B , IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva of vaccinated naïve and SARS - CoV - 2 - Exp subjects 7 - 10 days after second dose ( T2 ) and the antibody response was compared to that obtained in COVID - 19 patients ( plasma : n = 28 ; saliva : n = 26 ) and in non - vaccinated subjects ( n = 19 , Control ) .

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B, IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva of vaccinated naïve and SARS-CoV-2-Exp subjects 7-10 days after second dose (T2) and the antibody response was compared to that obtained in COVID-19 patients (plasma: n=28; saliva: n=26) and in non-vaccinated subjects (n=19, Control).


( b ) AlPcS2a can be specifically targeted onto lysosomes . Using our staining procedure , AlPcS2a signals colocalized with Lysotracker Red ( top panels ) and lamp1 - RFP ( middle panels ) , but not with other cellular organelle markers such as EGFP - LC3B ( bottom panels , marker for autophagic structures ) , in HeLa cells . Scale bars , 10 μm .

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(b) AlPcS2a can be specifically targeted onto lysosomes. Using our staining procedure, AlPcS2a signals colocalized with Lysotracker Red (top panels) and lamp1-RFP (middle panels), but not with other cellular organelle markers such as EGFP-LC3B (bottom panels, marker for autophagic structures), in HeLa cells. Scale bars, 10 μm.


( a ) Simultaneous loss of Lysotracker and AlPcS2a fluorescence immediately following light activation . A HeLa cell labelled with Lysotracker Red and AlPcS2a was 635 - nm illuminated within the white square region ( left panels ) . Lysotracker fluorescence within the illuminated region was immediately lost after light illumination ( middle panels ) , indicating the successful triggering of LMP . Right panels : magnified view of Lysotracker fluorescence within the 635 - nm illuminated region pre - and post light activation . ( b ) 635 nm illumination did not lead to complete lysosome rupture . A HeLa cell labelled with FITC - dextran ( molecular weight = 10 , 000 ) and AlPcS2a was 635 - nm illuminated within the white square region ( left panels ) . FITC - dextran remained within illuminated lysosomes ( an increase in FITC fluorescence was observed , in accordance with an increase in pH in damaged lysosomes ; middle panels ) . Right panels : magnified view of FITC - dextran fluorescence within the 635 - nm illuminated region pre - and post light activation . ( c ) Light activation did not affect other cellular structures , such as mitochondria . A HeLa cell labelled with TMRE and AlPcS2a was 635 - nm illuminated within the white square region ( left panels ) . Illumination did not affect TMRE fluorescence , indicating that mitochondria within the same region were not affected / impaired . Right panels : magnified view of TMRE fluorescence within the 635 - nm illuminated region pre - and post light activation . Scale bars , 10 μm .

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(a) Simultaneous loss of Lysotracker and AlPcS2a fluorescence immediately following light activation. A HeLa cell labelled with Lysotracker Red and AlPcS2a was 635-nm illuminated within the white square region (left panels). Lysotracker fluorescence within the illuminated region was immediately lost after light illumination (middle panels), indicating the successful triggering of LMP. Right panels: magnified view of Lysotracker fluorescence within the 635-nm illuminated region pre- and post light activation. (b) 635 nm illumination did not lead to complete lysosome rupture. A HeLa cell labelled with FITC-dextran (molecular weight=10,000) and AlPcS2a was 635-nm illuminated within the white square region (left panels). FITC-dextran remained within illuminated lysosomes (an increase in FITC fluorescence was observed, in accordance with an increase in pH in damaged lysosomes; middle panels). Right panels: magnified view of FITC-dextran fluorescence within the 635-nm illuminated region pre- and post light activation. (c) Light activation did not affect other cellular structures, such as mitochondria. A HeLa cell labelled with TMRE and AlPcS2a was 635-nm illuminated within the white square region (left panels). Illumination did not affect TMRE fluorescence, indicating that mitochondria within the same region were not affected/impaired. Right panels: magnified view of TMRE fluorescence within the 635-nm illuminated region pre- and post light activation. Scale bars, 10 μm.


( a ) A HeLa cell expressing EGFP - Ub and lamp1 - RFP , and labelled with AlPcS2a , was 635 - nm illuminated within the white square

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(a) A HeLa cell expressing EGFP-Ub and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square region


b ) Punctate structures containing ubiquitin appeared within the 635 - nm illuminated region ( white arrow ) 30 - 50 min after light activation .

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b) Punctate structures containing ubiquitin appeared within the 635-nm illuminated region (white arrow) 30-50 min after light activation.


( c ) Ubiquitin puncta colocalized with damaged lysosomes . Magnified view of the 635 - nm illuminated cell region showed that ubiquitin signals localized on lysosomes ( lamp1 - labelled ) lacking AlPcS2a fluorescence ( indicating that it had been 635 - nm illuminated ) . White arrows indicate examples of Ub on damaged lysosomes .

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(c) Ubiquitin puncta colocalized with damaged lysosomes. Magnified view of the 635-nm illuminated cell region showed that ubiquitin signals localized on lysosomes (lamp1-labelled) lacking AlPcS2a fluorescence (indicating that it had been 635-nm illuminated). White arrows indicate examples of Ub on damaged lysosomes.


( d ) Quantification of ubiquitin signals within three independent cellular regions ( A1 , A2 and A3 ) revealed a specific recruitment of ubiquitin to the site of light activation ( inset , right , A1 ) . Scale bars , 10 μm ( a , b , d ) ; 3 μm ( c ) .

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(d) Quantification of ubiquitin signals within three independent cellular regions (A1, A2 and A3) revealed a specific recruitment of ubiquitin to the site of light activation (inset, right, A1). Scale bars, 10 μm (a,b,d); 3 μm (c).


( a ) A HeLa cell expressing EGFP - Ub and TagRFP - p62 , and labelled with AlPcS2a , was 635 - nm illuminated within the white square region , resulting in ubiquitination of and p62 recruitment to the photodamaged lysosomes , as shown in b . ( b ) Magnified view of the 635 - nm illuminated cell region in a . Ubiquitin and p62 initially distributed uniformly within the region ( t = 0 min , top panels ) , but formed distinct foci after light activation ( bottom panels ) .

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(a) A HeLa cell expressing EGFP-Ub and TagRFP-p62, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in ubiquitination of and p62 recruitment to the photodamaged lysosomes, as shown in b. (b) Magnified view of the 635-nm illuminated cell region in a. Ubiquitin and p62 initially distributed uniformly within the region (t=0 min, top panels), but formed distinct foci after light activation (bottom panels).


( c ) A HeLa cell expressing EGFP - LC3B and lamp1 - RFP , and labelled with AlPcS2a , was 635 - nm illuminated within the white square region , resulting in LC3 recruitment onto photodamaged lysosomes , as shown in d . ( d ) Magnified view of the 635 - nm illuminated cell region in c . LC3 is initially distributed away from lysosomes ( lamp1 , t = 0 min , top panels ) , but appears on lamp1 - positive structures 40 min after light activation ( bottom panels ) . Scale bars , 10 μm ( a , c ) ; 3 μm ( b , d ) .

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(c) A HeLa cell expressing EGFP-LC3B and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in LC3 recruitment onto photodamaged lysosomes, as shown in d. (d) Magnified view of the 635-nm illuminated cell region in c. LC3 is initially distributed away from lysosomes (lamp1, t=0 min, top panels), but appears on lamp1-positive structures 40 min after light activation (bottom panels). Scale bars, 10 μm (a,c); 3 μm (b,d).


