( 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 .

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

(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 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 .

O O O 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 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

(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 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 .

O O O O O O O O O O O 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

(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 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 .

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

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


( 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (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 cells were immunoprecipitated with anti - GRWD1 antibody or control IgG . Immunoprecipitates ( IPs ) and 2 . 7 % of inputs were immunoblotted with the indicated 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

(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 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 .

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 O

(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 and bound proteins analyzed by immunoblotting with the indicated antibodies . I , input . P , pull - down .

O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 fluorescent microscopy with MitoTracker Orange straining . Scale bar for all micrographs 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 O O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O 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 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 .

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

(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 .

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


( 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 .

O O O O O O O O O O O O O O O O O O O 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 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 .

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 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 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 .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 .

O O 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

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 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 .

O O 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 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 cells overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H and analyzed by immunoblotting ( IB ) with anti - HA 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

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

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

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 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 .

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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O 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 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 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 .

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

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 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 )

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

(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 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 ) .

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

(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 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 )

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 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 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 ) 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 )

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

(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 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 ) .

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

(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 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 ) .

O O O O O O 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

(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 ) 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

O O O O O O O O O O O O O O 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 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 ) 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

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

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

(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 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 .

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 O O B-CONTROLLED_VAR 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 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

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


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

O 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 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

(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 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 .

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 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

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


( b ) TR3 mutant Flag - ΔNLS but not Flag - NLS promotes the induction of autophagy in response to THPN treatment . The siRNA - resistant versions of Flag - ΔNLS and Flag - NLS were transfected into TR3 - knockdown A375 cells . Top , the cells cotransfected with GFP - LC3 were further treated with THPN for 24 h before microscopic analysis ( scale bars , 10 μm ) . Middle , the conversion of LC3 - I to LC3 - II was analyzed by western blotting . The rate of cell survival was determined after THPN treatment for 48 h ( bottom ) . ' - ' represents vehicle treatment only .

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

O 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 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

(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 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 .

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

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


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

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

(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 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 .

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

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


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

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


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

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


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

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(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 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 .

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


( b ) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM . Endogenous Toms were separately knocked down by their corresponding shRNAs , and the TR3 expression of mitochondrial fractions was detected as described above . Ctrl , control .

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(b) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM. Endogenous Toms were separately knocked down by their corresponding shRNAs, and the TR3 expression of mitochondrial fractions was detected as described above. Ctrl, control.


( c ) Tom40 and Tom70 are required for the THPN - associated dissipation of ΔΨm ( left ) , conversion of LC3 - I to LC3 - II ( right and top ) and cell death ( right and bottom ) . Endogenous Toms were separately knocked down by the corresponding shRNAs . ΔΨm , LC3 - II and cell death were detected as described above . Images of full blots are shown in Supplementary Figure 7 .

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(c) Tom40 and Tom70 are required for the THPN-associated dissipation of ΔΨm (left), conversion of LC3-I to LC3-II (right and top) and cell death (right and bottom). Endogenous Toms were separately knocked down by the corresponding shRNAs. ΔΨm, LC3-II and cell death were detected as described above. Images of full blots are shown in Supplementary Figure 7.


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

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(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 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 .

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


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

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


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

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


B . Comparisons of MTAP mRNA and protein expression between CL1 - 0 and CL1 - 5 cells detected by RT - qPCR ( top , mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) and Western blot assays using anti - MTAP antibody ( bottom , representative of three independent experiments ) .

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B. Comparisons of MTAP mRNA and protein expression between CL1-0 and CL1-5 cells detected by RT-qPCR (top, mean ± SD, Student t test, n=3, biological replicates, *p < 0.05) and Western blot assays using anti-MTAP antibody (bottom, representative of three independent experiments).


C . Cell invasion abilities of MTAP - overexpressing CL1 - 5 or MTAP - knockout CL1 - 0 and H1650 were determined by Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) . sgCTRL indicates cells transduced with a non - targeting control sgRNA , and sgMTAP indicates cells transduced with a sgRNA targeting MTAP . Bottom : the protein expression levels of V5 - MTAP and endogenous MTAP were detected by Western blots .

