( c ) Rates of arm - level and focal SCNVs ( Online Methods ) across different types of malignancies . Data regarding SCNVs of the following types were summarized from Zack et al . : RCC , renal cell carcinoma ; GBM , glioblastoma multiforme ; UCEC , uterine cervix ; COAD , colorectal adenocarcinoma ; BRCA , breast ; HNSC , head and neck squamous cell ; OV , ovary ; LUAD , lung adenocarcinoma ; BLCA , bladder ; LUSC , lung squamous cell .

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(c) Rates of arm-level and focal SCNVs (Online Methods) across different types of malignancies. Data regarding SCNVs of the following types were summarized from Zack et al.: RCC, renal cell carcinoma; GBM, glioblastoma multiforme; UCEC, uterine cervix; COAD, colorectal adenocarcinoma; BRCA, breast; HNSC, head and neck squamous cell; OV, ovary; LUAD, lung adenocarcinoma; BLCA, bladder; LUSC, lung squamous cell.


Data matrix showing frequent SCNVs , significantly mutated genes ( bold ; Benjamini - Hochberg FDR 0 . 2 calculated with CHASM ) and their associated pathway or family mutations discovered in cases with NPC analyzed by WES , targeted deep sequencing ( TS ) or SNP array ( SNP - a ) . The frequencies of the alterations are plotted on the right . The colors and shapes denoting different types of somatic events are also applied in Figure 5a . Columns , examined cases ; rows , genes ; hot spot , identical mutations have been registered in COSMIC ( see URLs ) ; LOH , loss of heterozygosity . The asterisk indicates that this tumor was also subjected to SNP array profiling . PIK3CA amplification in C666 cells has been previously described .

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Data matrix showing frequent SCNVs, significantly mutated genes (bold; Benjamini-Hochberg FDR 0.2 calculated with CHASM) and their associated pathway or family mutations discovered in cases with NPC analyzed by WES, targeted deep sequencing (TS) or SNP array (SNP-a). The frequencies of the alterations are plotted on the right. The colors and shapes denoting different types of somatic events are also applied in Figure 5a. Columns, examined cases; rows, genes; hot spot, identical mutations have been registered in COSMIC (see URLs); LOH, loss of heterozygosity. The asterisk indicates that this tumor was also subjected to SNP array profiling. PIK3CA amplification in C666 cells has been previously described.


( b , c ) Significant correlations between dysregulation of chromatin modification and total number of somatic mutations ( b ) and overall survival probability ( c ) . Mann - Whitney test ( b ) and log - rank test ( c ) were used to calculate statistical significance . Horizontal bars , mean value ; HR , hazard ratio .

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(b,c) Significant correlations between dysregulation of chromatin modification and total number of somatic mutations (b) and overall survival probability (c). Mann-Whitney test (b) and log-rank test (c) were used to calculate statistical significance. Horizontal bars, mean value; HR, hazard ratio.


( a - d ) NPC cells stably expressing either negative control shRNA ( Scramble ) or pooled shRNAs targeting ARID1A ( shARID1A ) were subjected to anchorage - independent colony formation assay ( a ) , migration assay ( b ) , xenograft growth assay ( horizontal lines , mean values ) and western blot ( WB ) assay with the indicated antibodies ( d ) .

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(a-d) NPC cells stably expressing either negative control shRNA (Scramble) or pooled shRNAs targeting ARID1A (shARID1A) were subjected to anchorage-independent colony formation assay (a), migration assay (b), xenograft growth assay (horizontal lines, mean values) and western blot (WB) assay with the indicated antibodies (d).


( e - g ) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation assay ( e )

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(e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e)


( e - g ) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation assay ( e ) , migration assay ( f )

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(e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e), migration assay (f)


e - g ) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation assay ( e ) , migration assay ( f ) and WB assay with the indicated antibodies ( g ) .

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e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e), migration assay (f) and WB assay with the indicated antibodies (g).


( h , i ) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage - independent colony formation assay ( h )

(h,i) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage-independent colony formation assay (h)


h , i ) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage - independent colony formation assay ( h ) and WB assay with indicated antibodies ( i ) . β - actin was examined as a loading control . The data shown in a , b , e , f and h represent the mean ± s . d . ; n = 3 for a , b , e and f ; n = 4 for h . * P 0 . 05 , * * P 0 . 01 . Student ' s t - test was used to calculate statistical significance in a - c , e , f and h ; the blots shown in d , g and i are representative of three total blots run .

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h,i) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage-independent colony formation assay (h) and WB assay with indicated antibodies (i). β-actin was examined as a loading control. The data shown in a, b, e, f and h represent the mean ± s.d.; n = 3 for a, b, e and f; n = 4 for h. *P 0.05, **P 0.01. Student's t-test was used to calculate statistical significance in a-c, e, f and h; the blots shown in d, g and i are representative of three total blots run.