( a ) Two HeLa cells expressing RFP - EGFP - LC3 and labelled with AlPcS2a was 635 - nm illuminated within the white rectangular region , resulting in local LC3 puncta formation , as shown in b . ( b ) LC3 puncta were specifically induced within the 635 - nm illuminated cell regions in a ( LC3 initially distributed uniformly at t = 0 ) . ( c ) The loss of EGFP fluorescence from light - induced LC3 puncta preceded the disappearance of RFP fluorescence ( white arrows indicate examples of LC3 puncta that exhibited diminished EGFP fluorescence ) , indicating their successful transition from autophagosomes to autolysosomes . Scale bars , 10 μm .

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(a) Two HeLa cells expressing RFP-EGFP-LC3 and labelled with AlPcS2a was 635-nm illuminated within the white rectangular region, resulting in local LC3 puncta formation, as shown in b. (b) LC3 puncta were specifically induced within the 635-nm illuminated cell regions in a (LC3 initially distributed uniformly at t=0). (c) The loss of EGFP fluorescence from light-induced LC3 puncta preceded the disappearance of RFP fluorescence (white arrows indicate examples of LC3 puncta that exhibited diminished EGFP fluorescence), indicating their successful transition from autophagosomes to autolysosomes. Scale bars, 10 μm.


( left ) representative photomicrographs of H & E stained tibialis anterior muscle sections from control and iNOS KO mice injected with or without LPS . Scale bars = 100 μm . ( right ) frequency histogram showing the distribution of muscle fiber minimum feret diameter in the tibialis anterior muscles from saline or LPS treated ( top ) WT control and ( bottom ) iNOS KO mice ( n = 4 ) . A total of 300 fibers per muscle were used for the analysis . Statistical comparisons , mean , and standard deviation of the mean are shown in the histogram legend .

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(left) representative photomicrographs of H&E stained tibialis anterior muscle sections from control and iNOS KO mice injected with or without LPS. Scale bars = 100 μm. (right) frequency histogram showing the distribution of muscle fiber minimum feret diameter in the tibialis anterior muscles from saline or LPS treated (top) WT control and (bottom) iNOS KO mice (n = 4). A total of 300 fibers per muscle were used for the analysis. Statistical comparisons, mean, and standard deviation of the mean are shown in the histogram legend.


( left ) Western blot analysis of pThr172 - AMPK ( pAMPK ) and total AMPK ( AMPK ) in quadriceps muscle . ( right ) Quantification of the pAMPK to AMPK ratio . Ratios are expressed relative to the saline treated controls .

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(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle. (right) Quantification of the pAMPK to AMPK ratio. Ratios are expressed relative to the saline treated controls.


Relative concentrations of metabolites involved in glycolysis and TCA cycle . Relative concentration of arginine .

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Relative concentrations of metabolites involved in glycolysis and TCA cycle. Relative concentration of arginine.


( D - G ) Weight of tibialis anterior ( D ) , gastrocnemius ( E ) , quadricep ( F ) and soleus ( G ) muscle normalized to initial body weight .

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(D-G) Weight of tibialis anterior (D), gastrocnemius (E), quadricep (F) and soleus (G) muscle normalized to initial body weight.


( left ) Western blot analysis of pThr172 - AMPK ( pAMPK ) and total AMPK ( AMPK ) in quadriceps muscle ( n = 3 ) . ( right ) Quantification of the pAMPK to AMPK ratio relative to the saline treated control ( n = 3 ) .

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(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle (n = 3). (right) Quantification of the pAMPK to AMPK ratio relative to the saline treated control (n = 3).


Relative concentrations of metabolites involved in glycolysis . Relative concentrations of amino acids .

Relative concentrations of metabolites involved in glycolysis. Relative concentrations of amino acids.


Representative immunofluorescence imaging for myoglobin and myosin heavy chain ( MyHC ) in not treated ( NT ) controls and IFNγ / TNFα ( IT ) samples treated with or without GW . Nuclei were visualized with DAPI staining ( n = 4 ) .

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Representative immunofluorescence imaging for myoglobin and myosin heavy chain (MyHC) in not treated (NT) controls and IFNγ/TNFα (IT) samples treated with or without GW. Nuclei were visualized with DAPI staining (n=4).


Western blot analysis for pThr172 - AMPK ( pAMPK ) , total AMPK ( AMPK ) , pSer79 - ACC ( pACC ) , and total ACC ( ACC ) .

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Western blot analysis for pThr172-AMPK (pAMPK), total AMPK (AMPK), pSer79-ACC (pACC), and total ACC (ACC).


Western blot analysis for pThr389 - S6K ( pS6K ) , total S6K ( S6K ) , pSer235 / 236 - S6 ( pS6 ) , and total S6 ( S6 ) ( n = 5 ) .

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Western blot analysis for pThr389-S6K (pS6K), total S6K (S6K), pSer235/236-S6 (pS6), and total S6 (S6) (n=5).


Glycolytic index of basal metabolism.


Western blot analysis for ETC protein complex subunits .

Western blot analysis for ETC protein complex subunits.


Male C57BL / 6 wildtype ( WT ) and iNOS knockout ( KO ) mice were intraperitoneally injected with 1mg kg - 1 LPS or an equivalent volume of carrier solution . Control WT , Control KO , and LPS - treated KO cohorts were pair - fed ( PF ) to the WT LPS - treated cohorts . After 18h , mice were euthanized , and tibialis anterior muscles were imaged by transmission electron microscopy . ( left ) Representative micrograph of tibialis anterior muscle ( n = 2 ) . Scale bar = 1 µm . ( right ) Zoomed section of representative image to highlight mitochondria . Scale bar = 0 . 5 µm .

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Male C57BL/6 wildtype (WT) and iNOS knockout (KO) mice were intraperitoneally injected with 1mg kg-1 LPS or an equivalent volume of carrier solution. Control WT, Control KO, and LPS-treated KO cohorts were pair-fed (PF) to the WT LPS-treated cohorts. After 18h, mice were euthanized, and tibialis anterior muscles were imaged by transmission electron microscopy. (left) Representative micrograph of tibialis anterior muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria. Scale bar = 0.5 µm.


Male BALB / C mice were injected subcutaneously with C26 cells ( 1 . 25x106 cells ) or an equivalent volume of saline . After 5 days , and everyday thereafter , saline and C26 injected mice were treated with or without GW ( 5mg kg - 1 ) . After 16 days , mice were euthanized and gastrocnemius muscles were imaged by transmission electron microscopy . ( left ) Representative micrograph of gastrocnemius muscle ( n = 2 ) . Scale bar = 1 µm . ( right ) Zoomed section of representative image to highlight mitochondria mitochondria . Scale bar = 0 . 5 µm .

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Male BALB/C mice were injected subcutaneously with C26 cells (1.25x106 cells) or an equivalent volume of saline. After 5 days, and everyday thereafter, saline and C26 injected mice were treated with or without GW (5mg kg-1). After 16 days, mice were euthanized and gastrocnemius muscles were imaged by transmission electron microscopy. (left) Representative micrograph of gastrocnemius muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria mitochondria. Scale bar = 0.5 µm.


Heatmap of overlapped expressed genes in response to sorafenib - resistance .

Heatmap of overlapped expressed genes in response to sorafenib-resistance.


D The mRNA expression level of METTL3 in sorafenib - sensitive ( n = 3 ) and sorafenib - resistant ( n = 3 ) human liver tumors . Data information : In all relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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D The mRNA expression level of METTL3 in sorafenib-sensitive (n=3) and sorafenib-resistant (n=3) human liver tumors. Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


E Expression of METTL3 was significantly down - regulated in advanced stage of liver cancer from TCGA .

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E Expression of METTL3 was significantly down-regulated in advanced stage of liver cancer from TCGA.


