( B ) Expression of GFP , lncRNA - c1 , GFP - lncRNA - c1 , lncRNA - c2 GFP - lncRNA - c2 ( x - axis ) in 8 day 4 - OHT treated , miRNA depleted ( KO ) relative to ethanol treated ( WT ) mESC ( y - axis ) 24h hours post - transfection . Four independent biological replicates were treated , transfected analyzed by RT - qPCR . Statistical significance represented on figure on comparison of KO / WT fold change in expression of GFP , with GFP - lncRNA - c1 with GFP - lncRNA - c2 , ( paired two - t - p - value = 0 . 034 0 . 032 respectively ) on comparison of KO / WT fold change in expression of GFP - lncRNAc1 with lncRNA - c1 GFP - lncRNA - c2 with lncRNA - c2 ( paired two - t - p - value = 0 . 012 0 . 018 respectively ) . Data is represented as mean ± SD Each point corresponds to results of one independent biological replicate . Data information : For RTqPCR analyses , transcript expression was first normalized by amount of Act - β PolII next by total amount of transfected vectors per estimated on of relative Neomycin expression . Each point corresponds to results of one independent biological replicate . Statistics : NS - p - value > 0 . 05 , * - p - value < 0 . 05 , * * - p - value < 0 . 01 * * * - p - value < 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of GFP, lncRNA-c1, GFP-lncRNA-c1, lncRNA-c2 and GFP-lncRNA-c2 (x-axis) in 8 day 4-OHT treated, miRNA depleted cells (KO) relative to ethanol treated (WT) mESC (y-axis) 24h hours post-transfection. Four independent biological replicates were treated, transfected and analyzed by RT-qPCR. Statistical significance represented on the figure based on comparison of KO/WT fold change in expression of GFP, with GFP-lncRNA-c1 and with GFP-lncRNA-c2, (paired two-tailed t-test p-value= 0.034 and 0.032 respectively) and based on comparison of KO/WT fold change in expression of GFP-lncRNAc1 with lncRNA-c1 and GFP-lncRNA-c2 with lncRNA-c2 (paired two-tailed t-test p-value= 0.012 and 0.018 respectively). Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.


( C ) Expression of GFP - lncRNA - c1 ΔMRE relative to GFP - lncRNA - c1 ( y - axis ) in ethanol treated ( WT , circles ) or 4 - OHT treated , miRNA - depleted ( KO , triangles , x - axis ) . Four independent biological replicates were treated , transfected analyzed by RTqPCR . Data is represented as mean ± SD Each point corresponds to results of one independent biological replicate . Paired two - t - p - value = 0 . 0071 Data information : For RTqPCR analyses , transcript expression was first normalized by amount of Act - β PolII next by total amount of transfected vectors per estimated on of relative Neomycin expression . Each point corresponds to results of one independent biological replicate . Statistics : NS - p - value > 0 . 05 , * - p - value < 0 . 05 , * * - p - value < 0 . 01 * * * - p - value < 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Expression of GFP-lncRNA-c1 ΔMRE relative to GFP-lncRNA-c1 (y-axis) in ethanol treated (WT, circles) or 4-OHT treated, miRNA-depleted cells (KO, triangles, x-axis). Four independent biological replicates were treated, transfected and analyzed by RTqPCR. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0071 Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.


( F ) Immunoblot of GFP ( GFP ) in extracts from mESCs transfected with mock , BoxB ( - 30 ) - GFP GFP expressing vectors . ACTIN - β ( ACT - β ) was used as an internal control . One representative blot is depicted ( G ) Fold - change ( FC ) in normalized expression of GFP - lncRNA - c1 , GFP - lncRNA - c2 ( noBoxB , circles ) BoxB ( - 30 ) - GFP - lncRNA - c1 , BoxB ( - 30 ) - GFP - lncRNA - c2 ( BoxB ( - 30 ) , triangles ) ( x - axis ) 4 - OHT treated , miRNA depleted mESCs ( KO ) relative to ethanol treated mESCs ( WT ) ( y - axis ) . Four independent biological replicates were analyzed . Data is represented as mean ± SD Each point corresponds to results of one independent biological replicate . Paired two - t - p - value = 0 . 0494 for GFP - lncRNA - c1 p - value = 0 . 0355 for GPF - lncRNA - c2Data information : Uncropped blots used for assembly of panels F are provided in Figure 4 Source Data .