( a ) Data matrix showing alterations of genes involved in the ERBB - PI3K signaling pathway and their related targeting molecules . Considering space limitations , the numbers of targeting agents available for each protein are shown in parentheses . Drugs currently in phase 2 clinical trials are marked in blue , those in phase 3 are in purple , and FDA - approved drugs are in brown ( see URLs ) .

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(a) Data matrix showing alterations of genes involved in the ERBB-PI3K signaling pathway and their related targeting molecules. Considering space limitations, the numbers of targeting agents available for each protein are shown in parentheses. Drugs currently in phase 2 clinical trials are marked in blue, those in phase 3 are in purple, and FDA-approved drugs are in brown (see URLs).


( b ) Kaplan - Meier survival curves of NPC subjects either with or without genetic lesions in the ERBB - PI3K signaling pathway . Log - rank test was used to calculate statistical significance .

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(b) Kaplan-Meier survival curves of NPC subjects either with or without genetic lesions in the ERBB-PI3K signaling pathway. Log-rank test was used to calculate statistical significance.


C Relative mRNA expression of Zeb1 , Acp5 , and Ctsk during the differentiation from BMDMs to osteoclasts in culture ( n = 3 ) .

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C Relative mRNA expression of Zeb1, Acp5, and Ctsk during the differentiation from BMDMs to osteoclasts in culture (n = 3).


D Western blot of Zeb1 , c - Src , and Ctsk expression in BMDMs and mature osteoclasts ( n = 3 ) .

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D Western blot of Zeb1, c-Src, and Ctsk expression in BMDMs and mature osteoclasts (n = 3).


F Zeb1 ( red ) and TRAP ( green ) immunofluorescence of a femur section including the growth plate ( GP ) and metaphysis from a wild - type mouse . The multinucleated cells associated with the surface of the bone marrow cavity ( BM cavity ) that are shown in the red box are further enlarged in the panels to the right . Scale bar , 10 μm .

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F Zeb1 (red) and TRAP (green) immunofluorescence of a femur section including the growth plate (GP) and metaphysis from a wild-type mouse. The multinucleated cells associated with the surface of the bone marrow cavity (BM cavity) that are shown in the red box are further enlarged in the panels to the right. Scale bar, 10 μm.


A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3 - month - old wild - type and Zeb1ΔM / ΔM male mice are shown . The genotype of Zeb1 + / + , Zeb1f / + , Zebf / f mice is defined as " wild - type " control . Scale bar , 500 μm . B Quantification of BV / TV , Tb . Th , Tb . N , and Tb . Sp as determined by nanoCT in 3 - month - old wild - type and Zeb1ΔM / ΔM male mice ( n = 8 ) .

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A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown. The genotype of Zeb1+/+, Zeb1f/+, Zebf/f mice is defined as "wild-type" control. Scale bar, 500 μm. B Quantification of BV/TV, Tb.Th, Tb.N, and Tb.Sp as determined by nanoCT in 3-month-old wild-type and Zeb1ΔM/ΔM male mice (n = 8). C


C H & E and TRAP staining of the distal femurs of 3 - month - old male wild - type and Zeb1ΔM / ΔM mice . Scale bar , 200 μm . D Quantification of osteoclast number per bone surface ( N . Oc / BS ) and eroded surface per bone surface ( ES / BS ) , as well as serum CTX - I and OCN levels in 3 - month - old wild - type and Zeb1ΔM / ΔM male mice are shown ( n = 6 ) .

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C H&E and TRAP staining of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice. Scale bar, 200 μm. D Quantification of osteoclast number per bone surface (N.Oc/BS) and eroded surface per bone surface (ES/BS), as well as serum CTX-I and OCN levels in 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown (n = 6). E


E Golden ' s trichrome staining illuminating howship ' s lacunae ( arrow ) of the distal femurs of 3 - month - old male wild - type and Zeb1ΔM / ΔM mice were performed along with double - calcein bone labeling and confocal imaging to assess bone formation . Scale bar , 10 μm . F Quantification of MAR and BFR as assessed in 3 - month - old male wild - type and Zeb1ΔM / ΔM mice ( n = 6 ) .

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E Golden's trichrome staining illuminating howship's lacunae (arrow) of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice were performed along with double-calcein bone labeling and confocal imaging to assess bone formation. Scale bar, 10 μm. F Quantification of MAR and BFR as assessed in 3-month-old male wild-type and Zeb1ΔM/ΔM mice (n = 6). D


B Zeb1 , NFATc1 , c - Fos , c - Src , β3 integrin , Mmp9 , Mmp14 , and Ctsk expression as assessed by Western blot in wild - type and Zeb1ΔM / ΔM BMDMs during osteoclast differentiation ( n = 3 ) .