F METTL3 RNA levels between naïve HepG - 2 cells ( n = 3 ) and sorafenib - resistant HepG - 2 cells ( n = 10 ) . Data information : In all relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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F METTL3 RNA levels between naïve HepG-2 cells (n=3) and sorafenib-resistant HepG-2 cells (n=10). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


G The IC50 of sorafenib - resistant HepG - 2 cells treated with sorafenib under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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G The IC50 of sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


H The protein level of METTL3 in sorafenib - resistant HepG - 2 cells treated with sorafenib under hypoxia condition ( 1 % O2 ) .

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H The protein level of METTL3 in sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2).


I The global RNA m6A level in naïve HepG - 2 and sorafenib - resistant HepG - 2 determined by dot - blotting assay under hypoxia condition ( 1 % O2 ) .

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I The global RNA m6A level in naïve HepG-2 and sorafenib-resistant HepG-2 determined by dot-blotting assay under hypoxia condition (1%O2).


A - B Clonogenic survival of METTL3 - knockdown ( A ) and METTL3 - overexpression ( B ) in normal liver cell line WRL68 cells for 7 days in normoxia condition ( 21 % O2 ) and quantification of clusters in A - 1 and B - 1 , respectively . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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A-B Clonogenic survival of METTL3-knockdown (A) and METTL3-overexpression (B) in normal liver cell line WRL68 cells for 7 days in normoxia condition (21% O2) and quantification of clusters in A-1 and B-1, respectively. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


C - D The IC50 of METTL3 - knockdown SMMC - 7721 cells ( C ) and Bel - 7402 cells ( D ) after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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C-D The IC50 of METTL3-knockdown SMMC-7721 cells (C) and Bel-7402 cells (D) after treated with sorafenib for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


E Overexpression of wild type METTL3 or catalytic mutant METTL3 in sorafenib - resistant HepG - 2 cells .

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E Overexpression of wild type METTL3 or catalytic mutant METTL3 in sorafenib-resistant HepG-2 cells.


F The global RNA m6A level in sorafenib - resistant HepG - 2 cells with wild type METTL3 - overexpression or catalytic mutant METTL3 - overexpression by dot - blotting assay .

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F The global RNA m6A level in sorafenib-resistant HepG-2 cells with wild type METTL3-overexpression or catalytic mutant METTL3-overexpression by dot-blotting assay.


G - I Rescue of shRNA - resistant wild type METTL3 but not catalytic mutant METTL3 sensitized METTL3 - knockdown SMMC - 7721 cells ( G ) , Bel - 7402 cells ( H ) and sorafenib - resistant HepG - 2 cells ( I ) to sorafenib treatment . The IC50 of the cells was measured after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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G-I Rescue of shRNA-resistant wild type METTL3 but not catalytic mutant METTL3 sensitized METTL3-knockdown SMMC-7721 cells (G), Bel-7402 cells (H) and sorafenib-resistant HepG-2 cells (I) to sorafenib treatment. The IC50 of the cells was measured after treated with sorafenib for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


J Cell survival assay of sorafenib - resistant HepG - 2 cells with wild type METTL3 overexpression or catalytic mutant METTL3 overexpression after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Scale bar , 1mm .

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

J Cell survival assay of sorafenib-resistant HepG-2 cells with wild type METTL3 overexpression or catalytic mutant METTL3 overexpression after treated with sorafenib for 24h under hypoxia condition (1%O2). Scale bar, 1mm.


K Capillary - like structures in HUVECs treated with the tumor - conditioned medium ( TCM ) from control HCCs and METTL3 - knockdown HCCs cultured under hypoxic conditions for 48 h . Quantification of the number of tubes with image J in K - 1 . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

K Capillary-like structures in HUVECs treated with the tumor-conditioned medium (TCM) from control HCCs and METTL3-knockdown HCCs cultured under hypoxic conditions for 48 h. Quantification of the number of tubes with image J in K-1. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


L - M The relative mRNA expression levels of angiogenesis genes were detected in SMMC - 7721 cells ( L ) and Bel - 7402 cells ( M ) with METTL3 - knockdown by RT - PCR assay . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

O O O O O O 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

L-M The relative mRNA expression levels of angiogenesis genes were detected in SMMC-7721 cells (L) and Bel-7402 cells (M) with METTL3-knockdown by RT-PCR assay. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


N The protein levels of VEGF - A in METTL3 - knockdown HCCs . O The protein levels of PDGF - B in METTL3 - knockdown HCCs .

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

N The protein levels of VEGF-A in METTL3-knockdown HCCs. O The protein levels of PDGF-B in METTL3-knockdown HCCs.


A Electron micrographs of METTL3 - knockdown SMMC - 7721 cells under hypoxia for 48 h .

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

A Electron micrographs of METTL3-knockdown SMMC-7721 cells under hypoxia for 48 h.


Protein levels of LC3 I / II in METTL3 - knockdown SMMC - 7721 ( B ) , Bel - 7402 ( C ) cells under hypoxia condition for 48h .

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

Protein levels of LC3 I/II in METTL3-knockdown SMMC-7721 (B), Bel-7402 (C) cells under hypoxia condition for 48h.


Protein levels of LC3 I / II in HepG - 2 ( D ) cells under hypoxia condition for 48h .

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

Protein levels of LC3 I/II in HepG-2 (D) cells under hypoxia condition for 48h.


E Protein level of LC3 I / II in HepG - 2 and sorafenib - resistant HepG - 2 cells under hypoxia condition for 48h .

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

E Protein level of LC3 I/II in HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia condition for 48h.


F - H Overexpression of shRNA - resistant wild type METTL3 but not catalytic mutant METTL3 rescue autophagy phenotype in METTL3 - knockdown SMMC - 7721 cells ( F ) , Bel - 7402 cells ( G ) and sorafenib - resistant HepG2 cells ( H ) by representative immunostaining images of LC3 under hypoxia condition for 48h . Scale bar , 200μm . Quantification of the numbers of GFP - LC3 puncta / cell by Imaris in F - 1 , G - 1 and H - 1 , respectively . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Overexpression of shRNA-resistant wild type METTL3 but not catalytic mutant METTL3 rescue autophagy phenotype in METTL3-knockdown SMMC-7721 cells (F), Bel-7402 cells (G) and sorafenib-resistant HepG2 cells (H) by representative immunostaining images of LC3 under hypoxia condition for 48h. Scale bar, 200μm. Quantification of the numbers of GFP-LC3 puncta/cell by Imaris in F-1, G-1 and H-1, respectively. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


I - K Protein levels of LC3 I / II in METTL3 - knockdown SMMC - 7721 cells ( I ) , Bel - 7402 cells ( J ) and HepG - 2 cells ( K ) treated with or without 3 - MA under hypoxia condition for 24h .

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

I-K Protein levels of LC3 I/II in METTL3-knockdown SMMC-7721 cells (I), Bel-7402 cells (J) and HepG-2 cells (K) treated with or without 3-MA under hypoxia condition for 24h.


L - N The IC50 of sorafenib in SMMC - 7721 cells ( L ) , Bel - 7402 cells ( M ) and HepG - 2 cells ( N ) treated with or without 3 - MA for 24h under hypoxia condition ( 1 % O2 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

L-N The IC50 of sorafenib in SMMC-7721 cells (L), Bel-7402 cells (M) and HepG-2 cells (N) treated with or without 3-MA for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


A Gene Set Enrichment Analysis ( GSEA ) output images of cellular response to stress pathways displaying a correlation of differentially regulated genes in METTL3 - knockdown HepG2 , SMMC - 7721 and Bel - 7402 cells with the C5 . biological process symbol set . ( p < 0 . 001 ) .