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

(F) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(-30)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted (G) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(-30)-GFP-lncRNA-c1, BoxB (-30)-GFP-lncRNA-c2 (BoxB(-30), triangles) (x-axis) 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates were analyzed. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0494 for GFP-lncRNA-c1 and p-value=0.0355 for GPF-lncRNA-c2Data information: Uncropped blots used for assembly of panels F are provided in Figure 4 Source Data.


( H ) Immunoblot of GFP ( GFP ) in extracts from mESCs transfected with mock , BoxB ( + 339 ) - GFP GFP expressing vectors . ACTIN - β ( ACT - β ) was used as an internal control . One representative blot is depicted . ( I ) Fold - change ( FC ) in normalized expression of GFP - lncRNA - c1 , GFP - lncRNA - c2 ( noBoxB , circles ) BoxB ( + 339 ) - GFP - lncRNA - c1 , BoxB ( + 339 ) - GFP - lncRNA - c2 ( BoxB ( + 339 ) , triangles ) ( x - axis ) in 8 day 4 - OHT treated , miRNA depleted mESCs ( KO ) relative to ethanol treated mESCs ( WT ) ( y - axis ) . Four independent biological replicates analyzed . Data information : Uncropped blots used for assembly of panels H are provided in Figure 4 Source Data .

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

(H) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(+339)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted. (I) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(+339)-GFP-lncRNA-c1, BoxB (+339)-GFP-lncRNA-c2 (BoxB(+339), triangles) (x-axis) in 8 day 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates analyzed.Data information: Uncropped blots used for assembly of panels H are provided in Figure 4 Source Data.


Antibody response in plasma saliva at different time points ( T0 , T1 , T2 ) from BNT162b2 vaccination in naïve ( plasma : n = 64 ; saliva : n = 66 ) SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects . Subjects received first vaccine dose at day 0 ( T0 ) , 2nd dose at day 21 ( T1 ) ( indicated with an arrow ) . T2 corresponds to 7 - 10 days after 2nd dose . Plasma saliva were tested for IgG to full - spike its receptor binding domain ( RBD ) . For plasma samples titers of antigen specific Ig are expressed in IU / ml . LoD is indicated by a dotted : LoD ( spike ) = 12 , LoD ( RBD ) = 13 . 8 . For saliva samples titers of antigen specific Ig were normalized by dividing values of SARS - CoV - 2 specific Ig by total IgG of each sample . normalization was applied only to values higher than LoD . adjusted values are expressed in AU . box plots show interquartile range , horizontal lines show median values , whiskers indicate minimum - to - maximum range .

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

Antibody response in plasma and saliva at different time points (T0, T1, T2) from BNT162b2 vaccination in naïve (plasma: n=64; saliva: n=66) and SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. Plasma and saliva were tested for IgG to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike)=12, LoD (RBD)=13.8. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgG concentration of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU. The box plots show the interquartile range, the horizontal lines show the median values, and the whiskers indicate the minimum-to-maximum range.


Antibody response in plasma saliva at different time points ( T0 , T1 , T2 ) from BNT162b2 vaccination in naïve ( n = 66 ) SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects . Subjects received first vaccine dose at day 0 ( T0 ) , 2nd dose at day 21 ( T1 ) ( indicated with an arrow ) . T2 corresponds to 7 - 10 days after 2nd dose . Plasma saliva were tested for IgA to full - spike its receptor binding domain ( RBD ) . For plasma samples titers of antigen specific Ig are expressed in IU / ml . LoD is indicated by a dotted : LoD ( spike ) = 54 . 22 , LoD ( RBD ) = 54 . 08 . For saliva samples titers of antigen specific Ig were normalized by dividing values of SARS - CoV - 2 specific Ig by total IgA of each sample . normalization was applied only to values higher than LoD .

