubated with FlaC for pull - down assays analyzed by immunoblotting ( IB ) . Vector wild type ( WT ) were used as controls . Relative band ratios were analyzed by ImageJ ( n = 3 ) . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used for n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing mutants were incubated with FlaC for pull-down assays and analyzed by immunoblotting (IB). Vector and wild type (WT) were used as controls. Relative band intensity ratios were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for n represents biological replicates. *P < 0.05, **P < 0.01.


Lysates of FHM overexpressing CiTLR5a CiTLR5b ( including region deletants ) were incubated with biotin - poly ( I : C ) . Vector was used as a control . Pull - down immunoblotting ( IB ) were performed .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b (including region deletants) were incubated with biotin-poly(I:C). Vector was used as a control. Pull-down and immunoblotting (IB) were performed.


H - K Lysates of FHM overexpressing CiTLR5a CiTLR5b site mutants were incubated with biotin - poly ( I : C ) . Vector wild type were used as controls . Pull - down immunoblotting were carried out ( H , J ) . ( I , K ) Relative band ratios of ( H ) ( J ) were analyzed by ImageJ , respectively ( n = 3 ) . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used for ( I ) , ( K ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-K Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b site mutants were incubated with biotin-poly(I:C). Vector and wild type were used as controls. Pull-down and immunoblotting were carried out (H, J). (I, K) Relative band intensity ratios of (H) and (J) were analyzed by ImageJ, respectively (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (I), and (K). n represents biological replicates. *P < 0.05, **P < 0.01.


Lysates of FHM overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H analyzed by immunoblotting ( IB ) with anti - HA antibody .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H and analyzed by immunoblotting (IB) with anti-HA antibody.


Mutants with predicted N - glycosylation sites were transfected into FHM examined via immunoblotting to show change of molecular .

Mutants with the predicted N-glycosylation sites were transfected into FHM cells and examined via immunoblotting to show the change of molecular weight.


Lysates of FHM overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with biotin - poly ( I : C ) ( I ) Vector wild type were used as controls . Pull - down immunoblotting assays were conducted ( I , ( J , Relative band ratios of ( I ) were analyzed by ImageJ ( n = 3 ) . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used for ( J ) , n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with biotin-poly(I:C) (I) Vector and wild type were used as controls. Pull-down and immunoblotting assays were conducted (I, (J, Relative band intensity ratios of (I) were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (J), n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Lysates of FHM overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with FlaC ( K ) . Vector wild type were used as controls . Pull - down immunoblotting assays were conducted K ) . L ) Relative band ratios of ( K ) were analyzed by ImageJ ( n = 3 ) . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used for ( L ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with FlaC (K). Vector and wild type were used as controls. Pull-down and immunoblotting assays were conducted K). L) Relative band intensity ratios of (K) were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (L). n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Lysates of FHM overexpressing LoTLR5 ( A ) , DrTLR5a ( B ) , DrTLR5b ( B ) , IpTLR5 - 1 ( C ) , IpTLR5 - 2 ( C ) , IpTLR5S ( C ) , OmTLR5M ( D ) , OmTLR5S ( D ) , were respectively incubated with FlaC - His ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by Anti - GFP antibody coupling beads analyzed by immunoblotting ( IB ) with anti - His antibody .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing LoTLR5 (A), DrTLR5a (B), DrTLR5b (B), IpTLR5-1 (C), IpTLR5-2 (C), IpTLR5S (C), OmTLR5M (D), OmTLR5S (D), were respectively incubated with FlaC-His (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by Anti-GFP antibody coupling beads and analyzed by immunoblotting (IB) with anti-His antibody.


Lysates of FHM overexpressing HsTLR5 ( E ) were respectively incubated with FlaC - His ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by Anti - GFP antibody coupling beads analyzed by immunoblotting ( IB ) with anti - His antibody .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing HsTLR5 (E) were respectively incubated with FlaC-His (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by Anti-GFP antibody coupling beads and analyzed by immunoblotting (IB) with anti-His antibody.


Lysates of FHM overexpressing LoTLR5 ( F ) , DrTLR5a ( G ) , DrTLR5b ( G ) , IpTLR5 - 1 ( H ) , IpTLR5 - 2 ( H ) , IpTLR5S ( H ) , were respectively incubated with biotin - poly ( I : C ) ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by streptavidin beads analyzed via immunoblotting with anti - GFP antibody .

O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing LoTLR5 (F), DrTLR5a (G), DrTLR5b (G), IpTLR5-1 (H), IpTLR5-2 (H), IpTLR5S (H), were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti-GFP antibody.