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

A Gene Set Enrichment Analysis (GSEA) output images of cellular response to stress pathways displaying a correlation of differentially regulated genes in METTL3-knockdown HepG2, SMMC-7721 and Bel-7402 cells with the C5. biological process symbol set. (p < 0.001).


C Protein level of FOXO3 in METTL3 - knockdown HCCs under hypoxia for 48h .

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

C Protein level of FOXO3 in METTL3-knockdown HCCs under hypoxia for 48h.


D Half - life of FOXO3 mRNA in Bel - 7402 cells and correspondent METTL3 - knockdown Bel - 7402 cells treated with actinomycin D .

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

D Half-life of FOXO3 mRNA in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with actinomycin D.


E Half - life of FOXO3 protein in Bel - 7402 cells and correspondent METTL3 - knockdown Bel - 7402 cells treated with cycloheximide . Quantification of the protein optical density by Image J in E - 1 .

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

E Half-life of FOXO3 protein in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with cycloheximide. Quantification of the protein optical density by Image J in E-1.


F Analysis of FOXO3 mRNA in non - ribosome portion ( < 40S ) , 40S , 60S , 80S and polysome for the METTL3 - knockdown Bel - 7402 cells compare to control cells under hypoxia for 48h . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

F Analysis of FOXO3 mRNA in non-ribosome portion (<40S), 40S, 60S, 80S and polysome for the METTL3-knockdown Bel-7402 cells compare to control cells under hypoxia for 48h. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


G Knockdown of YTHDF1 and YTHDF2 in Bel - 7402 cells by lentiviral shRNAs .

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

G Knockdown of YTHDF1 and YTHDF2 in Bel-7402 cells by lentiviral shRNAs.


H Knockdown of YTHDF1 but not YTHDF2 abolished the increase of FOXO3 protein level in METTL3 - overexpressing Bel - 7402 cells . Quantification of the protein optical density by Image J in H - 1 and H - 2 . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-MEASURED_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 O O O O O O O O O O O O O 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 Knockdown of YTHDF1 but not YTHDF2 abolished the increase of FOXO3 protein level in METTL3-overexpressing Bel-7402 cells. Quantification of the protein optical density by Image J in H-1 and H-2. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


I RNA level of FOXO3 in YTHDF1 - knockdown Bel - 7402 cells by RT - PCR . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

I RNA level of FOXO3 in YTHDF1-knockdown Bel-7402 cells by RT-PCR. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


J The m6A - IP sequencing under hypoxia proved the m6A modification participated in regulation of FOXO3 . The YTHDF1 binding site locates at the 3 ' UTR of FOXO3 .

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

J The m6A-IP sequencing under hypoxia proved the m6A modification participated in regulation of FOXO3. The YTHDF1 binding site locates at the 3'UTR of FOXO3.


L Relative luciferase activity of the wild - type and mutant form of FOXO3 3 ' UTR reporter vectors in Bel - 7402 cells with various levels of METTL3 and YTHDF1 . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

L Relative luciferase activity of the wild-type and mutant form of FOXO3 3'UTR reporter vectors in Bel-7402 cells with various levels of METTL3 and YTHDF1. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


M Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the m6A MeRIP analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

M Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the m6A MeRIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


N Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the YTHDF1 - RIP analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

N Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the YTHDF1-RIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


O Correlation analysis of the RNA expression levels of METTL3 and FOXO3 in liver tumor cohort ( Kim - 90 ) by Pearson .

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

O Correlation analysis of the RNA expression levels of METTL3 and FOXO3 in liver tumor cohort (Kim-90) by Pearson.


P Correlation analysis of the RNA expression levels of YTHDF1 and FOXO3 in liver tumor cohort ( Kim - 90 ) by Pearson .

P Correlation analysis of the RNA expression levels of YTHDF1 and FOXO3 in liver tumor cohort (Kim-90) by Pearson.


A Validation of biomarker genes related to autophagy in FOXO3 - knockdown Bel - 7402 cells by RT - PCR . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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

A Validation of biomarker genes related to autophagy in FOXO3-knockdown Bel-7402 cells by RT-PCR. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


B Protein level of FOXO3 in naïve HepG - 2 and sorafenib - resistant HepG - 2 cells under hypoxia for 48h .

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

B Protein level of FOXO3 in naïve HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia for 48h.


C Protein levels of LC3 I / II in FOXO3 - knockdown SMMC - 7721 cells .

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

C Protein levels of LC3 I/II in FOXO3-knockdown SMMC-7721 cells.


D Overexpression of FOXO3 in sorafenib - resistant HepG - 2 cells by Western blot .

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

D Overexpression of FOXO3 in sorafenib-resistant HepG-2 cells by Western blot.


E Protein levels of LC3 I / II in METTL3 - knockdown & FOXO3 - overexpression SMMC - 7721 cells under hypoxia for 48 h .

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

E Protein levels of LC3 I/II in METTL3-knockdown & FOXO3-overexpression SMMC-7721 cells under hypoxia for 48 h.


F Electron micrographs of METTL3 - knockdown SMMC7721 cells and METTL3 - knockdown & FOXO3 - overexpression SMMC7721 cells under hypoxia for 48 h . G Representative Immunostaining images of LC3 in METTL3 - knockdown SMMC7721 cells and METTL3 - knockdown & FOXO3 - overexpression SMMC7721 cells under hypoxia for 48 h . Scale bar , 200μm . The quantification of the numbers of GFP - LC3 puncta / cell by Imaris in G - 1 . H Representative Immunostaining images of LC3 in naïve HepG - 2 cells and sorafenib - resistant HepG - 2 cells under hypoxia for 48 h . Scale bar , 200μm . The quantification of the numbers of GFP - LC3 puncta / cell by Imaris in H - 1 . I The IC50 of METTL3 - knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

O O O O B-CONTROLLED_VAR 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 O O O O B-MEASURED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Electron micrographs of METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. G Representative Immunostaining images of LC3 in METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in G-1. H Representative Immunostaining images of LC3 in naïve HepG-2 cells and sorafenib-resistant HepG-2 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in H-1. I The IC50 of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


J The IC50 of naïve HepG - 2 cells and sorafenib - resistant HepG - 2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD and are representative of 3 independent experiments .

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 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

J The IC50 of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.


K Cell survival assay of METTL3 - knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition .

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

K Cell survival assay of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.


L Cell survival assay of naïve HepG - 2 cells and sorafenib - resistant HepG - 2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition .

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 B-CONTROLLED_VAR O O O O O O

L Cell survival assay of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.


A Depletion of METTL3 in Hepa1 - 6 cells by CRISPR - Cas9 knockout enhanced sorafenib - resistance in vivo .

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

A Depletion of METTL3 in Hepa1-6 cells by CRISPR-Cas9 knockout enhanced sorafenib-resistance in vivo.


Growth curve ( B ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days ( n = 5 ) .

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Growth curve (B) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days (n=5).


tumor weight ( C ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days ( n = 5 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD .

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tumor weight (C) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days (n=5). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.


D Knockdown of METTL3 slightly increased liver tumor growth treated with sorafenib in orthotopic xenograft mouse model . Stable METTL3 - knockdown Bel - 7402 cells and control cells were injected into the liver of each NOD / SCID mouse . 3 weeks after injection , livers were separated for pathological analysis . Red circle indicates tumors in the livers .

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D Knockdown of METTL3 slightly increased liver tumor growth treated with sorafenib in orthotopic xenograft mouse model. Stable METTL3-knockdown Bel-7402 cells and control cells were injected into the liver of each NOD/SCID mouse. 3 weeks after injection, livers were separated for pathological analysis. Red circle indicates tumors in the livers.