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

Antibody response in plasma and saliva at different time points (T0, T1, T2) from BNT162b2 vaccination in naïve (n=66) and SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. Plasma and saliva were tested for IgA to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike)=54.22, LoD (RBD)=54.08. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA concentration of each sample. The normalization was applied only to values higher than LoD.


antibody response of vaccinated naïve ( plasma : n = 64 ; saliva n = 66 ) or SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects at 7 - 10 days after second dose ( T2 ) was compared to that obtained in COVID - 19 ( plasma n = 28 ; saliva n = 26 ) . As a control , SARS - CoV - 2 specific antibody response was analyzed in non - vaccinated subjects ( n = 19 , Control ) . Plasma saliva were tested for IgA to full - spike its receptor binding domain ( RBD ) . For plasma samples titers of antigen specific Ig are expressed in IU / ml . LoD is indicated by a dotted : LoD ( spike IgG ) = 12 , LoD ( RBD IgG ) = 13 . 8 , LoD ( spike IgA ) = 54 . 22 , LoD ( RBD IgA ) = 54 . 08 . For saliva samples titers of antigen specific Ig were normalized by dividing values of SARS - CoV - 2 specific Ig by total IgA or total IgG concentrations of each sample . normalization was applied only to values higher than LoD . adjusted values are expressed in AU .

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

The antibody response of vaccinated naïve (plasma: n=64; saliva n=66) or SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects at 7-10 days after the second dose (T2) was compared to that obtained in COVID-19 patients (plasma n=28; saliva n=26). As a control, SARS-CoV-2 specific antibody response was analyzed in non-vaccinated subjects (n=19, Control). Plasma and saliva were tested for IgA to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike IgG)=12, LoD (RBD IgG)=13.8, LoD (spike IgA)=54.22, LoD (RBD IgA)=54.08. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA or total IgG concentrations of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU.


A , IgA1 IgA2 subtypes specific for spike were measured in plasma saliva at different time point ( T0 , T1 , T2 ) of vaccinated naïve ( plasma : n = 64 ; saliva : n = 63 ( IgA1 ) , n = 55 ( IgA2 ) ) or SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects . Subjects received first vaccine dose at day 0 ( T0 ) , 2nd dose at day 21 ( T1 ) ( indicated with an arrow ) . T2 corresponds to 7 - 10 days after 2nd dose . For plasma samples titers of antigen specific IgA1 IgA2 are expressed in IU / ml AU , respectively ( see Methods ) . LoD is indicated by a dotted : LoD ( IgA1 ) = 74 . 64 ; LoD ( IgA2 ) = 0 . 009 . For saliva samples titers of antigen specific Ig were normalized by dividing values of SARS - CoV - 2 specific Ig by total IgA of each sample . normalization was applied only to values higher than LoD . adjusted values are expressed in AU .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva at different time point (T0,T1, T2) of vaccinated naïve (plasma: n=64; saliva: n=63 (IgA1), n=55 (IgA2)) or SARS-CoV-2-Exp (plasma: n=21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. For plasma samples the titers of antigen specific IgA1 and IgA2 are expressed in IU/ml and AU, respectively (see Methods). LoD is indicated by a dotted line: LoD (IgA1)=74.64; LoD (IgA2)=0.009. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA concentration of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 procedure , AlPcS2a signals colocalized with Lysotracker Red ( top panels ) lamp1 - RFP ( middle panels ) , but not with other organelle markers such as EGFP - LC3B ( bottom panels , marker for autophagic structures ) , in HeLa . Scale bars , 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 O O O O O 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) 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 AlPcS2a immediately following activation . A HeLa labelled with Lysotracker Red AlPcS2a was 635 - nm illuminated within square region ( left panels ) . Lysotracker within illuminated region was immediately lost after illumination ( middle panels ) , indicating successful triggering of LMP . Right panels : magnified view of Lysotracker within 635 - nm illuminated region pre - post activation . ( b ) 635 nm illumination did not lead to complete lysosome rupture . A HeLa labelled with FITC - dextran ( molecular = 10 , 000 ) AlPcS2a was 635 - nm illuminated within square region ( left panels ) . FITC - dextran remained within illuminated lysosomes ( an increase in FITC was observed , in accordance with an increase in pH in damaged lysosomes ; middle panels ) . Right panels : magnified view of FITC - dextran within 635 - nm illuminated region pre - post activation . ( c ) activation did not affect other structures , such as mitochondria . A HeLa labelled with TMRE AlPcS2a was 635 - nm illuminated within square region ( left panels ) . Illumination did not affect TMRE , indicating that mitochondria within same region were not affected / impaired . Right panels : magnified view of TMRE within 635 - nm illuminated region pre - post activation . Scale bars , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 expressing EGFP - Ub lamp1 - RFP , labelled with AlPcS2a , was 635 - nm illuminated within square