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B Zeb1, NFATc1, c-Fos, c-Src, β3 integrin, Mmp9, Mmp14, and Ctsk expression as assessed by Western blot in wild-type and Zeb1ΔM/ΔM BMDMs during osteoclast differentiation (n = 3).


G Supernatant CTX - I determined by ELISA ( n = 6 ) .

G Supernatant CTX-I determined by ELISA (n = 6).


H , I After a 6 - day culture atop bone slice , phalloidin staining ( red ) was performed in wild - type versus Zeb1ΔM / ΔM osteoclasts ( I ) , and actin ring area per cell quantified ( H ) . Scale bar , 20 μm .

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H, I After a 6-day culture atop bone slice, phalloidin staining (red) was performed in wild-type versus Zeb1ΔM/ΔM osteoclasts (I), and actin ring area per cell quantified (H). Scale bar, 20 μm.


E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice .

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E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice.


F MtCK1 , Ckb , VDAC , and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice ( n = 3 ) .

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F MtCK1, Ckb, VDAC, and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).


H MtCK1 ( green ) immunofluorescence of wild - type and Zeb1ΔM / ΔM osteoclasts . Scale bar , 20

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H MtCK1 (green) immunofluorescence of wild-type and Zeb1ΔM/ΔM osteoclasts. Scale bar, 20 μ


A Phosphocreatine ( PCr ) and creatine ( Cr ) level , as well as PCr / Cr ratio of wild - type or Zeb1ΔM / ΔM osteoclasts cultured atop plastic substrata as determined by LC / MS analysis ( n = 3 ) .

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A Phosphocreatine (PCr) and creatine (Cr) level, as well as PCr/Cr ratio of wild-type or Zeb1ΔM/ΔM osteoclasts cultured atop plastic substrata as determined by LC/MS analysis (n = 3).


B , C Oxygen consumption rate ( OCR ) profile plot ( B ) and mitochondrial function parameters ( C ) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild - type or Zeb1ΔM / ΔM mice after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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B, C Oxygen consumption rate (OCR) profile plot (B) and mitochondrial function parameters (C) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild-type or Zeb1ΔM/ΔM mice after sequential treatment of compounds modulating mitochondrial function (n = 4).


C Wild - type BMDMs were transduced with either a mock vector , wild - type human MtCK1 , or a catalytically - inactive MtCK1C316G mutant expression vector , differentiated into osteoclasts , and cell lysates collected for MtCK1 , Ckb , Tomm20 , VDAC , Zeb1 , c - Src , and Ctsk immunoblotting ( n = 3 ) .

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C Wild-type BMDMs were transduced with either a mock vector, wild-type human MtCK1, or a catalytically-inactive MtCK1C316G mutant expression vector, differentiated into osteoclasts, and cell lysates collected for MtCK1, Ckb, Tomm20, VDAC, Zeb1, c-Src, and Ctsk immunoblotting (n = 3).


E PCr / Cr ratio of mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts cultured on plastic substrata as determined by LC / MS analysis ( n = 3 ) .

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E PCr/Cr ratio of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts cultured on plastic substrata as determined by LC/MS analysis (n = 3).


OCR profile plot ( H ) analyzed by XF Cell Mito Stress Assay in mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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OCR profile plot (H) analyzed by XF Cell Mito Stress Assay in mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).


J , K Mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type pre - osteoclasts were cultured atop bone slices for 3 days , stained with phalloidin ( red ) . Osteoclasts were removed and resorption pits visualized by WGA - DAB staining ( J ) . The actin ring area per cell and resorption pit area were quantified ( K ; n = 6 ) . Scale bar , upper 20 μm , lower 100 μm .

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J, K Mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type pre-osteoclasts were cultured atop bone slices for 3 days, stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (J). The actin ring area per cell and resorption pit area were quantified (K; n = 6). Scale bar, upper 20 μm, lower 100 μm.


A , B Human monocyte - derived macrophages ( hMDMs ) were transfected with either shCTRL or siRNA targeting ZEB1 , differentiated into osteoclasts , and relative mRNA expression of CKMT1 ( A ) and mitochondrial creatine kinase activity determined ( B ; n = 3 ) .

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A, B Human monocyte-derived macrophages (hMDMs) were transfected with either shCTRL or siRNA targeting ZEB1, differentiated into osteoclasts, and relative mRNA expression of CKMT1 (A) and mitochondrial creatine kinase activity determined (B; n = 3).


F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non - targeting siCTRL - or siZEB1 - transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non-targeting siCTRL- or siZEB1-transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).


G , H siCTRL - or siZEB1 - transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin ( red ) . Osteoclasts were removed and resorption pits visualized by WGA - DAB staining ( G ) . Scale bar , upper 20 μm , lower 100 μm . The actin ring area per cell , resorption pit area , and the number of TRAP + MNCs were quantified ( H ; n = 6 ) .

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G, H siCTRL- or siZEB1-transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (G). Scale bar, upper 20 μm, lower 100 μm. The actin ring area per cell, resorption pit area, and the number of TRAP+ MNCs were quantified (H; n = 6).


I - L Calvaria isolated from wild - type and Zeb1ΔM / ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine , and stained for phalloidin ( red ) and Vpp3 ( green ) ( I ) . Scale bar , 10 μm . The TRAP activity of whole cell lysates , actin ring area per cell , and supernatant CTX - I were quantified ( J - L ; n = 6 ) .

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I-L Calvaria isolated from wild-type and Zeb1ΔM/ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine, and stained for phalloidin (red) and Vpp3 (green) (I). Scale bar, 10 μm. The TRAP activity of whole cell lysates, actin ring area per cell, and supernatant CTX-I were quantified (J-L; n = 6).


( B ) Density of HEAP - AGO2 peaks across cytosolic lncRNAs ( n = 62 , blue ) and the 3 ' UTR regions of mRNAs ( n = 8798 , red ) with experimental evidence for AGO2 binding in mESCs ( > 0 AGO2 peaks ) . Central band of boxplot represents median , box depicts 25 - 75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution .

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(B) Density of HEAP-AGO2 peaks across cytosolic lncRNAs (n=62, blue) and the 3'UTR regions of mRNAs (n=8798, red) with experimental evidence for AGO2 binding in mESCs (>0 AGO2 peaks). Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.


( D ) Distribution of the fold - change ( FC ) in degradation rate of mRNAs ( n = 13296 , red ) , micropeptide encoding transcripts ( n = 43 , pink ) , bona fide cytosolic ( n = 759 , blue ) and nuclear ( n = 4299 , grey ) lncRNAs in 4 - OHT treated ( KO ) relative to ethanol treated ( WT ) cells after 8 days treatment , horizontal dashed line represents a KO / WT FC in degradation rate of 1 . Statistics : NS - p > 0 . 05 , * - p - value < 0 . 05 . Two - tailed Mann - Whitney U test : p - value = 0 . 044 . Central band of boxplot represents median , box depicts 25 - 75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution .

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(D) Distribution of the fold-change (FC) in degradation rate of mRNAs (n=13296, red), micropeptide encoding transcripts (n=43, pink), bona fide cytosolic (n=759, blue) and nuclear (n=4299, grey) lncRNAs in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days treatment, horizontal dashed line represents a KO/WT FC in degradation rate of 1. Statistics: NS-p>0.05, *-p-value<0.05. Two-tailed Mann-Whitney U test: p-value=0.044. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.


( F ) Distribution of the fold - change ( FC ) in degradation rates of mRNAs ( n = 29900 , red ) , cytosolic ( n = 474 , blue ) and nuclear ( n = 2348 , grey ) lncRNAs , in 4 - OHT treated ( KO ) relative to ethanol treated ( WT ) cells after 8 days of treatment ( estimated based on the 10 minutes 4sU pulse ) , horizontal dashed line represents a KO / WT FC in degradation rate of 1 Statistics : NS - p - value > 0 . 05 anband of boxplot represents median box depicts quantiles distribution and whiskers represent the > th and 95th quantiles of the distribution . ( two - tailed Mann - Whitney U test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of the fold-change (FC) in degradation rates of mRNAs (n=29900, red), cytosolic (n=474, blue) and nuclear (n=2348, grey) lncRNAs, in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days of treatment (estimated based on the 10 minutes 4sU pulse), horizontal dashed line represents a KO/WT FC in degradation rate of 1 Statistics: NS-p-value>0.05 anband of boxplot represents median box depicts quantiles distribution and whiskers represent the>th and 95th quantiles of the distribution. (two-tailed Mann-Whitney U test).


( B ) Fraction of cytosolic lncRNAs with experimental evidence for ribosomal binding with ( red ) or without ( blue ) an overlapping conserved short open reading frame .

O O O O O O 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) Fraction of cytosolic lncRNAs with experimental evidence for ribosomal binding with (red) or without (blue) an overlapping conserved short open reading frame.


( 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 .

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

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

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

(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 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 .

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

(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 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 .

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

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


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 .

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

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

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

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cell expressing EGFP - Ub and lamp1 - RFP , and labelled with AlPcS2a , was 635 - nm illuminated within the white square

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


Glycolytic index of basal metabolism.


Western blot analysis for ETC protein complex subunits .

Western blot analysis for ETC protein complex subunits.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O B-CONTROLLED_VAR O O 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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O 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).


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 .

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

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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 and eosin staining .

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


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

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

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


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

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


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

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


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

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

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

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.