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C. Cell invasion abilities of MTAP-overexpressing CL1-5 or MTAP-knockout CL1-0 and H1650 were determined by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). sgCTRL indicates cells transduced with a non-targeting control sgRNA, and sgMTAP indicates cells transduced with a sgRNA targeting MTAP. Bottom: the protein expression levels of V5-MTAP and endogenous MTAP were detected by Western blots.


E . Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays . Top : representative photographs of lungs and H & E staining of the lung sections . The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows . Bottom : quantification of averaged primary tumor sizes , metastatic incidence and nodule number .

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E. Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays. Top: representative photographs of lungs and H&E staining of the lung sections. The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows. Bottom: quantification of averaged primary tumor sizes, metastatic incidence and nodule number.


F . Kaplan - Meier analyses of overall survival ( top ) and progression - free survival ( bottom ) for 101 patients with lung adenocarcinoma grouped into high - or low - MTAP mRNA expression measured by RT - qPCR . p values were obtained by log - rank test .

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F. Kaplan-Meier analyses of overall survival (top) and progression-free survival (bottom) for 101 patients with lung adenocarcinoma grouped into high- or low-MTAP mRNA expression measured by RT-qPCR. p values were obtained by log-rank test.


A . The necessity of MTAP activity in cell invasion . CL1 - 5 and A549 cells were introduced with indicated plasmids and subjected to Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 compared to the vector control group ) . The protein expression levels of exogenous wild - type and mutant V5 - MTAP were detected by Western blots .

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A. The necessity of MTAP activity in cell invasion. CL1-5 and A549 cells were introduced with indicated plasmids and subjected to Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05 compared to the vector control group). The protein expression levels of exogenous wild-type and mutant V5-MTAP were detected by Western blots.


B . Western blot analysis of the symmetric dimethylarginine ( sDMA ) , monomethylarginine ( mMA ) and asymmetric dimethylarginine ( aDMA ) levels in MTAP - overexpressing and MTAP - knockout cells . V5 - MTAP wild - type and D220A mutant were restored in H1650 MTAP - knockout cells . Data shown are representative of three independent experiments .

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B. Western blot analysis of the symmetric dimethylarginine (sDMA), monomethylarginine (mMA) and asymmetric dimethylarginine (aDMA) levels in MTAP-overexpressing and MTAP-knockout cells. V5-MTAP wild-type and D220A mutant were restored in H1650 MTAP-knockout cells. Data shown are representative of three independent experiments.


C . In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti - sDMA antibody . Arrow marks dimethyl - vimentin .

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C. In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti-sDMA antibody. Arrow marks dimethyl-vimentin.


D . Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells . Arrow marks dimethyl - vimentin . Data shown are representative of three independent experiments .

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D. Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells. Arrow marks dimethyl-vimentin. Data shown are representative of three independent experiments.


E - F . Immunoprecipitation analysis for vimentin dimethylation in control and MTAP - knockout cells . Arrows mark vimentin , asterisks mark heavy chains , and arrowheads mark nonspecific bands . Data shown are representative of three independent experiments .

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E-F. Immunoprecipitation analysis for vimentin dimethylation in control and MTAP-knockout cells. Arrows mark vimentin, asterisks mark heavy chains, and arrowheads mark nonspecific bands. Data shown are representative of three independent experiments.


G . Vimentin - knockdown counteracts MTAP - loss - induced effect on cell invasion . MTAP - knockout and control H1650 cells were transfected with vimentin - specific siRNAs for 72 hours and analyzed by Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) . The silence efficiency of vimentin was examined by Western blots .

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G. Vimentin-knockdown counteracts MTAP-loss-induced effect on cell invasion. MTAP-knockout and control H1650 cells were transfected with vimentin-specific siRNAs for 72 hours and analyzed by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). The silence efficiency of vimentin was examined by Western blots.


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

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


B . Reconstitution of vimentin - knockout cells with wild - type , 4RK - and 4RF - mutated V5 - vimentin . Both protein and mRNA levels of vimentin were determined by Western blots and RT - qPCR . Data shown are representative of three independent experiments .

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B. Reconstitution of vimentin-knockout cells with wild-type, 4RK- and 4RF-mutated V5-vimentin. Both protein and mRNA levels of vimentin were determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.