E Liver tumors in orthotopic xenograft mouse model were confirmed by hematoxylin and eosin staining .

E Liver tumors in orthotopic xenograft mouse model were confirmed by hematoxylin and eosin staining.


F Overexpression of FOXO3 rescued sorafenib - resistance mediated by METTL3 - knockdown in xenograft liver tumors . Representative xenograft tumors at endpoint , containing 6 groups .

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F Overexpression of FOXO3 rescued sorafenib-resistance mediated by METTL3-knockdown in xenograft liver tumors. Representative xenograft tumors at endpoint, containing 6 groups.


Growth curve ( G ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days ( n = 5 ) .

Growth curve (G) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days (n=5).


tumor weight ( H ) of Hepa1 - 6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days ( n = 5 ) . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD .

tumor weight (H) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days (n=5). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.


I H & E and immunohistochemical images of Ki67 , LC3 - B , VEGF - A in Hepa1 - 6 cells derived xenografts in 6 treatment groups .

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I H&E and immunohistochemical images of Ki67, LC3-B, VEGF-A in Hepa1-6 cells derived xenografts in 6 treatment groups.


J - L Quantification of Ki67 ( K ) , LC3 - B ( K ) , and VEGF - A ( L ) expression in immunohistochemical images by Image Pro Plus ( IPP ) analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD .

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J-L Quantification of Ki67 (K), LC3-B (K), and VEGF-A (L) expression in immunohistochemical images by Image Pro Plus (IPP) analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.


In vivo analyses of tumor ( B ) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups .

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In vivo analyses of tumor (B) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups.


In vivo analyses of tumor growth ( C ) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups .

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In vivo analyses of tumor growth (C) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups.


In vivo analyses of tumor weight ( D ) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD .

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In vivo analyses of tumor weight (D) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.


E H & E and immunohistochemical images of Ki67 , LC3 - B , VEGF - A in randomly selected PDX # 1 and PDX # 2 tumors .

E H&E and immunohistochemical images of Ki67, LC3-B, VEGF-A in randomly selected PDX#1 and PDX#2 tumors.


Quantification of Ki67 ( F ) expression in immunohistochemical images from PDX tumors by Image Pro Plus ( IPP ) analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD .

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Quantification of Ki67 (F) expression in immunohistochemical images from PDX tumors by Image Pro Plus (IPP) analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.


Quantification of LC3 - B ( G ) , and VEGF - A ( H ) expression in immunohistochemical images from PDX tumors by Image Pro Plus ( IPP ) analysis . Data information : In all relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - tailed t - test . Data are presented as mean ± SD .

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Quantification of LC3-B (G), and VEGF-A (H) expression in immunohistochemical images from PDX tumors by Image Pro Plus (IPP) analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.


I Knockdown of METTL3 by siRNA in PDX # 1 and PDX # 2 tissues were confirmed by Western blot .

I Knockdown of METTL3 by siRNA in PDX#1 and PDX#2 tissues were confirmed by Western blot.


( F ) ChIP - seq tracks Brd4 on select genes ( Foxp3 , Nr4a2 , Med1 , Taf10 , Ar , and Fbxw7 ) in Th2 cells , treated with or without JQ1 .

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(F) ChIP-seq tracks Brd4 on select genes (Foxp3, Nr4a2, Med1, Taf10, Ar, and Fbxw7) in Th2 cells, treated with or without JQ1.


( B ) qPCR analysis of Il4 , Il5 , Il13 , Gata3 , Foxp3 and Fbxw7 in mouse Th2 cells infected with sh - Ctrl , sh - Foxp3 , and sh - Fbxw7 lenti - virus .

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(B) qPCR analysis of Il4, Il5, Il13, Gata3, Foxp3 and Fbxw7 in mouse Th2 cells infected with sh-Ctrl, sh-Foxp3, and sh-Fbxw7 lenti-virus.


( C ) Immunoprecipitation of Flag - tagged Fbxw7 with anti - Flag affinity gel , followed by Western blotting of Myc to detect Myc - Gata3 in HEK293T cells transfected with Myc - Gata3 and / or Flag - Fbxw7 plasmids .

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(C) Immunoprecipitation of Flag-tagged Fbxw7 with anti-Flag affinity gel, followed by Western blotting of Myc to detect Myc-Gata3 in HEK293T cells transfected with Myc-Gata3 and/or Flag-Fbxw7 plasmids.


( F ) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 ( 500nM ) , and then treated with cycloheximide ( CHX ; 25μg / ml ) for 0h , 0 . 5h , 1h and 2h before harvest to stop protein synthesis .

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(F) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 (500nM), and then treated with cycloheximide (CHX; 25μg/ml) for 0h, 0.5h, 1h and 2h before harvest to stop protein synthesis.


( C ) ChIP - qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 ( 250nM ) .

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(C) ChIP-qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 (250nM).


( D ) Immunoprecipitation of Brd4 in mouse Th2 cells , followed by Western blotting of Ezh2 , Suz12 , EED and G9a .

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(D) Immunoprecipitation of Brd4 in mouse Th2 cells, followed by Western blotting of Ezh2, Suz12, EED and G9a.


( F ) ChIP - qPCR analysis of Brd4 , Ezh2 , Suz12 and EED binding on the gene loci of Il4 , Il5 , Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 ( 250nM ) .

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(F) ChIP-qPCR analysis of Brd4, Ezh2, Suz12 and EED binding on the gene loci of Il4, Il5, Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 (250nM).


( B ) HEK293T cells are transfected with Flag - Brd4 and HA - EED , HA - Ezh2 , and Suz12 plasmids . Lysates are immunoprecipitated with Flag - tagged Brd4 , treated in - vitro with JQ1 ( 5μM ) , MS402 ( 5μM ) , and ABBV - 744 ( 5μM ) , followed by Western blotting of HA .

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(B) HEK293T cells are transfected with Flag-Brd4 and HA-EED, HA-Ezh2, and Suz12 plasmids. Lysates are immunoprecipitated with Flag-tagged Brd4, treated in-vitro with JQ1 (5μM), MS402 (5μM), and ABBV-744 (5μM), followed by Western blotting of HA.


( F ) HEK293T cells are transfected with Flag - Brd4 , HA - EED and HA - EED ( K19R ) plasmids . Lysates are immunoprecipitated with Flag followed by Western blotting of HA , and vice versa .

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(F) HEK293T cells are transfected with Flag-Brd4, HA-EED and HA-EED(K19R) plasmids. Lysates are immunoprecipitated with Flag followed by Western blotting of HA, and vice versa.


( B ) qPCR analysis of Il4 , Il5 , Gata3 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM .

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(B) qPCR analysis of Il4, Il5, Gata3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM.


( D ) Western blotting of Gata3 of mouse Th2 cells treated with ABBV - 744 ( 500nM ) and MG132 ( 20μM ) .

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(D) Western blotting of Gata3 of mouse Th2 cells treated with ABBV-744 (500nM) and MG132 (20μM).


( E ) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM . ( F ) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM . (

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(E) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (F) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (


( A , B ) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles ( Dataset EV1 ) . A , all mitochondrial proteins detected . Grey area indicates proteins that changed less than 2 - fold in both directions . Subunits of selected mitochondrial machineries are highligted as follows : magenta , pyruate dehydrogenase complex ( PDH ) ; blue , mitochondial carriers ( Carrier ) ; brown , translocase of the outer membrane ( TOM ) ; yellow , translocase of the inner membrane ( TIM ) ; green , mitochondrial contact site and cristae organizing system ( MICOS ) ; yellow - green , mitochondrial fusion - fission system ( Dynamics ) ; red , porin or voltage dependent anion channel ( VDAC ) ; B , subunits of OXPHOS complexes ( Complex I - V ) and mitoribosomes ( Ribosome ) . Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex . For the full gene name , add NDU ( complex I ) , SDH ( complex II ) , UQC ( R10 ) , COX ( complex IV ) , ATP ( 5L , 5J2 ) , MRP ( Ribosome ) in front of the abbreviated gene names given in the figure .