Lysates of FHM overexpressing OmTLR5M ( I ) , OmTLR5S ( I ) , HsTLR5 ( J ) were respectively incubated with biotin - poly ( I : C ) ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by streptavidin beads analyzed via immunoblotting with anti - GFP antibody .

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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing OmTLR5M (I), OmTLR5S (I), and HsTLR5 (J) were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti-GFP antibody.


Lysates of FHM overexpressing DrTLR5a DrTLR5b mutants were incubated with biotin - poly ( I : C ) analyzed by immunoblotting ( N ) , relative band ratios were analyzed by ImageJ ( O ) ( n = 3 ) . Vector wild type ( WT ) were used as controls . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used for ( O ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates of FHM cells overexpressing DrTLR5a and DrTLR5b mutants were incubated with biotin-poly(I:C) and analyzed by immunoblotting (N), and relative band intensity ratios were analyzed by ImageJ (O) (n = 3). Vector and wild type (WT) were used as controls. Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (O). n represents biological replicates. *P < 0.05, **P < 0.01.


( A ) Huh7 . 5 were pre - incubated with neutralizing antibody to ACE2 SARS - CoV - 2 pseudovirus was pre - incubated with isolated mAb COVA1 - 18 , COVA1 - 21 COVA2 - 15 ( 10 µg / mL ) or UF heparin ( 250 IU / mL ) for 30 min at 37 ° C . SARS - CoV - 2 pseudovirus alone or with blocks was added to for 4h at 4 ° C binding was determined by ELISA . Data information : Data show mean values error bars are . Statistical was performed using ordinary one - way ANOVA with Tukey multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( n = 3 )

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Huh7.5 cells were pre-incubated with neutralizing antibody to ACE2 and SARS-CoV-2 pseudovirus was pre-incubated with patient isolated mAb COVA1-18, COVA1-21 and COVA2-15 (10 µg/mL) or UF heparin (250 IU/mL) for 30 min at 37°C. SARS-CoV-2 pseudovirus alone or with blocks was added to the cells for 4h at 4°C and binding was determined by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way ANOVA with Tukey multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001(n=3)


( D ) Control EXT1 - / - XG1 were exposed to SARS - CoV - 2 pseudovirus or SARS - CoV - 2 pseudovirus pre - treated with 250 IU / mL UF heparin for 30 min at 37 ° C . After incubation for 4h at 4 ° C , were lysed binding was measured by ELISA . Data information : Data show mean values error bars are . Statistical was performed using two - way ANOVA with Dunnett ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 3 ) .

O O O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Control and EXT1-/- XG1 cells were exposed to SARS-CoV-2 pseudovirus or SARS-CoV-2 pseudovirus pre-treated with 250 IU/mL UF heparin for 30 min at 37°C. After incubation for 4h at 4°C, cells were lysed and binding was measured by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using two-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=3).


( B ) SARS - CoV - 2 pseudovirus was pre - incubated for 30 min at 37 ° C with UF heparin ( 250 IU / mL ) or LMWHs tinzaparin ( 250 IU / mL ) or dalteparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) or nadroparin ( 250 IU / mL ) . Huh7 . 5 were exposed to SARS - CoV - 2 pseudovirus , alone or treated with different LMWHs for 4 hours at 4 ° C , washed , lysed binding was determined by ELISA . ( C ) SARS - CoV - 2 pseudovirus was pre - incubated for 30 min at 37 ° C with UF heparin ( 250 IU / mL ) or LMWHs tinzaparin ( 250 IU / mL ) or dalteparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) or nadroparin ( 250 IU / mL ) . Huh7 . 5 were infected with SARS - CoV - 2 pseudovirus in presence or absence of different LMWHsSARS - CoV - 2 infection was determined after 5 days by luciferase reporter activity . Data information : Data show mean values error bars are . Statistical was performed using ( B ) ordinary one - way ANOVA with Tukey ' s multiple - comparison ( n = 3 donors measured in monoplo ) , ( C ) ordinary one - way ANOVA with Dunnett ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( n = 3 donors measured in triplicate )