C . Vimentin protein and mRNA levels in indicated paired cells were measured by Western blots and RT - qPCR . Data shown are representative of three independent experiments .

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C. Vimentin protein and mRNA levels in indicated paired cells were measured by Western blots and RT-qPCR. Data shown are representative of three independent experiments.


D . Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT - qPCR . Data shown are representative of three independent experiments .

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D. Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.


E . Western blot analysis of vimentin protein levels in MTAP - knockout and control cells transfected with PRMT5 siRNAs ( left ) or Flag - PRMT5 ( right ) . Arrows mark the site of Flag - PRMT5 and endogenous PRMT5 . Data shown are representative of three independent experiments .

E. Western blot analysis of vimentin protein levels in MTAP-knockout and control cells transfected with PRMT5 siRNAs (left) or Flag-PRMT5 (right). Arrows mark the site of Flag-PRMT5 and endogenous PRMT5. Data shown are representative of three independent experiments.


F . Effect of PRMT5 on the levels of ectopically expressed wild - type , 4RK - and 4RF - mutated V5 - vimentin in HEK293T cells assessed by Western blots . Data shown are representative of three independent experiments . G . Effects of wild - type and catalytically dead R368A mutant PRMT5 on the level of V5 - vimentin in HEK293T cells detected by Western blot assays . Data shown are representative of three independent experiments .

F. Effect of PRMT5 on the levels of ectopically expressed wild-type, 4RK- and 4RF-mutated V5-vimentin in HEK293T cells assessed by Western blots. Data shown are representative of three independent experiments. G. Effects of wild-type and catalytically dead R368A mutant PRMT5 on the level of V5-vimentin in HEK293T cells detected by Western blot assays. Data shown are representative of three independent experiments.


A . Measurement of V5 - vimentin wild - type , 4RK and 4RF mutant protein levels in CL1 - 5 vimentin - knockout cells treated with 10 μM MG132 by Western blots . Data shown are representative of three independent experiments .

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A. Measurement of V5-vimentin wild-type, 4RK and 4RF mutant protein levels in CL1-5 vimentin-knockout cells treated with 10 μM MG132 by Western blots. Data shown are representative of three independent experiments.


B . Western blot analysis of vimentin protein levels in control and MTAP - knockout cells treated with 10 μM MG132 . Data shown are representative of three independent experiments .

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B. Western blot analysis of vimentin protein levels in control and MTAP-knockout cells treated with 10 μM MG132. Data shown are representative of three independent experiments.


Immunoprecipitation analysis of ubiquitination ( C ) of V5 - vimentin in HEK293T cells transfected with wild - type , 4RK - or 4RF - mutated vimentin in the presence of HA - tagged ubiquitin . Arrows mark the sites of V5 - vimentin . Data shown are representative of three independent experiments .

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Immunoprecipitation analysis of ubiquitination (C) of V5-vimentin in HEK293T cells transfected with wild-type, 4RK- or 4RF-mutated vimentin in the presence of HA-tagged ubiquitin. Arrows mark the sites of V5-vimentin. Data shown are representative of three independent experiments.


K48 - linked polyubiquitination ( D ) of V5 - vimentin in HEK293T cells transfected with wild - type , 4RK - or 4RF - mutated vimentin in the presence of HA - tagged ubiquitin . Arrows mark the sites of V5 - vimentin . Data shown are representative of three independent experiments .

K48-linked polyubiquitination (D) of V5-vimentin in HEK293T cells transfected with wild-type, 4RK- or 4RF-mutated vimentin in the presence of HA-tagged ubiquitin. Arrows mark the sites of V5-vimentin. Data shown are representative of three independent experiments.


E . Immunoprecipitation analysis for K48 - linked polyubiquitination of vimentin in CL1 - 0 control and MTAP - knockout cells . Arrow marks the site of vimentin . Data shown are representative of three independent experiments .

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E. Immunoprecipitation analysis for K48-linked polyubiquitination of vimentin in CL1-0 control and MTAP-knockout cells. Arrow marks the site of vimentin. Data shown are representative of three independent experiments.