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(A, B) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles (Dataset EV1). A, all mitochondrial proteins detected. Grey area indicates proteins that changed less than 2-fold in both directions. Subunits of selected mitochondrial machineries are highligted as follows: magenta, pyruate dehydrogenase complex (PDH); blue, mitochondial carriers (Carrier); brown, translocase of the outer membrane (TOM); yellow, translocase of the inner membrane (TIM); green, mitochondrial contact site and cristae organizing system (MICOS); yellow-green, mitochondrial fusion-fission system (Dynamics); red, porin or voltage dependent anion channel (VDAC); B, subunits of OXPHOS complexes (Complex I-V) and mitoribosomes (Ribosome). Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex. For the full gene name, add NDU (complex I), SDH (complex II), UQC (R10), COX (complex IV), ATP (5L, 5J2), MRP (Ribosome) in front of the abbreviated gene names given in the figure.


( A , B ) Selected proteins involved in DNA ( A ) and RNA ( B ) maintenance of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , for POLG , UNG and TWNK , only abundances > 1 MDa were quantified . Plots and gene names are in corresponding colors . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test for individual proteins and two - way ANOVA for changes of multiple proteins in a complex ) .

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(A, B) Selected proteins involved in DNA (A) and RNA (B) maintenance of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, for POLG, UNG and TWNK, only abundances >1 MDa were quantified. Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test for individual proteins and two-way ANOVA for changes of multiple proteins in a complex).


( A - F ) OXPHOS complexes ( A - C ) and selected enzymes involved in the energy metabolism of control ( 143B , solid lines ) and ρ0 cells ( dashed lines ) . A , Complex ( C ) I , III and IV ; B , F1 , FO domain and inhibitor protein IF1 of complex V ; C , subunits SDHA , SDHB - D and assembly factor SDHAF2 of complex II ; D , oxoglutarate ( OGDH ; average of OGDHL , OGDH , DLST ) and pyruvate dehydrogenase complex ( PDH ; average of PDHA1 , PDHB , PDHX , DLAT ) ; E , subunits of GDP forming ( SUCLG2 ) and ADP forming ( SUCLA2 ) succinyl - CoA ligase ; F , urea cycle enzymes and glutaminase ( GLS ) . Plots and gene names are in corresponding colors .

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(A-F) OXPHOS complexes (A-C) and selected enzymes involved in the energy metabolism of control (143B, solid lines) and ρ0 cells (dashed lines). A, Complex (C) I, III and IV; B, F1, FO domain and inhibitor protein IF1 of complex V; C, subunits SDHA, SDHB-D and assembly factor SDHAF2 of complex II; D, oxoglutarate (OGDH; average of OGDHL, OGDH, DLST) and pyruvate dehydrogenase complex (PDH; average of PDHA1, PDHB, PDHX, DLAT); E, subunits of GDP forming (SUCLG2) and ADP forming (SUCLA2) succinyl-CoA ligase; F, urea cycle enzymes and glutaminase (GLS). Plots and gene names are in corresponding colors.


( A - D ) Selected metabolite carriers of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , isoforms of the nucleotide carrier ( ANT1 - 3 ) and phosphate carrier ( PiC ) ; B , pyruvate carrier ; C , dicarboxylate carriers ; D , mitoferrins ( MFRN1 - 2 ) and glycine transporter ( GlyC ) . Plots and gene names are in corresponding colors . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) .

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(A-D) Selected metabolite carriers of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, isoforms of the nucleotide carrier (ANT1-3) and phosphate carrier (PiC); B, pyruvate carrier; C, dicarboxylate carriers; D, mitoferrins (MFRN1-2) and glycine transporter (GlyC). Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01 (Student's t-test).


( A - D ) Components of the mitochondrial protein import machinery and selected proteins related to this process of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored and dark - grey bars ) . A and B , components of the translocase of the outer membrane ( TOM ) machinery ( TOM avg . : average of TOMM40 , TOMM22 , TOMM5 , TOMM6 , TOMM7 ) ; C , components of the trans locase of the inner membrane ( TIM ) machinery ( TIM22 avg . : TIMM22 , AGK , TIMM10B ; TIM23 avg . : TIMM23 , TIMM17A , TIMM17B ) ; D , components of the TIM23 - associated TIMM21 assembly machinery ( left panel ) and its association with prohibitin ( PHB ) and components of the mitochondrial quality control system ( right panel ) . Plots and gene names are in corresponding colors , except for TIMM44 and TIMM50 ( no migration profiles shown ) . In ( A - C ) data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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(A-D) Components of the mitochondrial protein import machinery and selected proteins related to this process of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored and dark-grey bars). A and B, components of the translocase of the outer membrane (TOM) machinery (TOM avg.: average of TOMM40, TOMM22, TOMM5, TOMM6, TOMM7); C, components of the translocase of the inner membrane (TIM) machinery (TIM22 avg.: TIMM22, AGK, TIMM10B; TIM23 avg.: TIMM23, TIMM17A, TIMM17B); D, components of the TIM23-associated TIMM21 assembly machinery (left panel) and its association with prohibitin (PHB) and components of the mitochondrial quality control system (right panel). Plots and gene names are in corresponding colors, except for TIMM44 and TIMM50 (no migration profiles shown). In (A-C) data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test).


Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , mitochondrial contact site and cristae organizing system ( MICOS : MIC60 , MIC19 , MIC25 ) and SAM complex ( SAM : SAMM50 , MTX2 , MTX3 ) together forming the MIB

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

Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, mitochondrial contact site and cristae organizing system (MICOS: MIC60, MIC19, MIC25) and SAM complex (SAM: SAMM50, MTX2, MTX3) together forming the MIB complex


Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . B , proteins involved in controlling mitochondrial morphology ( orange ) and dynamics ( teal ) ;

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Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). B, proteins involved in controlling mitochondrial morphology (orange) and dynamics (teal);


Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . C , DNM1L / DRP1 and interacting proteins . For DNM1L , MTFR1 and ARL2BP , quantification of the entire migra tion profiles ( Overall ) and the range above 1 , 400 kDa only ( > 1 , 400 kDa ) are shown . Plots and gene names are in corresponding colors , except for ARL2 ( dark - grey ) not shown in the plot . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - test ) .

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Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). C, DNM1L/DRP1 and interacting proteins. For DNM1L, MTFR1 and ARL2BP, quantification of the entire migration profiles (Overall) and the range above 1,400 kDa only (>1,400 kDa) are shown. Plots and gene names are in corresponding colors, except for ARL2 (dark-grey) not shown in the plot. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).


B Vero E6 cells were inoculated with SARS - CoV - 2 , fixed with paraformaldehyde after two days , stained with 30 nM of the indicated Alexa Fluor 488 ( AF488 ) - labeled anti - RBD VHH antibodies , and imaged by confocal laser scanning microscopy ( CLSM ) . C Immunofluorescence ( IF ) staining as in B , but with the indicated AF568 - labeled anti - S1ΔRBD VHH antibodies .