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SARS-CoV-2 pseudovirus was pre-incubated for 30 min at 37°C with UF heparin (250 IU/mL) or LMWHs tinzaparin (250 IU/mL) or dalteparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) or nadroparin (250 IU/mL). Huh7.5 were exposed to the SARS-CoV-2 pseudovirus, alone or treated with different LMWHs for 4 hours at 4°C, washed, lysed and binding was determined by ELISA. (C) SARS-CoV-2 pseudovirus was pre-incubated for 30 min at 37°C with UF heparin (250 IU/mL) or LMWHs tinzaparin (250 IU/mL) or dalteparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) or nadroparin (250 IU/mL). Huh7.5 cells were infected with SARS-CoV-2 pseudovirus in presence or absence of different LMWHsSARS-CoV-2 and infection was determined after 5 days by luciferase reporter activity. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (B) ordinary one-way ANOVA with Tukey's multiple-comparison test (n=3 donors measured in monoplo), (C) ordinary one-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=3 donors measured in triplicate)


( C , D ) SARS - CoV - 2 binding was measured in polarized Caco - 2 ( C ) Calu - 3 ( D ) in presence or absence of antibodies against ACE2 ( incubation ) , UF heparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) ( incubation ) . Data information : Data show mean values error bars are . Statistical was performed using ordinary one - way ANOVA with Tukey ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( C ) ( n = 3 Caco - 2 donors measured in triplicate ) , ( D ) ( n = 3 Calu - 3 donors measured in triplicate )

O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) SARS-CoV-2 binding was measured in polarized Caco-2 (C) and Calu-3 (D) cells in presence or absence of antibodies against ACE2 (cell incubation), and UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) (virus incubation). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3 Caco-2 donors measured in triplicate), (D)(n=3 Calu-3 donors measured in triplicate)


( A ) Namalwa ectopically expressing either Syndecan 1 or 4 were exposed to either SARS - CoV - 2 pseudovirus alone or SARS - Cov - 2 pseudovirus pre - treated with UF heparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . Binding was measured after 4 hours at 4 ° C by ELISA . ( B ) SARS - CoV - 2 isolate ( hCoV - 19 / Italy ) was pre - incubated with LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . Namalwa expressing Syndecan 1 4 were exposed to either SARS - CoV - 2 isolate ( 100 TCID / mL ) or SARS - CoV - 2 isolate ( 100 TCID / mL ) pre - treated with LMWH LMWH enoxaparin ( 250 IU / mL ) for 4 hours at 4 ° C binding was determined by quantitative real - time PCR . Data show mean values error bars are . Statistical was performed using ( A ) 2way - ANOVA with Dunnett ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 7 ) , ( B ) 2way - ANOVA with Sidak ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( n = 3 measured in triplicate ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Namalwa cells ectopically expressing either Syndecan 1 or 4 were exposed to either SARS-CoV-2 pseudovirus alone or SARS-Cov-2 pseudovirus pre-treated with UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Binding was measured after 4 hours at 4°C by ELISA. (B) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-incubated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Namalwa cells expressing Syndecan 1 and 4 were exposed to either SARS-CoV-2 isolate (100 TCID/mL) or SARS-CoV-2 isolate (100 TCID/mL) pre-treated with LMWH LMWH enoxaparin (250 IU/mL) for 4 hours at 4°C and binding was determined by quantitative real-time PCR. Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) 2way-ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n = 7), (B) 2way-ANOVA with Sidak's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n = 3 measured in triplicate).


( B ) SARS - CoV - 2 isolate ( hCoV - 19 / Italy ) was pre - treated for 30 min at 37 ° C with either LMWH enoxaparin ( 250 IU / mL ) or one of following neutralizing antibodies ( COVA1 - 18 , 1 - 21 2 - 15 ) , a non - neutralizing antibody ( COVA1 - 27 ) or a human IgG1 isotype control , at of 1 pg / mL . SARS - CoV - 2 isolate alone or with blocks was added at a of 100 TICD / mL . Detection of binding to Syndecan 1 expressing Namalwa was measured by quantitative real - time PCR . Data information : Data show mean values error bars are . Statistical was performed using ordinary one - way with Tukey ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 2 measured in duplicates ) .

O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated for 30 min at 37°C with either LMWH enoxaparin (250 IU/mL) or one of the following neutralizing antibodies (COVA1-18, 1-21 and 2-15), a non-neutralizing antibody (COVA1-27) or a human IgG1 isotype control, all at the concentration of 1 pg/mL. SARS-CoV-2 isolate alone or with blocks was added at a concentration of 100 TICD/mL. Detection of virus binding to Syndecan 1 expressing Namalwa was measured by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01 (n=2 measured in duplicates).


( F , G ) DCs ( F ) LCs ( G ) were pre - incubated with SARS - CoV - 2 pseudovirus for 4 hours at 37 ° C in presence or absence of UF heparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) , extensively washed co - cultured with Huh7 . 5 . Transmission by DCs or LCs to Huh7 . 5 was determined by luciferase reporter activity . Data information : Data show mean values error bars are . ( F ) ordinary one - way ANOVA with Dunnett ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 4 measured in triplicate ) , ( G ) ordinary one - way ANOVA with Tukey ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( n = 3 measured in triplicate ) . DCs : Dendritic , LCs : Langerhans , RLU : relative

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) DCs (F) and LCs (G) were pre-incubated with SARS-CoV-2 pseudovirus for 4 hours at 37°C in presence or absence of UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL), extensively washed and co-cultured with Huh7.5 cells. Transmission by DCs or LCs to Huh7.5 cells was determined by luciferase reporter activity. Data information: Data show the mean values and error bars are the SEM. (F) ordinary one-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=4 measured in triplicate), (G) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=3 measured in triplicate). DCs: Dendritic cells, LCs: Langerhans cells, RLU: relative light units


( H , I ) SARS - CoV - 2 isolate ( hCoV - 19 / Italy ) was pre - treated with LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . DCs ( H ) LCs ( I ) were exposed to either untreated or pre - treated SARS - CoV - 2 isolate ( 100 TCID / mL ) for 24h , washed thoroughly co - cultured with Huh7 . 5 . of viral was measured by quantitative real - time PCR . Data information : Data show mean values error bars are . ( H , I ) ordinary one - way ANOVA with Tukey ' s multiple - comparison . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( H ) ( n = 3 in duplicates ) , ( I ) ( n = 4 in duplicates ) . DCs : Dendritic , LCs : Langerhans

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H, I) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. DCs (H) and LCs (I) were exposed to either the untreated or pre-treated SARS-CoV-2 isolate (100 TCID/mL) for 24h, washed thoroughly and co-cultured with Huh7.5 cells. Quantification of viral RNA was measured by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. (H, I) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (H) (n=3 in duplicates), (I) (n=4 in duplicates). DCs: Dendritic cells, LCs: Langerhans cells


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

O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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).


( b ) THPN - induced is prevented by autophagic inhibitors wortmannin ( 100 nM ) chloroquine ( 20 μM ) but not by apoptotic inhibitor zVAD ( 20 μM ) or necrotic inhibitors butylated hydroxyanisole ( 100 μM ) necrostatin - 1 ( 30 μM ) . were pretreated with each inhibitor for 1 h separately before THPN treatment ( 20 μM , 48 h ) . Ctrl , control .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O 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) 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 . Top , THPN ( 20 μM , 24 h ) induces a change in localization of GFP - LC3 fusion from a ubiquitous , diffuse pattern to . Serum starvation ( SS , 24 h ) was used as a positive control ( scale bars , 10 μm ) . Middle , GFP - LC3 - positive with were calculated from five randomly picked regions . Bottom , conversion of LC3 - I to LC3 - II induced by THPN was determined by western blotting . of full blots are shown in Supplementary Figure 7 . of data are presented as mean ± s . e . m . of three independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; NS , not significant .

O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( a ) THPN induces TR3 translocation from cytoplasm to mitochondria , detected by western blotting for various fractionations ( top ) by confocal microscopy ( bottom ) . were treated with THPN at times indicated detected by TR3 ( red ) Hsp60 ( green ) . nuclei were stained with DAPI ( blue ) ( scale bars , 10 μm ) . PARP , Tubulin Hsp60 were used as loading controls for extracts ( NE ) , extracts ( CE ) mitochondrial fractions ( Mito ) , respectively . IB , immunoblotting .

O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O 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-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 induction of autophagy in response to THPN treatment . siRNA - resistant versions of Flag - ΔNLS Flag - NLS were transfected into TR3 - knockdown A375 . Top , cotransfected with GFP - LC3 were further treated with THPN for 24 h before microscopic ( scale bars , 10 μm ) . Middle , conversion of LC3 - I to LC3 - II was analyzed by western blotting . rate of survival was determined after THPN treatment for 48 h ( bottom ) . ' - ' represents vehicle treatment only .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(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 TR3 mitochondrial targeting . with Nix , Pink1 Bnip3 knockdown were treated with THPN ( 20 μM , 6 h ) . Top , mitochondrial TR3 was assayed by western blotting . Nix knockdown abolishes THPN - induced ( 20 μM , 24 h ) formation of LC3 foci ( middle bottom ; scale bars , 10 μm ) . of full blots are shown in Supplementary Figure 7 . of data are presented as mean ± s . e . m . of three independent experiments . * * P 0 . 01 ; NS , not significant ; ctrl , control .

O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( b ) Site C of LBD is critical for Nix binding , as determined by in vitro pull - down . purified bead - bound GST or GST - Nix were incubated with different mutants as indicated THPN ( 20 μM ) for 3 h ; were then analyzed by western blotting . IB , immunoblotting .

O O O O O O O O O O B-MEASURED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(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 , structure of DPDO . Middle , DPDO binding could not form interacting surface for Nix , unlike THPN . structures of Arg563 Ser553 are same as those in native structure . Bottom , DPDO does not enhance interaction between TR3 Nix . Different plasmids were transfected into A375 as indicated , were treated with DPDO ( 20 μM , 3 h ) . TR3 interaction with Nix was determined by co - immunoprecipitation . of full blots are shown in Supplementary Figure 7 . IP , immunoprecipitation ; HA , hemagglutinin .

O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( a ) THPN induces dissipation of ΔΨm in both time - dose - dependent manners . compromised ΔΨm is shifted to right upon THPN treatment in flow cytometry . ' - ' represents vehicle treatment only .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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 THPN - induced dissipation of ΔΨm . Top , endogenous TR3 was knocked down by shRNA , siRNA - resistant version of TR3 , TR3 - NLS , TR3 - ΔNLS or TAD ( Supplementary Fig . 2c ) were separately transfected into as indicated . Bottom , similarly , siRNA - resistant version of Nix or ΔTM were separately transfected into Nix knockdown . Transfected were treated with THPN ( 20 μM , 12 h ) , their ΔΨm was assessed by flow cytometry . Ctrl , control .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(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 VDAC1 are required for THPN - induced autophagy . ANT1 VDAC1 shRNA - expressing plasmids , which also encoded EGFP for evaluating transfection efficiency , were separately transfected into ; mCherry - LC3 was cotransfected to display autophagy . Green are those transfected with ANT1 or VDAC1 shRNA , with red are autophagic ( top ; scale bars , 10 μm ) .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(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 VDAC1 are required for THPH - induced dissipation of ΔΨm . Endogenous ANT1 or VDAC1 was separately knocked down by shRNA , dissipation of ΔΨm was determined after THPN ( 20 μM , 12 h ) treatment ( left ) . ANT1 VDAC1 knockdown abolishes THPN - induced ( 20 μM , 48 h ) ( right ) . of data are presented as mean ± s . e . m . of three independent experiments . * * P 0 . 01 .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( a ) Endogenous TR3 is localized in MIM in response to THPN stimulation . Left , isolated mitochondria from treated with THPN ( 20 μM , 6 h ) were incubated with proteinase K ( 20 μM 40 μM ) for 30 min . TR3 expression was determined . Tom20 in MOM Tim23 in MIM were used as controls . Right , mitochondria were further subjected to submitochondrial fractionation . VDAC1 , Tim23 Hsp60 were used to represent MOM , MIM mitochondrial matrix , respectively . ' - ' represents vehicle treatment only .

O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 Tom70 blocks TR3 transit to MIM . Endogenous Toms were separately knocked down by their corresponding shRNAs , TR3 expression of mitochondrial fractions was detected as described above . Ctrl , control .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(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 Tom70 are required for THPN - associated dissipation of ΔΨm ( left ) , conversion of LC3 - I to LC3 - II ( right top ) ( right bottom ) . Endogenous Toms were separately knocked down by corresponding shRNAs . ΔΨm , LC3 - II were detected as described above . of full blots are shown in Supplementary Figure 7 .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(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 ) mitochondria were markedly eliminated after treatment with THPN for 24 h . Right , endogenous Tom40 Tom70 were knocked down , amount of mitochondria was measured by of Tom20 ( green ) . Left , volume of mitochondria from five randomly picked regions was quantified by ImageJ software . of data are presented as mean ± s . e . m . of three independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; NS , not significant .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( a ) THPN inhibits formation of tumors in liver lung in a TR3 - dependent manner . B16 transfected with either control shRNA , TR3 shRNA or ATG7 shRNA ( 5 × 105 ) were separately injected into lateral tail vein of 6 - week - old C57BL / 6J mice ( n = 7 ) . THPN were administered 3 d after inoculation . tumors formed in lung liver are indicated ( top ; scale bars , 5 mm ) were counted ( bottom ) . Ctrl , control .

O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 + / + Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 − / − mice ( n = 12 ) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms , mice were treated with THPN as described in Online Methods . representative of dorsal melanomas ( top ) numbers ( bottom ) are presented .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(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 ) survival curves of crossed Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 + / + Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 − / − mice are shown ( n > 16 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(c) The survival curves of crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice are shown (n > 16).