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) 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 635 - nm illuminated region ( arrow ) 30 - 50 min after activation .

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

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


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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 expressing EGFP - LC3B lamp1 - RFP , labelled with AlPcS2a , was 635 - nm illuminated within square region , resulting in LC3 recruitment onto photodamaged lysosomes , as shown in d . ( d ) Magnified view of 635 - nm illuminated 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 activation ( bottom panels ) . Scale bars , 10 μm ( a , c ) ; 3 μm ( b , d ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 expressing RFP - EGFP - LC3 labelled with AlPcS2a was 635 - nm illuminated within rectangular region , resulting in local LC3 formation , as shown in b . ( b ) LC3 were specifically induced within 635 - nm illuminated regions in a ( LC3 initially distributed uniformly at t = 0 ) . ( c ) loss of EGFP from - induced LC3 preceded disappearance of RFP ( arrows indicate examples of LC3 that exhibited diminished EGFP ) , indicating their successful transition from autophagosomes to autolysosomes . Scale bars , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 iNOS KO mice injected with or without LPS . Scale bars = 100 μm . ( right ) frequency histogram showing distribution of muscle fiber minimum feret diameter in tibialis anterior from saline or LPS treated ( top ) WT control ( bottom ) iNOS KO mice ( n = 4 ) . A total of 300 per muscle were used for . Statistical comparisons , mean , standard deviation of mean are shown in histogram legend .

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 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

(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 of pThr172 - AMPK ( pAMPK ) total AMPK ( AMPK ) in quadriceps muscle . ( right ) of pAMPK to AMPK . Ratios are expressed relative to saline treated controls .

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

(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 TCA cycle . Relative of arginine .

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

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


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

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


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

(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 .

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


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

O O O O O 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

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 for pThr172 - AMPK ( pAMPK ) , total AMPK ( AMPK ) , pSer79 - ACC ( pACC ) , total ACC ( ACC ) .

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

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


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

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.


C57BL / 6 wildtype ( WT ) iNOS knockout ( KO ) mice were intraperitoneally injected with 1mg kg - 1 LPS or an equivalent volume of carrier solution . Control WT , Control KO , LPS - treated KO cohorts were pair - fed ( PF ) to WT LPS - treated cohorts . After 18h , mice were euthanized , tibialis anterior 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 .

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

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.


BALB / C mice were injected subcutaneously with C26 ( 1 . 25x106 ) or an equivalent volume of saline . After 5 days , everyday thereafter , saline C26 injected mice were treated with or without GW ( 5mg kg - 1 ) . After 16 days , mice were euthanized gastrocnemius 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 .

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

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.


of overlapped expressed genes in response to sorafenib - resistance .

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


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

D The 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 .

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


F METTL3 between naïve HepG - 2 ( n = 3 ) sorafenib - resistant HepG - 2 ( n = 10 ) . Data information : In relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O 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 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 IC50 of sorafenib - resistant HepG - 2 treated with sorafenib under hypoxia condition ( 1 % O2 ) . Data information : In relevant panels , * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD are representative of 3 independent experiments .

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

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

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

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


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

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

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 ) METTL3 - overexpression ( B ) in normal liver WRL68 for 7 days in normoxia condition ( 21 % O2 ) of clusters in A - 1 B - 1 , respectively . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 IC50 of METTL3 - knockdown SMMC - 7721 ( C ) Bel - 7402 ( D ) after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 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

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 .

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


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

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

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 ( G ) , Bel - 7402 ( H ) sorafenib - resistant HepG - 2 ( I ) to sorafenib treatment . IC50 of was measured after treated with sorafenib for 24h under hypoxia condition ( 1 % O2 ) . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 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

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 survival of sorafenib - resistant HepG - 2 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 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

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 - conditioned medium ( TCM ) from control HCCs METTL3 - knockdown HCCs cultured under hypoxic conditions for 48 h . of number of tubes with image J in K - 1 . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 relative mRNA expression of angiogenesis genes were detected in SMMC - 7721 ( L ) Bel - 7402 ( M ) with METTL3 - knockdown by RT - PCR . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of VEGF - A in METTL3 - knockdown HCCs . O of PDGF - B in METTL3 - knockdown HCCs .

O O O O O O O O O O O O O O O O O O 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 of METTL3 - knockdown SMMC - 7721 under hypoxia for 48 h .

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


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

O O O O O O 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.


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

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


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

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


F - H Overexpression of shRNA - resistant wild type METTL3 but not catalytic mutant METTL3 rescue autophagy phenotype in METTL3 - knockdown SMMC - 7721 ( F ) , Bel - 7402 ( G ) sorafenib - resistant HepG2 ( H ) by representative immunostaining of LC3 under hypoxia condition for 48h . Scale bar , 200μm . of numbers of GFP - LC3 / by Imaris in F - 1 , G - 1 H - 1 , respectively . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of LC3 I / II in METTL3 - knockdown SMMC - 7721 ( I ) , Bel - 7402 ( J ) HepG - 2 ( K ) treated with or without 3 - MA under hypoxia condition for 24h .

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

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

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 ( GSEA ) output of response to stress pathways displaying a correlation of differentially regulated genes in METTL3 - knockdown HepG2 , SMMC - 7721 Bel - 7402 with 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 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 level of FOXO3 in METTL3 - knockdown HCCs under hypoxia for 48h .

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


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

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

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 in Bel - 7402 correspondent METTL3 - knockdown Bel - 7402 treated with cycloheximide . of optical density by Image J in E - 1 .

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

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 of FOXO3 mRNA in non - ribosome portion ( < 40S ) , 40S , 60S , 80S polysome for METTL3 - knockdown Bel - 7402 compare to control under hypoxia for 48h . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 YTHDF2 in Bel - 7402 by lentiviral shRNAs .

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


H Knockdown of YTHDF1 but not YTHDF2 abolished increase of FOXO3 level in METTL3 - overexpressing Bel - 7402 . of optical density by Image J in H - 1 H - 2 . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 level of FOXO3 in YTHDF1 - knockdown Bel - 7402 by RT - PCR . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O 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 m6A - IP sequencing under hypoxia proved m6A modification participated in regulation of FOXO3 . YTHDF1 binding site locates at 3 ' UTR of FOXO3 .

O B-MEASURED_VAR 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

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 wild - type mutant form of FOXO3 3 ' UTR reporter vectors in Bel - 7402 with various of METTL3 YTHDF1 . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 m6A methylation in FOXO3 mRNA by m6A MeRIP . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD are representative of 3 independent experiments .

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

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 m6A methylation in FOXO3 mRNA by YTHDF1 - RIP . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD are representative of 3 independent experiments .

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

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 of expression of METTL3 FOXO3 in liver cohort ( Kim - 90 ) by Pearson .

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


P Correlation of expression of YTHDF1 FOXO3 in liver 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 by RT - PCR . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 level of FOXO3 in naïve HepG - 2 sorafenib - resistant HepG - 2 under hypoxia for 48h .

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


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

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


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

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


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

O O O O O O O O O O O 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 of METTL3 - knockdown SMMC7721 METTL3 - knockdown & FOXO3 - overexpression SMMC7721 under hypoxia for 48 h . G Representative Immunostaining of LC3 in METTL3 - knockdown SMMC7721 METTL3 - knockdown & FOXO3 - overexpression SMMC7721 under hypoxia for 48 h . Scale bar , 200μm . of numbers of GFP - LC3 / by Imaris in G - 1 . H Representative Immunostaining of LC3 in naïve HepG - 2 sorafenib - resistant HepG - 2 under hypoxia for 48 h . Scale bar , 200μm . of numbers of GFP - LC3 / by Imaris in H - 1 . I IC50 of METTL3 - knockdown SMMC7721 with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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 IC50 of naïve HepG - 2 sorafenib - resistant HepG - 2 with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . Data are presented as mean ± SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 survival of METTL3 - knockdown SMMC7721 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 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 survival of naïve HepG - 2 sorafenib - resistant HepG - 2 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 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 by CRISPR - Cas9 knockout enhanced sorafenib - resistance in vivo .

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


Growth curve ( B ) of Hepa1 - 6 derived xenografts in subcutaneous implantation mice model in six treatment groups for 12 days ( 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

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


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

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

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 growth treated with sorafenib in orthotopic xenograft mouse model . Stable METTL3 - knockdown Bel - 7402 control were injected into liver of each NOD / SCID mouse . 3 after injection , livers were separated for pathological . Red circle indicates tumors in livers .

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

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 eosin .

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 .

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

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 derived xenografts in 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).


( H ) of Hepa1 - 6 derived xenografts in subcutaneous implantation mice model in 6 treatment groups for 10 days ( n = 5 ) . Data information : In relevant panels , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; Two - t - . 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 immunohistochemical of Ki67 , LC3 - B , VEGF - A in Hepa1 - 6 derived xenografts in 6 treatment groups .

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


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

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 ( B ) in mice that were subcutaneously implanted with tissues from two human liver cancer divided into four groups .

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

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 growth ( C ) in mice that were subcutaneously implanted with tissues from two human liver cancer divided into four groups .

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


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

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

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 immunohistochemical of Ki67 , LC3 - B , VEGF - A in randomly selected PDX # 1 PDX # 2 tumors .

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


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

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

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.


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

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

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 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 , Fbxw7 ) in Th2 , treated with or without JQ1 .

O O O O O O O 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

(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 of Il4 , Il5 , Il13 , Gata3 , Foxp3 Fbxw7 in mouse Th2 infected with sh - Ctrl , sh - Foxp3 , sh - Fbxw7 lenti - .

O O O O O O O O O O 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) 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 transfected with Myc - Gata3 / or Flag - Fbxw7 plasmids .

O O O O O O O O O O O O O O O O O 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) 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 treated with or without JQ1 ( 500nM ) , then treated with cycloheximide ( CHX ; 25μg / ml ) for 0h , 0 . 5h , 1h 2h before harvest to stop synthesis .

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

(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 of histone modifications on gene loci of Foxp3 Fbxw7 in mouse Th2 treated with or without JQ1 ( 250nM ) .

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

(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 , followed by Western blotting of Ezh2 , Suz12 , EED G9a .

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

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


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

O O O O O O O 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

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

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

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

O O O O O O O O O O O O O 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) 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 of Il4 , Il5 , Gata3 in mouse Th2 treated with ABBV - 744 at 500nM 1μM .

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

(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 treated with ABBV - 744 ( 500nM ) MG132 ( 20μM ) .

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

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


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

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

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

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

(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 involved in ( A ) ( B ) maintenance of control ( 143B , solid lines , grey bars ) ρ0 ( dashed lines , colored bars ) . A , for POLG , UNG TWNK , only abundances > 1 MDa were quantified . Plots 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 - for two - way ANOVA for changes of multiple in a ) .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 mitochondrial import machinery selected related to this process of control ( 143B , solid lines , grey bars ) ρ0 ( dashed lines , colored - grey bars ) . A B , components of translocase of outer ( TOM ) machinery ( TOM avg . : average of TOMM40 , TOMM22 , TOMM5 , TOMM6 , TOMM7 ) ; C , components of trans locase of inner ( TIM ) machinery ( TIM22 avg . : TIMM22 , AGK , TIMM10B ; TIM23 avg . : TIMM23 , TIMM17A , TIMM17B ) ; D , components of TIM23 - associated TIMM21 assembly machinery ( left panel ) its association with prohibitin ( PHB ) components of mitochondrial quality control system ( right panel ) . Plots gene names are in corresponding colors , except for TIMM44 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 - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 complexes involved in controlling mitochondrial fusion / fission machinery related processes of control ( 143B , solid lines , grey bars ) ρ0 ( dashed lines , colored bars ) . A , mitochondrial contact site cristae organizing system ( MICOS : MIC60 , MIC19 , MIC25 ) SAM ( SAM : SAMM50 , MTX2 , MTX3 ) together forming 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 O O O O O O 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 complexes involved in controlling mitochondrial fusion / fission machinery related processes of control ( 143B , solid lines , grey bars ) ρ0 ( dashed lines , colored bars ) . B , involved in controlling mitochondrial ( orange ) dynamics ( teal ) ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). B, proteins involved in controlling mitochondrial morphology (orange) and dynamics (teal);


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

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

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 indicated concentrations for 300 s , followed by dipping in buffer for 600 s . response curves ( black ) were fitted using either a 1 : 1 ( for VHH - 72 ) or 2 : 1 ( for Re6B06 Re9F06 ) binding models to obtain KDs . Fitted curves are shown in red . For Re6B06 Re9F06 , some secondary binding with weaker affinity at high VHH concentrations was observed ( denoted as KD2 ) .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 indicated VHH antibodies at nanomolar ( A B ) or picomolar ( C D ) concentrations . neutralization experiment was performed ; n " indicates number of independent biological

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 thermostable Re5D06 variants R15 R28 at indicated concentrations . neutralization experiment was

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

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

O O O O O O O O O O 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 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 indicated VHH antibodies . neutralization experiment was performed Note gain in neutralization efficacy when a VHH is trimerized . Re6B06 , for example , is 30 000 - fold more potent when symmetry - matched with Spike .

O O O O O O O O O O O O O O O O O O O O O O O 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 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 quadruple RBD mutant was monitored by BLI . Immobilized RBD was incubated with 100 nM of nanobodies for 450 s , followed by buffer for 900 s .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 - monoubiquitin resembles SARS PLpro activity ( Békés et al , 2015 ; 2016 ) . See Source Data for uncropped gels , Appendix Fig S1B - D for gel - cleavage .

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 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

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

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

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

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

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 polarisation with PLpro wild - type ( reproduced from Appendix Fig S1A ) , PLpro F69S . ( Left ) Using same range of SARS2 PLpro , F69S mutant activity could not be fitted ; ( middle ) a higher 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 S2 site contributes difference in binding for N - terminal Ubl - fold . Experiments for F69S were performed in technical triplicate biological duplicate ; wild - type data is reproduced from experiments shown in Fig 1D , Appendix Fig S1A .

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

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 showing hydrolysis time course of triubiquitin ( D ) , proISG15CTD ( E ) 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 ) reduces proISG15 cleavage to same as proISG15CTD ( compare E , F ) , Experiments were performed in duplicate , see Source Data for uncropped gels .

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 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

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.