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B Vero E6 cells were inoculated with SARS-CoV-2, fixed with paraformaldehyde after two days, stained with 30 nM of the indicated Alexa Fluor 488 (AF488)-labeled anti-RBD VHH antibodies, and imaged by confocal laser scanning microscopy (CLSM). C Immunofluorescence (IF) staining as in B, but with the indicated AF568-labeled anti-S1ΔRBD VHH antibodies.


BLI sensorgrams of intermediate VHHs A SARS - CoV - 2 RBD - loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s , followed by dipping in assay buffer for 600 s . The response curves ( black ) were fitted using either a 1 : 1 ( for VHH - 72 ) or 2 : 1 ( for Re6B06 and Re9F06 ) binding models to obtain KDs . Fitted curves are shown in red . For Re6B06 and Re9F06 , some secondary binding with weaker affinity at high VHH concentrations was observed ( denoted as KD2 ) .

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BLI sensorgrams of intermediate VHHs A SARS-CoV-2 RBD-loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s, followed by dipping in assay buffer for 600 s. The response curves (black) were fitted using either a 1:1 (for VHH-72) or 2:1 (for Re6B06 and Re9F06) binding models to obtain KDs. Fitted curves are shown in red. For Re6B06 and Re9F06, some secondary binding with weaker affinity at high VHH concentrations was observed (denoted as KD2).


A Vero E6 cells were infected with SARS - CoV - 2 , pre - incubated with indicated concentrations of the neutralizing VHH - 72 Cells were fixed two days after inoculation , stained with sets of anti - RBD ( green ) and anti - S1ΔRBD ( red ) nanobodies and analyzed by CLSM . We used a cocktail of such fluorophore - labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody .

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A Vero E6 cells were infected with SARS-CoV-2, pre-incubated with indicated concentrations of the neutralizing VHH-72 Cells were fixed two days after inoculation, stained with sets of anti-RBD (green) and anti-S1ΔRBD (red) nanobodies and analyzed by CLSM. We used a cocktail of such fluorophore-labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody.


B Vero E6 cells were infected as in A . Viral RNA in the culture supernatants was quantified by reverse transcription ( RT ) - qPCR . RNA signals are normalized to the respective minus - VHH controls ( 100 % ) . Note the log10 scale of the y - axis . " Inoculum " ( red dotted line ) marks the RNA level detected at the time of infection .

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B Vero E6 cells were infected as in A. Viral RNA in the culture supernatants was quantified by reverse transcription (RT)-qPCR. RNA signals are normalized to the respective minus-VHH controls (100%). Note the log10 scale of the y-axis. "Inoculum" (red dotted line) marks the RNA level detected at the time of infection.


Neutralization of SARS - CoV - 2 by the indicated VHH antibodies at nanomolar ( A and B ) or picomolar ( C and D ) concentrations . The neutralization experiment was performed ; n " indicates the number of independent biological

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Neutralization of SARS-CoV-2 by the indicated VHH antibodies at nanomolar (A and B) or picomolar (C and D) concentrations. The neutralization experiment was performed ;n" indicates the number of independent biological replicates


A HeLa cells were transiently transfected to express the SARS - CoV - 2 Spike protein . Following fixation , cells were stained for 1 hour with fluorophore - labeled Re10B10 ( 5 nM , green ) and Re7E02 ( 15 nM , red ) in the presence of the indicated unlabeled VHH competitors . Competitor ( 150 nM ) was added 20 min prior to the labeled nanobodies . The weakly binding competitors Re5A08 , Re9F06 , Re6B06 , and , Re6D06 were added as trimers ( see below ) . Cells were imaged by CLSM . For each competitor , the sequence class and the binding site on the RBD ( epitope 1 or 2 ) are indicated .

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A HeLa cells were transiently transfected to express the SARS-CoV-2 Spike protein. Following fixation, cells were stained for 1 hour with fluorophore-labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and, Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated.


F Neutralization of SARS - CoV - 2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations . The neutralization experiment was

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F Neutralization of SARS-CoV-2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations. The neutralization experiment was performed


B Re6B06 monomer and trimer variants ( 20 nM ) were analyzed by BLI for RBD - binding , with 900 s association and 1500 s dissociation steps . Note the avidity effect of Re6B06 trimerization .

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B Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD-binding, with 900 s association and 1500 s dissociation steps. Note the avidity effect of Re6B06 trimerization.


C Neutralization of SARS - CoV - 2 by the indicated VHH antibodies . The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized . Re6B06 , for example , is 30 000 - fold more potent when symmetry - matched with the virus Spike .

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C Neutralization of SARS-CoV-2 by the indicated VHH antibodies. The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized. Re6B06, for example, is 30 000-fold more potent when symmetry-matched with the virus Spike.


C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI . Immobilized RBD was incubated with 100 nM of nanobodies for 450 s , followed by assay buffer for 900 s .

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C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI. Immobilized RBD was incubated with 100 nM of nanobodies for 450 s, followed by assay buffer for 900 s.


A and B Neutralization of SARS - CoV - 2 B . 1 . 351 / Beta by the indicated VHH antibody constructs . The neutralization experiment was performed as described in Fig 3A , with the difference being that a mutant - optimized anti - RBD / S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown , corresponding to the standard ( upper row ) or a five - fold higher virus inoculum ( lower row ) that was used for even more stringent neutralization conditions .

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A and B Neutralization of SARS-CoV-2 B.1.351/Beta by the indicated VHH antibody constructs. The neutralization experiment was performed as described in Fig 3A, with the difference being that a mutant-optimized anti-RBD/S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown, corresponding to the standard (upper row) or a five-fold higher virus inoculum (lower row) that was used for even more stringent neutralization conditions.


C ) Time course analysis of triubiquitin ( 2 µM ) hydrolysis using 250 nM SARS2 PLpro , resolved on a Coomassie stained SDS - PAGE gel . Linkage specific cleavage of Lys48 - linked triubiquitin to di - and monoubiquitin resembles SARS PLpro activity ( Békés et al , 2015 ; 2016 ) . See Source Data for uncropped gels , and Appendix Fig S1B - D for gel - based cleavage quantification .

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C) Time course analysis of triubiquitin (2 µM) hydrolysis using 250 nM SARS2 PLpro, resolved on a Coomassie stained SDS-PAGE gel. Linkage specific cleavage of Lys48-linked triubiquitin to di- and monoubiquitin resembles SARS PLpro activity (Békés et al, 2015; 2016). See Source Data for uncropped gels, and Appendix Fig S1B-D for gel-based cleavage quantification.


B ) Fluorescence polarisation assays against ubiquitin - TAMRA and ISG15 - TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n = 2 for each mutant , and compared to wild - type PLpro as shown in Appendix Fig S1A . Catalytic efficiencies were calculated as described in Methods .

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B) Fluorescence polarisation assays against ubiquitin-TAMRA and ISG15-TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n=2 for each mutant, and compared to wild-type PLpro as shown in Appendix Fig S1A. Catalytic efficiencies were calculated as described in Methods.


C ) Gel based analysis of PLpro variant activity against Lys48 - triubiquitin and proISG15 . Experiments were performed in duplicate , see Fig EV3 and Source Data for uncropped gels .

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C) Gel based analysis of PLpro variant activity against Lys48-triubiquitin and proISG15. Experiments were performed in duplicate, see Fig EV3 and Source Data for uncropped gels.


C ) Calculated enzymatic efficiencies for K48 - diUb - TAMRA cleavage , followed by fluorescence polarisation with PLpro wild - type ( reproduced from Appendix Fig S1A ) , and PLpro F69S . ( Left ) Using the same concentration range of SARS2 PLpro , the F69S mutant activity could not be fitted ; ( middle ) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin - TAMRA ( 45 vs . 86 M - 1s - 1 , compare Fig 1D , Appendix Fig S1A ) . ( Right ) A ~ 3 - fold lower efficiency for PLpro F69S cleaving ISG15 - TAMRA yields values similar to cleavage of ISG15CTD - TAMRA ( Appendix Fig S1A ) , suggesting that the S2 site contributes the difference in binding for the N - terminal Ubl - fold . Experiments for F69S were performed in technical triplicate and biological duplicate ; wild - type data is reproduced from experiments shown in Fig 1D , Appendix Fig S1A .

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C) Calculated enzymatic efficiencies for K48-diUb-TAMRA cleavage, followed by fluorescence polarisation with PLpro wild-type (reproduced from Appendix Fig S1A), and PLpro F69S. (Left) Using the same concentration range of SARS2 PLpro, the F69S mutant activity could not be fitted; (middle) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin-TAMRA (45 vs. 86 M-1s-1, compare Fig 1D, Appendix Fig S1A). (Right) A ~3-fold lower efficiency for PLpro F69S cleaving ISG15-TAMRA yields values similar to cleavage of ISG15CTD-TAMRA (Appendix Fig S1A), suggesting that the S2 site contributes the difference in binding for the N-terminal Ubl-fold. Experiments for F69S were performed in technical triplicate and biological duplicate; wild-type data is reproduced from experiments shown in Fig 1D, Appendix Fig S1A.


D - F ) Gel based analysis showing hydrolysis time course of triubiquitin ( D ) , proISG15CTD ( E ) and proISG15 ( F ) using wild - type PLpro ( left , wild - type gels reproduced from Appendix Fig S1B - D to enable direct comparison ) or PLpro F69S ( right ) . PLpro F69S has a strong effect on triubiquitin hydrolysis ( D ) , no marked effect on hydrolysis of proISG15CTD ( E ) and reduces proISG15 cleavage to the same levels as proISG15CTD ( compare E , F ) , Experiments were performed in duplicate , see Source Data for uncropped gels .

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D-F) Gel based analysis showing hydrolysis time course of triubiquitin (D), proISG15CTD (E) and proISG15 (F) using wild-type PLpro (left, wild-type gels reproduced from Appendix Fig S1B-D to enable direct comparison) or PLpro F69S (right). PLpro F69S has a strong effect on triubiquitin hydrolysis (D), no marked effect on hydrolysis of proISG15CTD (E) and reduces proISG15 cleavage to the same levels as proISG15CTD (compare E, F), Experiments were performed in duplicate, see Source Data for uncropped gels.


B ) Hit compounds and compound rac5c ( see Fig 5 ) , were further assessed in 10 - point IC50 titrations using the Ub - Rhodamine assay , using a starting concentration of 100 µM serially diluted in 1 : 3 steps . Degree of inhibition is shown as a titration heat - map from dark ( full inhibition ) to light blue ( low / no inhibition ) . The catalytic domain of human USP21 ( Ye et al , 2011 ) was used as a counterscreen . Each PLpro hit compound showed either no activity in the titration analysis , or an identical inhibition profile against PLpro and USP21 , suggesting assay interference . Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM . IC50 assays were performed in technical triplicate in two independent experiments .

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B) Hit compounds and compound rac5c (see Fig 5), were further assessed in 10-point IC50 titrations using the Ub-Rhodamine assay, using a starting concentration of 100 µM serially diluted in 1:3 steps. Degree of inhibition is shown as a titration heat-map from dark (full inhibition) to light blue (low/no inhibition). The catalytic domain of human USP21 (Ye et al, 2011) was used as a counterscreen. Each PLpro hit compound showed either no activity in the titration analysis, or an identical inhibition profile against PLpro and USP21, suggesting assay interference. Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM. IC50 assays were performed in technical triplicate in two independent experiments.


C ) In vitro inhibition ( IC50 ) for rac5c inhibiting SARS2 PLpro . Experiments were performed using the HTS assay ( Fig 4 ) , in technical triplicate in three independent experiments . A geometric mean was used to determine IC50 .

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C) In vitro inhibition (IC50) for rac5c inhibiting SARS2 PLpro. Experiments were performed using the HTS assay (Fig 4), in technical triplicate in three independent experiments. A geometric mean was used to determine IC50.


D ) Full - length nsp3 was expressed from a C - terminally GFP - tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h . GFP is released from the C - terminus , presumably by nsp3 protease activity . Nsp3 can be detected by a SARS / SARS2 PLpro antibody ( see Fig EV5E for antibody validation ) . Lysates were blotted for Lys48 - linked polyubiquitin with a linkage specific antibody ( K48 ) . Experiments were performed in duplicate with similar results . Also see Fig EV5F , G and Source Data for uncropped blots .

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D) Full-length nsp3 was expressed from a C-terminally GFP-tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h. GFP is released from the C-terminus, presumably by nsp3 protease activity. Nsp3 can be detected by a SARS/SARS2 PLpro antibody (see Fig EV5E for antibody validation). Lysates were blotted for Lys48-linked polyubiquitin with a linkage specific antibody (K48). Experiments were performed in duplicate with similar results. Also see Fig EV5F, G and Source Data for uncropped blots.


B ) Reduction in SARS - CoV - 2 induced cytopathic effect with rac5c , Remdesivir ( RDV ) and hydroxychloroquine ( HCQ ) treatment . DMSO 0 . 3 % ( v / v ) was required to keep 33 µM rac5c in solution ( see Fig EV6A , B ) . Mean ( black line ) is provided for 18 samples in each group , representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c . HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats . P values were calculated using a one - way ANOVA , with regular Dunnet ' s post - hoc test for multiple comparisons between treatment arms and infected / vehicle treated control using a single pooled variance .

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B) Reduction in SARS-CoV-2 induced cytopathic effect with rac5c, Remdesivir (RDV) and hydroxychloroquine (HCQ) treatment. DMSO 0.3% (v/v) was required to keep 33 µM rac5c in solution (see Fig EV6A, B). Mean (black line) is provided for 18 samples in each group, representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c. HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats. P values were calculated using a one-way ANOVA, with regular Dunnet's post-hoc test for multiple comparisons between treatment arms and infected/vehicle treated control using a single pooled variance.


C ) TCID50 data , mean and SD , for one representative experiment from B with 6 technical replicates .

C) TCID50 data, mean and SD, for one representative experiment from B with 6 technical replicates.


Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right . Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope - tagged proteins in the anti - Mta1 , Mta2 or Mta3 panels ; and a grey triangle shows the location of IgG bands in the anti - Mbd3 panel . 10 % of the input was loaded into " Input " lanes . Molecular weights in kDa are shown at left .

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

Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right. Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope-tagged proteins in the anti-Mta1, Mta2 or Mta3 panels; and a grey triangle shows the location of IgG bands in the anti-Mbd3 panel. 10% of the input was loaded into "Input" lanes. Molecular weights in kDa are shown at left.


Proteins co - purifying with Mta1 , Mta2 or Mta3 in IP - mass spectrometry experiments in wild type cells ( top ) or Mbd3 - null cells ( bottom ) . Proteins showing significant enrichment with the bait protein are located outside the dotted lines . For all panels the protein being immunoprecipitated is indicated in red , the other MTA proteins in purple , and other NuRD components in blue . Each IP / Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation .

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

Proteins co-purifying with Mta1, Mta2 or Mta3 in IP-mass spectrometry experiments in wild type cells (top) or Mbd3-null cells (bottom). Proteins showing significant enrichment with the bait protein are located outside the dotted lines. For all panels the protein being immunoprecipitated is indicated in red, the other MTA proteins in purple, and other NuRD components in blue. Each IP/Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation.