( A - C ' ' ' ) The midguts from adult female flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ with ( B - B ' ' ' ) or without ( A - A ' ' ' ) UAS - RasV12 or with UAS - RasV12 and UAS - stg - RNAi ( C - C ' ' ' ) were dissected and immunostained with antibodies against GFP , β - gal and Dl . The bigger GFP ─ nucleus with LacZ expression ( only red arrowheads ) indicate differentiating or mature ECs . Blue arrowheads indicate Dl + ISCs ; white arrows indicate GFP + EBs ; and red arrowheads indicate EB progeny cells ( LacZ + GFP ─ ) . ( D ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the indicated genotypes , n = 50 .

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(A-C''') The midguts from adult female flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ with (B-B''') or without (A-A''') UAS-RasV12 or with UAS-RasV12 and UAS-stg-RNAi (C-C''') were dissected and immunostained with antibodies against GFP, β-gal and Dl. The bigger GFP─ nucleus with LacZ expression (only red arrowheads) indicate differentiating or mature ECs. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the indicated genotypes, n=50.


( C - E ' ' ) The representative twin clones from midguts when EGFRA887T is overexpressed in EBs ( D - D ' ' , red and green arrows ) or adult flies were fed with P . e for 36h ( E - E ' ' , red and green arrows ) , but no twin clone is found in the control ( C - C ' ' ) . ( F ) Quantification of different types of twin clones in the control midguts , or in the midguts with EGFRA887T overexpression or P . e infection . n = 10 guts for each genotype .

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(C-E'') The representative twin clones from midguts when EGFRA887T is overexpressed in EBs (D-D'', red and green arrows) or adult flies were fed with P.e for 36h (E-E'', red and green arrows), but no twin clone is found in the control (C-C''). (F) Quantification of different types of twin clones in the control midguts, or in the midguts with EGFRA887T overexpression or P.e infection. n = 10 guts for each genotype.


( A - C ' ' ) ISCs from EBs upon RasV12 expression or P . e infection could differentiate to generate EE or pre - EE cells ( B - C ' ' , LacZ + Pros + GFP ─ , blue arrowheads ) , but no EE or pre - EE cells from EBs are found in the control ( A - A ' ' , white arrowheads ) . ( D ) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments . n = 9 guts for each genotype or treatment .

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(A-C'') ISCs from EBs upon RasV12 expression or P. e infection could differentiate to generate EE or pre-EE cells (B-C'', LacZ+Pros+GFP─, blue arrowheads), but no EE or pre-EE cells from EBs are found in the control (A-A'', white arrowheads). (D) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments. n = 9 guts for each genotype or treatment.


( B ) Kaplan - Meier survival curve based on n = 17 144DG11 - treated and n = 9 vehicle - treated animals . 144DG11 significantly increased survival ( log - rank test p - value < 0 . 000692 ) .

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(B) Kaplan-Meier survival curve based on n=17 144DG11-treated and n=9 vehicle-treated animals. 144DG11 significantly increased survival (log-rank test p-value<0.000692).


( M ) average duration in movement in an open field as a function of time after treating Gbeys / ys mice with vehicle ( n = 8 ) or 144DG11 ( n = 9 ) at 6 months of age ( at onset ) . Data information : Two way ANOVA with repeated measures show that , throughout the period , treatment values were not different than vehicle ( p < 0 . 15 ) .

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(M) average duration in movement in an open field as a function of time after treating Gbeys/ys mice with vehicle (n=8) or 144DG11 (n=9) at 6 months of age (at onset). Data information: Two way ANOVA with repeated measures show that, throughout the period, treatment values were not different than vehicle (p<0.15).


( A ) Left panel , Mice , treated as indicated , were sacrificed and the indicated tissues were collected and stained for PG ( arrows ) with PAS following diastase treatment . Scale bars , 200 µm ( liver , muscle ) , 50 µm ( cortex , heart , peripheral nerve ) . Right panel , PAS staining was quantified based on analysis of 4 sections from each tissue in n = 3 wt , n = 7 Gbeys / ys vehicle - treated , and n = 9 144DG11 - treated mice .

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(A) Left panel, Mice, treated as indicated, were sacrificed and the indicated tissues were collected and stained for PG (arrows) with PAS following diastase treatment. Scale bars, 200 µm (liver, muscle), 50 µm (cortex, heart, peripheral nerve). Right panel, PAS staining was quantified based on analysis of 4 sections from each tissue in n=3 wt, n=7 Gbeys/ys vehicle-treated, and n=9 144DG11-treated mice.


( C ) 144DG11 Pharmacokinetics . Gbeys / ys mice injected with 144DG1 were sacrificed 30 , 60 , 90 , and 210 min post injection and the indicated tissues were removed , as well as 200 uL of serum drawn . Graph shows means ( + / - SEM ) of 144DG11 levels in the different tissues determined by LC - MS / MS Results obtained from n = 3 mice at each time point .

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(C) 144DG11 Pharmacokinetics. Gbeys/ys mice injected with 144DG1 were sacrificed 30, 60, 90, and 210 min post injection and the indicated tissues were removed, as well as 200 uL of serum drawn. Graph shows means (+/- SEM) of 144DG11 levels in the different tissues determined by LC-MS/MS Results obtained from n = 3 mice at each time point.


A - F Mice were monitored over a 24 hr period . Effective mass was calculated by ANCOVA Data are mean ± SEM from nine month old mice ( n = 11 , wt vehicle - treated , n = 6 Gbeys / ys vehicle - treated , and n = 7 Gbeys / ys 144DG11 - treated ) . Vehicle - treated Gbeys / ys mice demonstrate lower respiratory quotient ( in the light ) ( A ) , total energy expenditure ( TEE ) ( B ) , and fat oxidation ( C ) compared to wt controls . 144DG11 treatment increased these parameters ( for fat oxidation only in the dark and total time ) . Carbohydrate oxidation and ambulatory activity , not significantly affected by the diseased state , were increased by 144DG11 even beyond wt control levels ( D and E ) ( note , while 144DG11 increased carbohydrate oxidation in the light ( D ) , p was only < 0 . 06 ) . 144DG11 also reversed the decrease in meal size and water sip volume observed in Gbeys / ys mice as compared to wt control ( F ) . Data information : * p < 0 . 05 v wt controls , # p < 0 . 05 v Gbeys / ys vehicle treated mice . Statistical differences were determined by two - tailed t - tests .

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A-F Mice were monitored over a 24 hr period. Effective mass was calculated by ANCOVA Data are mean±SEM from nine month old mice (n=11, wt vehicle-treated, n=6 Gbeys/ys vehicle-treated, and n=7 Gbeys/ys 144DG11-treated). Vehicle-treated Gbeys/ys mice demonstrate lower respiratory quotient (in the light) (A), total energy expenditure (TEE) (B), and fat oxidation (C) compared to wt controls. 144DG11 treatment increased these parameters (for fat oxidation only in the dark and total time). Carbohydrate oxidation and ambulatory activity, not significantly affected by the diseased state, were increased by 144DG11 even beyond wt control levels (D and E) (note, while 144DG11 increased carbohydrate oxidation in the light (D), p was only <0.06). 144DG11 also reversed the decrease in meal size and water sip volume observed in Gbeys/ys mice as compared to wt control (F). Data information: *p<0.05 v wt controls, #p<0.05 v Gbeys/ys vehicle treated mice. Statistical differences were determined by two-tailed t-tests.


( G ) Blood metabolic panel based on n = 3 , 9 . 5 month old mice treated as indicated . Blood glucose was increased and blood triglycerides decreased in Gbeys / ys cells by 144DG11 ( p < 0 . 05 ) .

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(G) Blood metabolic panel based on n=3, 9.5 month old mice treated as indicated. Blood glucose was increased and blood triglycerides decreased in Gbeys/ys cells by 144DG11 (p<0.05).


( A ) PAS staining for total glycogen in skin fibroblasts from different APBD patients . Staining fluorescence was quantified by InCell2200 ( see Methods ) . Means ( + / - SEM ) are statistically different from each other ( p < 0 . 0001 , One Way ANOVA ) . n = 3 technical replicates from each patient indicated at the x - axis were used .

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(A) PAS staining for total glycogen in skin fibroblasts from different APBD patients. Staining fluorescence was quantified by InCell2200 (see Methods). Means (+/- SEM) are statistically different from each other (p<0.0001, One Way ANOVA). n=3 technical replicates from each patient indicated at the x-axis were used.


( B ) PAS staining for total glycogen in APBD87 fibroblasts glucose - starved for 48 h ( left ) , or glucose starved and then replenished for the last 24 h to induce glycogen burden ( right ) . Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter . Scale bar , 100 µm .

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(B) PAS staining for total glycogen in APBD87 fibroblasts glucose-starved for 48 h (left), or glucose starved and then replenished for the last 24 h to induce glycogen burden (right). Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter. Scale bar, 100 µm.


( D ) Glycolytic ( red ) and mitochondrial ( blue ) ATP production determined by Agilent ' s Seahorse machine and real - time ATP rate assay kit . HC and APBD patient fibroblasts were serum / glucose - starved for 48 h and then full medium was replenished for 24 h without ( untreated ) , or with ( chronic ) 50 µM 144DG11 . Acute , 50 µM 144DG11 was added on assay for 20 min after 24 h of serum / glucose replenishment . Readings were normalized to cell number as determined by Crystal Violet staining . Shown are mean and SEM values based on n = 3 - 6 technical repeats . For all experiments ( HC , p < 0 . 01 ; APBD1 , p < 0 . 02 ; APBD2 , p < 0 . 09 ; APBD3 , p < 0 . 11 ; One Way ANOVA with Sidak ' s post - hoc correction for multiple comparisons ) glycolytic ATP production was increased , as compared to untreated control , by acute , but not chronic , supplementation of 144DG11 . Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts ( p < 0 . 06 ) .

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(D) Glycolytic (red) and mitochondrial (blue) ATP production determined by Agilent's Seahorse machine and real-time ATP rate assay kit. HC and APBD patient fibroblasts were serum/glucose-starved for 48 h and then full medium was replenished for 24 h without (untreated), or with (chronic) 50 µM 144DG11. Acute, 50 µM 144DG11 was added on assay for 20 min after 24 h of serum/glucose replenishment. Readings were normalized to cell number as determined by Crystal Violet staining. Shown are mean and SEM values based on n=3-6 technical repeats. For all experiments (HC, p<0.01; APBD1, p<0.02; APBD2, p<0.09; APBD3, p<0.11; One Way ANOVA with Sidak's post-hoc correction for multiple comparisons) glycolytic ATP production was increased, as compared to untreated control, by acute, but not chronic, supplementation of 144DG11. Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts (p<0.06).


( C ) Cellular thermal shift assay ( CETSA ) of different targets of the 144DG11hetero - assembly ( B ) . Only LAMP1 was significantly protected by 144DG11 from heat - mediated denaturation ( see right shift in its Tm in the lower panel ) suggesting its specific interaction with 144DG11 . n = 3 biological replicates . Error bars represent S . D .

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(C) Cellular thermal shift assay (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1 was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D.


( A ) Left panel , Autophagic flux , determined by the extent of lysosomal inhibitors - dependent increase in the ratio of lipidated to non - lipidated LC3 ( LC3II / LC3I ) , is increased by 144DG11 ( 50µM , 24h ) . Right panel , 144DG11 - mediated increase in autophagic flux , demonstrated by enhanced degradation of the autophagy substrate p62 . Shown are representative immunoblots ( grouped from different gels ) and densitometric quantifications + / - s . d . ( for LC3II / LC3I , n = 4 biological replicates , * p < 0 . 0023 ; for p62 , n = 5 biological replicates , * p < 0 . 0099 ; two - tailed t - test ) .

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(A) Left panel, Autophagic flux, determined by the extent of lysosomal inhibitors-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II/LC3I), is increased by 144DG11 (50µM, 24h). Right panel, 144DG11-mediated increase in autophagic flux, demonstrated by enhanced degradation of the autophagy substrate p62. Shown are representative immunoblots (grouped from different gels) and densitometric quantifications +/-s.d. (for LC3II/LC3I, n=4 biological replicates, *p<0.0023; for p62, n=5 biological replicates, *p<0.0099; two-tailed t-test).


( B ) Left panel , TEM images of liver tissue from vehicle or 144DG11 - treated Gbeys / ys mice . High - magnification ( right , scale bars = 200 nm ) and low - magnification ( left , scale bars = 1 µm ) images show higher levels of glycogen / polyglucosan in lysosomes and cytosol , respectively . Right panel , quantification ( + / - s . d ) of lysosomal glycogen particles ( n = 3 biological replicates , * p < 0 . 03 , two - tailed t - test ) . G , Glycogen / polyglucosan ; L , Lysosomes ; M , Mitochondria .

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(B) Left panel, TEM images of liver tissue from vehicle or 144DG11-treated Gbeys/ys mice. High-magnification (right, scale bars=200 nm) and low-magnification (left, scale bars=1 µm) images show higher levels of glycogen/polyglucosan in lysosomes and cytosol, respectively. Right panel, quantification (+/- s.d) of lysosomal glycogen particles (n=3 biological replicates, *p<0.03, two-tailed t-test). G, Glycogen/polyglucosan; L, Lysosomes; M, Mitochondria.


( C ) 144DG11 reduces LC3 and p62 in mouse liver , but not muscle ( n = 3 biological replicates , * p < 0 . 04 for LC3 , * * p < 0 . 0007 for p62 ; two - tailed t - test , SEM ) . Scale bars , 10

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(C) 144DG11 reduces LC3 and p62 in mouse liver, but not muscle (n=3 biological replicates, *p<0.04 for LC3, **p<0.0007 for p62; two-tailed t-test, SEM). Scale bars, 10 µm


( E ) LAMP1 - KD and 144DG11 treatment cause lysosomal acidification . Upper panel , flow cytometry results showing that 144DG11 slightly increased acidification ( yellow to blue median fluorescence ratio ( Y / B ) ) in control , GFP - transduced , APBD fibroblasts ( Y / B ( GFP / 144DG11 ) > Y / B ( GFP ) , p < 0 . 12 ) , but significantly acidified LAMP1 - KD , GFP - shLAMP1 - transduced , APBD fibroblasts ( Y / B ( LAMP1 - KD / 144DG11 ) > ( Y / B ( LAMP1 - KD ) , p < 0 . 03 ) . LAMP1 - KD itself led to the most significant acidification ( Y / B ( LAMP1 - KD ) > Y / B ( GFP ) , p < 0 . 007 ) . n = 3 , two - tailed t - tests . Middle panel Lysosensor staining of the corresponding cells . Yellow fluorescence intensity correlates with acidification . Lower panel , PAS ( glycogen ) staining of the corresponding cells . Scale bars , 50 µm .

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(E) LAMP1-KD and 144DG11 treatment cause lysosomal acidification. Upper panel, flow cytometry results showing that 144DG11 slightly increased acidification (yellow to blue median fluorescence ratio (Y/B)) in control, GFP-transduced, APBD fibroblasts (Y/B(GFP/144DG11)>Y/B(GFP), p<0.12), but significantly acidified LAMP1-KD, GFP-shLAMP1-transduced, APBD fibroblasts (Y/B(LAMP1-KD/144DG11)>(Y/B(LAMP1-KD), p<0.03). LAMP1-KD itself led to the most significant acidification (Y/B (LAMP1-KD)>Y/B(GFP), p<0.007). n=3, two-tailed t-tests. Middle panel Lysosensor staining of the corresponding cells. Yellow fluorescence intensity correlates with acidification. Lower panel, PAS (glycogen) staining of the corresponding cells. Scale bars, 50 µm.


( G ) 144DG11 reduces lysosomal ( LAMP1 positive ) area in liver , but not muscle , of Gbeys / ys mice ( n = 3 biological replicates , * p < 0 . 01 , two - tailed t - test , SEM ) . Scale bars , 5 µm for liver ( upper left panel ) , and 10 µm for muscle ( lower left panel ) . Right panel shows quantification of the left panel .

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(G) 144DG11 reduces lysosomal (LAMP1 positive) area in liver, but not muscle, of Gbeys/ys mice (n=3 biological replicates, *p<0.01, two-tailed t-test , SEM).Scale bars, 5 µm for liver (upper left panel), and 10 µm for muscle (lower left panel). Right panel shows quantification of the left panel.


( B ) Morphological characterization of lysosomes based on lysotracker staining of n = 4 HC and n = 4 ( biological replicates ) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h . Values are deviations from untreated HC . 144DG11 has reduced lysotracker mean intensity , integrated intensity ( DXA ) , and area in both HC and APBD fibroblasts ( * * * p < 0 . 0001 , One - Way ANOVA with Sidak ' s multi - comparison post - hoc test ) .

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(B) Morphological characterization of lysosomes based on lysotracker staining of n=4 HC and n=4 (biological replicates) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h. Values are deviations from untreated HC. 144DG11 has reduced lysotracker mean intensity, integrated intensity (DXA), and area in both HC and APBD fibroblasts (***p<0.0001, One-Way ANOVA with Sidak's multi-comparison post-hoc test).


( C ) Functional ( mitochondrial membrane potential ( TMRE parameters ) ) and biomass ( mitotracker ) characterization of mitochondria in HC and APBD cells treated and analyzed as in ( B ) . Mean ( * * * , p < 0 . 0001 ) and integrated ( * * * , p < 0 . 0006 ) TMRE intensities , and mitochondrial biomass ( * * * , p < 0 . 0001 ) , were reduced in APBD v HC . 144DG11 increased TMRE mean ( * * , p < 0 . 009 , * * , p < 0 . 006 ) and integrated ( * , p < 0 . 01 , * * , p < 0 . 001 ) intensities in APBD and HC cells , respectively and mitotracker intensity in APBD cells only ( * * * , p < 0 . 0001 ) . Data represent n = 3 biological replicates . Statistical differences analyzed by One - way ANOVA with multiple comparisons .

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(C) Functional (mitochondrial membrane potential (TMRE parameters)) and biomass (mitotracker) characterization of mitochondria in HC and APBD cells treated and analyzed as in (B). Mean (***, p<0.0001) and integrated (***, p<0.0006) TMRE intensities, and mitochondrial biomass (***, p<0.0001), were reduced in APBD v HC. 144DG11 increased TMRE mean (**, p<0.009, **, p<0.006) and integrated (*, p<0.01, **, p<0.001) intensities in APBD and HC cells, respectively and mitotracker intensity in APBD cells only (***, p<0.0001). Data represent n=3 biological replicates. Statistical differences analyzed by One-way ANOVA with multiple comparisons.


B Confocal images of HeLa cells transiently expressing GFP - CBPs or GFP . The dotted lines show each cell shape . Scale bars , 10 µm .

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B Confocal images of HeLa cells transiently expressing GFP-CBPs or GFP. The dotted lines show each cell shape. Scale bars, 10 µm.


C Confocal images of HeLa cells transiently expressing GFP - CbpCT4 and mCherry - LC3 ( upper ) . The fluorescence intensities of GFP - CbpCT4 ( green ) and mCherry - LC3 ( red ) along the arrow are shown in the graph at the bottom .

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C Confocal images of HeLa cells transiently expressing GFP-CbpCT4 and mCherry-LC3 (upper). The fluorescence intensities of GFP-CbpCT4 (green) and mCherry-LC3 (red) along the arrow are shown in the graph at the bottom.


D Lysates from 293T cells transiently expressing GFP - Cbps or GFP were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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D Lysates from 293T cells transiently expressing GFP-Cbps or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


E Lysates from 293T cells transiently expressing GFP - CbpCT4 , GFP - LytR , or GFP in the presence or absence of chloroquine were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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E Lysates from 293T cells transiently expressing GFP-CbpCT4, GFP-LytR, or GFP in the presence or absence of chloroquine were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


F MEFs were infected with S . pneumoniae R6 WT or ΔcbpF for the indicated periods , and the intracellular survival of bacteria expressed as the number of CFUs .

O O O O O O 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 MEFs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated periods, and the intracellular survival of bacteria expressed as the number of CFUs.


G p62 - KO MEF cells infected with S . pneumoniae R6 ΔcbpF / pCbpFR6 - FLAG for 2 h were fixed and stained with DAPI and an anti - FLAG antibody , and representative epifluorescence images are shown . 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

G p62-KO MEF cells infected with S. pneumoniae R6 ΔcbpF/pCbpFR6-FLAG for 2 h were fixed and stained with DAPI and an anti-FLAG antibody, and representative epifluorescence images are shown. Scale bars, 10 µm.


A Quantification of the band intensities following streptavidin - based pulldown assays using the in vitro - translated biotinylated CbpCT4 and FLAG - tagged autophagy - related proteins shown in Fig EV2A .

O O O O O O O O O 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 Quantification of the band intensities following streptavidin-based pulldown assays using the in vitro-translated biotinylated CbpCT4 and FLAG-tagged autophagy-related proteins shown in Fig EV2A.


B Quantification of the band intensities following GST - pulldown assays with 293T cell lysates expressing the autophagy - related proteins and recombinant GST - CbpFR6 shown in Fig EV2D .

O O O O O O O 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 Quantification of the band intensities following GST-pulldown assays with 293T cell lysates expressing the autophagy-related proteins and recombinant GST-CbpFR6 shown in Fig EV2D.


C Lysates from 293T cells transiently expressing 3Myc - Atg14 or Atg5 - 3Myc , and GFP - CbpCT4 or GFP were immunoprecipitated with GFP - Trap , and the bound proteins were analyzed by immunoblotting .

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

C Lysates from 293T cells transiently expressing 3Myc-Atg14 or Atg5-3Myc, and GFP-CbpCT4 or GFP were immunoprecipitated with GFP-Trap, and the bound proteins were analyzed by immunoblotting.


D Lysates from 293T cells transiently expressing GFP - CbpCT4 or GFP , and 6Myc - Atg14 were immunoprecipitated with an anti - Myc antibody and Protein G PLUS - Agarose . The bound proteins were analyzed by immunoblotting .

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

D Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP, and 6Myc-Atg14 were immunoprecipitated with an anti-Myc antibody and Protein G PLUS-Agarose. The bound proteins were analyzed by immunoblotting.


E Recombinant GST - CbpFR6 or GST , and 3Myc - Atg14 were used for GST - pulldown assays . The bound proteins were analyzed by immunoblotting using an anti - Myc antibody .

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

E Recombinant GST-CbpFR6 or GST, and 3Myc-Atg14 were used for GST-pulldown assays. The bound proteins were analyzed by immunoblotting using an anti-Myc antibody.


F MEFs infected with R6 ΔcbpF expressing CbpFR6 - FLAG for 2 h were subjected to IP and assayed using anti - HA agarose beads . The bound proteins were analyzed by immunoblotting using an anti - FLAG antibody .

F MEFs infected with R6 ΔcbpF expressing CbpFR6-FLAG for 2 h were subjected to IP and assayed using anti-HA agarose beads. The bound proteins were analyzed by immunoblotting using an anti-FLAG antibody.


A Lysates from MEFs stably expressing HA - Atg14 infected with S . pneumoniae TIGR4 WT or ΔcbpC for 1 , 2 , or 3 h in the presence or absence of cycloheximide were subjected to SDS - PAGE and analyzed by immunoblotting with the indicated antibodies .

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

A Lysates from MEFs stably expressing HA-Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


B MEFs / HA - Atg14 cells were infected with S . pneumoniae TIGR4 WT or ΔcbpC for 1 , 2 , or 3 h in the presence or absence of Bafilomycin A1 ( BafA1 ) . The lysates were subjected to SDS - PAGE and analyzed by immunoblotting with the indicated antibodies .

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B MEFs/HA-Atg14 cells were infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


( C A549 cells were infected with S . pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti - Atg14 Representative epifluorescence images are shown . Scale bars , 10 µm . The percentages of perinuclear - localizing Atg14 containing cells were quantified .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti-Atg14 Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of perinuclear-localizing Atg14 containing cells were quantified.


A549 cells were infected with S . pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and - GM130 antibody . Representative epifluorescence images are shown . Scale bars , 10 µm . The percentages of intact Golgi apparatus - containing cells were quantified .

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

A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and -GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of intact Golgi apparatus-containing cells were quantified.


G S . pneumoniae ( Sp ) invasion - permissive A549 cells ( marked with GFP ) and Sp invasion - non - permissive ( pIgR knocked down ) A549 cells were co - cultured at a 1 : 3 ratio . Atg14 - disappearance experiments were conducted , and representative epifluorescence images are shown . Scale bars , 10 µm . The lines show each cell shape and the arrows show invasion - permissive A549 cells marked with GFP . H The percentages of perinuclear - localizing Atg14 containing cells in ( G ) were quantified .

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G S. pneumoniae (Sp) invasion-permissive A549 cells (marked with GFP) and Sp invasion-non-permissive (pIgR knocked down) A549 cells were co-cultured at a 1:3 ratio. Atg14-disappearance experiments were conducted, and representative epifluorescence images are shown. Scale bars, 10 µm. The lines show each cell shape and the arrows show invasion-permissive A549 cells marked with GFP. H The percentages of perinuclear-localizing Atg14 containing cells in (G) were quantified.


I Lysates from 293T cells transiently expressing GFP - CbpC or GFP , and HA - Atg14 were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against HA or GFP .

I Lysates from 293T cells transiently expressing GFP-CbpC or GFP, and HA-Atg14 were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against HA or GFP.


J Lysates from 293T cells transiently expressing GFP - CbpCT4 or GFP were immunoprecipitated using GST - GFP - Nanobody . Additionally , beads were mixed with lysates from 293T cells transiently expressing p62 - 3Myc and HA - Atg14 , and bound proteins were analyzed by immunoblotting .

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

J Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP were immunoprecipitated using GST-GFP-Nanobody. Additionally, beads were mixed with lysates from 293T cells transiently expressing p62-3Myc and HA-Atg14, and bound proteins were analyzed by immunoblotting.


K A549 cells treated with the indicated siRNAs were infected with S . pneumoniae R6 WT or ΔcbpF for the indicated durations . The cells were fixed and stained with DAPI and an anti - Atg14 antibody , and percentages of perinuclear - localizing Atg14 containing cells were quantified .

K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti-Atg14 antibody, and percentages of perinuclear-localizing Atg14 containing cells were quantified.


L Lysates from MEFs stably expressing GFP - CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS - PAGE and immunoblotted using antibodies against LC3 or p62 .

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

L Lysates from MEFs stably expressing GFP-CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS-PAGE and immunoblotted using antibodies against LC3 or p62.


M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag - Stx17 and Nanoluc - Atg14 in the presence or absence of GFP - CbpC or GFP .

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M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag-Stx17 and Nanoluc-Atg14 in the presence or absence of GFP-CbpC or GFP.


B GST - pulldown assays performed using GST - CbpFR6 or GST and lysates from 293T cells expressing GFP - Atg14 derivatives or GFP . The bound proteins were analyzed by immunoblotting using an anti - GFP antibody .

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

B GST-pulldown assays performed using GST-CbpFR6 or GST and lysates from 293T cells expressing GFP-Atg14 derivatives or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


D , E GST - pulldown assays using GST - CbpFR6 derivatives and lysates from 293T cells expressing GFP - Atg14 CCD or GFP . The bound proteins were analyzed by immunoblotting using an anti - GFP antibody . Each GST - CbpFR6 derivative - bound bead was confirmed by Coomassie brilliant blue ( CBB ) staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E GST-pulldown assays using GST-CbpFR6 derivatives and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 derivative-bound bead was confirmed by Coomassie brilliant blue (CBB) staining.


H GST - pulldown assays performed using GST - CbpFR6 mutants or GST and lysates from 293T cells expressing GFP - Atg14 CCD or GFP . The bound proteins were analyzed by immunoblotting using an anti - GFP antibody . Each GST - CbpFR6 mutant - bound bead was confirmed by CBB staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GST-pulldown assays performed using GST-CbpFR6 mutants or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 mutant-bound bead was confirmed by CBB staining.


I Lysates of 293T cells transiently expressing GFP - CbpCT4 mutants or GFP were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

I Lysates of 293T cells transiently expressing GFP-CbpCT4 mutants or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP - CbpCT4 mutants . Scale bars , 10 µm .

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J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP-CbpCT4 mutants. Scale bars, 10 µm.


B Lysates from 293T cells transiently expressing p62 - 3Myc and GFP - CbpCT4 variants were immunoprecipitated using a GST - GFP - Nanobody fusion protein . Bound proteins were analyzed by immunoblotting .

B Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using a GST-GFP-Nanobody fusion protein. Bound proteins were analyzed by immunoblotting.


E Lysates from 293T cells transiently expressing p62 - 3Myc and GFP - CbpCT4 variants were immunoprecipitated using the GST - GFP - Nanobody protein . Bound proteins were analyzed by immunoblotting .

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

E Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting.


G Lysates of 293T cells transiently expressing GFP - CbpCT4 and p62 - 3Myc variants were immunoprecipitated with the GST - GFP - Nanobody protein . Bound proteins were analyzed by immunoblotting using the indicated antibodies .

G Lysates of 293T cells transiently expressing GFP-CbpCT4 and p62-3Myc variants were immunoprecipitated with the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting using the indicated antibodies.


H A549 cells infected with the indicated S . pneumoniae strains for 2 h were fixed and stained with DAPI and an anti - Atg14 antibody , and the percentages of perinuclear - localizing Atg14 containing cells were quantified .

H A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti-Atg14 antibody, and the percentages of perinuclear-localizing Atg14 containing cells were quantified.


I Representative epifluorescence images in ( H ) are shown . Scale bars , 10 µm .

I Representative epifluorescence images in (H) are shown. Scale bars, 10 µm.


D GST - pulldown assays using the indicated GST - fusion proteins or GST and lysates from 293T cells expressing GFP - Atg14 CCD or GFP . Bound proteins were analyzed by immunoblotting using an anti - GFP antibody .

D GST-pulldown assays using the indicated GST-fusion proteins or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


E Lysates from 293T cells expressing GFP - fusion proteins or GFP were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

O O O O O O O O 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 Lysates from 293T cells expressing GFP-fusion proteins or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


F Confocal images of HeLa cells transiently expressing GFP - fusion proteins . Scale bars , 10 µm .

F Confocal images of HeLa cells transiently expressing GFP-fusion proteins. Scale bars, 10 µm.


H , I GST - pulldown assays using the indicated GST - fused proteins or GST and lysates from 293T expressing GFP - Atg14 CCD or GFP . Bound proteins were analyzed by immunoblotting using an anti - GFP antibody .

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

H, I GST-pulldown assays using the indicated GST-fused proteins or GST and lysates from 293T expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


( B ) Volcano plot of 8 , 701 mRNA isoforms from 2 , 931 genes with multiple isoforms . X - axis indicates a difference in percentage of each mRNA isoform within the gene ( Δ % smg - 2 - N2 ) . Y - axis indicates - log10 of p - value in Fisher ' s exact tests . Blue symbols indicate 219 mRNA isoforms significantly depleted from the smg - 2 mutant ( p < 0 . 05 , Δ % smg - 2 - N2 < - 10 ) . Red symbols indicate 420 isoforms significantly enriched in the smg - 2 mutant ( p < 0 . 05 , Δ % smg - 2 - N2 > 10 ) .

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(B) Volcano plot of 8,701 mRNA isoforms from 2,931 genes with multiple isoforms. X-axis indicates a difference in percentage of each mRNA isoform within the gene (Δ%smg-2-N2). Y-axis indicates -log10 of p-value in Fisher's exact tests. Blue symbols indicate 219 mRNA isoforms significantly depleted from the smg-2 mutant (p<0.05, Δ%smg-2-N2<-10). Red symbols indicate 420 isoforms significantly enriched in the smg-2 mutant (p<0.05, Δ%smg-2-N2>10).


( C , D ) Length distribution of 3 ' UTRs ( C ) and proportions of putative PTC - containing isoforms ( D ) in 10 , 136 mRNA isoforms detected in N2 and / or smg - 2 ( All ) , the 219 isoforms depleted from and the 420 isoforms enriched in the smg - 2 mutant .

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

(C, D) Length distribution of 3'UTRs (C) and proportions of putative PTC-containing isoforms (D) in 10,136 mRNA isoforms detected in N2 and/or smg-2 (All), the 219 isoforms depleted from and the 420 isoforms enriched in the smg-2 mutant.


( B ) Alternative splicing of sams - 3 , sams - 4 and sams - 5 in unfed and fed worms without or with a protein synthesis inhibitor emetine . Synchronized L1 larvae of an NMD - deficient strain KH1668 : smg - 2 ( yb979 ) ( lanes 1 - 3 ) and a wild - type strain N2 ( lanes 4 - 6 ) were incubated in S - complete medium alone ( lanes 1 , 4 ) , with a standard E . coli strain OP50 ( OD600 = 10 . 0 ) ( lanes 2 , 5 ) or OP50 supplemented with 10 mg / ml emetine ( lanes 3 , 6 ) for 3 hours at 20 ° C . Total RNAs were extracted from whole animals and subjected to semi - quantitative RT - PCR , whose products were analyzed by capillary electrophoresis . Representative gel - like presentation is indicated ( n = 3 ) . Schematic structure of each PCR product is indicated on the right . Open reading frames ( ORFs ) for full - length and truncated proteins are in orange and cyan , respectively . rpl - 12 was used as an unaffected 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 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Alternative splicing of sams-3, sams-4 and sams-5 in unfed and fed worms without or with a protein synthesis inhibitor emetine. Synchronized L1 larvae of an NMD-deficient strain KH1668: smg-2 (yb979) (lanes 1-3) and a wild-type strain N2 (lanes 4-6) were incubated in S-complete medium alone (lanes 1, 4), with a standard E. coli strain OP50 (OD600=10.0) (lanes 2, 5) or OP50 supplemented with 10 mg/ml emetine (lanes 3, 6) for 3 hours at 20°C. Total RNAs were extracted from whole animals and subjected to semi-quantitative RT-PCR, whose products were analyzed by capillary electrophoresis. Representative gel-like presentation is indicated (n=3). Schematic structure of each PCR product is indicated on the right. Open reading frames (ORFs) for full-length and truncated proteins are in orange and cyan, respectively. rpl-12 was used as an unaffected control.


( A ) Alternative splicing of sams - 3 , sams - 4 and sams - 5 in smg - 2 ( yb979 ) and smg - 2 ( yb979 ) ; sams - 5 ( gk147 ) ; sams - 1 ( ok2946 ) mutants . Synchronized L1 larvae of each strain were incubated in S - complete medium alone ( lanes 1 and 5 ) , with OP50 ( OD600 = 10 . 0 ) ( lanes 2 and 6 ) and with OP50 supplemented with 25 mM L - Met ( lanes 3 and 7 ) or 25 mM cycloleucine ( cLeu ) ( lanes 4 and 8 ) for 3 hours at 20 ° C . The splicing patterns were analyzed and presented as in Figure 2B ( n = 3 ) .

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

(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3).


( B , C ) Western blot analysis of SAMS - 1 , SAMS - 3 and SAMS - 4 during larval development in the smg - 2 ( yb979 ) ( B ) and wild - type ( C ) backgrounds . Genotypes of the worms are smg - 2 ( w ) and smg - 2 ; sams - 5 ; sams - 1 ( s ) in ( B ) and wild - type ( w ) and sams - 1 ( s ) in ( C ) . Synchronized L1 larvae of each strain were incubated with OP50 at 20 ° C and subjected to Western blot analysis at indicated time points . Anti - β - tubulin ( B ) or coomassie brilliant blue ( CBB ) staining ( C ) was used as a loading control . Specificity of the antibodies is confirmed in Appendix Figure S12 . Note that upregulation of SAMS - 1 protein in the wild type during larval development in ( C ) is consistent with feeding - induced upregulation of sams - 1 mRNA in Figure EV3D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C) Western blot analysis of SAMS-1, SAMS-3 and SAMS-4 during larval development in the smg-2 (yb979) (B) and wild-type (C) backgrounds. Genotypes of the worms are smg-2 (w) and smg-2; sams-5; sams-1 (s) in (B) and wild-type (w) and sams-1 (s) in (C). Synchronized L1 larvae of each strain were incubated with OP50 at 20°C and subjected to Western blot analysis at indicated time points. Anti-β-tubulin (B) or coomassie brilliant blue (CBB) staining (C) was used as a loading control. Specificity of the antibodies is confirmed in Appendix Figure S12. Note that upregulation of SAMS-1 protein in the wild type during larval development in (C) is consistent with feeding-induced upregulation of sams-1 mRNA in Figure EV3D.


( C ) Alternative splicing of sams - 3 , sams - 4 and sams - 5 in smg - 2 ( yb979 ) and smg - 2 ( yb979 ) ; mett - 10 ( ok2204 ) mutants . Synchronized L1 larvae of each strain were incubated in S - complete medium alone ( lanes 1 and 3 ) or with OP50 ( lanes 2 and 4 ) for 3 hours at 20 ° C and the splicing patterns were analyzed and presented as in Figure 2B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); mett-10 (ok2204) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 3) or with OP50 (lanes 2 and 4) for 3 hours at 20°C and the splicing patterns were analyzed and presented as in Figure 2B.


( A ) Mass chromatograms of RNA fragments to detect m6A ( top ) or unmodified A ( bottom ) after in vitro incubation of 127 - nt sams - 3 / sams - 4 pre - mRNA ( sequence available in Fig EV2 ) with recombinant full - length METT - 10 protein in the presence ( left ) or absence ( right ) of 1 mM SAM . The sequence , m / z value , and charge state for each fragment are indicated on the right . Asterisks indicate non - specific signals .

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 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

(A) Mass chromatograms of RNA fragments to detect m6A (top) or unmodified A (bottom) after in vitro incubation of 127-nt sams-3/sams-4 pre-mRNA (sequence available in Fig EV2) with recombinant full-length METT-10 protein in the presence (left) or absence (right) of 1 mM SAM. The sequence, m/z value, and charge state for each fragment are indicated on the right. Asterisks indicate non-specific signals.


( B ) The negatively - charged ions of RNase T1 fragment is decomposed in the instrument by collision - induced dissociation ( CID ) using helium gas . The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel . Nomenclature of the product ions are described in the literature ( McLuckey et al , 1992 ) . Product ions of c and y series derive from the 5 ' and 3 ' termini of the fragment , respectively .

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(B) The negatively-charged ions of RNase T1 fragment is decomposed in the instrument by collision-induced dissociation (CID) using helium gas. The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel. Nomenclature of the product ions are described in the literature (McLuckey et al, 1992). Product ions of c and y series derive from the 5' and 3' termini of the fragment, respectively.


A , B ) m6A - IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal / productive 3 ' SS . ( A ) Representative gel - like images ( n = 3 ) of semi - quantitative RT - PCR analysis of sams - 3 , sams - 4 and sams - 5 in input and immunoprecipitated ( IP ) RNAs from the smg - 2 ( yb979 ) mutant . The splicing patterns were analyzed and presented as in Figure 2B . ( B ) Relative enrichment of total and each of mRNA isoforms compared to eef - 1A . 1 mRNA by RT - qPCR . Error bars indicate standard error of mean ( n = 3 ) . (

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

A, B) m6A-IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal/productive 3'SS. (A) Representative gel-like images (n=3) of semi-quantitative RT-PCR analysis of sams-3, sams-4 and sams-5 in input and immunoprecipitated (IP) RNAs from the smg-2 (yb979) mutant. The splicing patterns were analyzed and presented as in Figure 2B. (B) Relative enrichment of total and each of mRNA isoforms compared to eef-1A.1 mRNA by RT-qPCR. Error bars indicate standard error of mean (n=3). (


C ) Examples of normalized Nanopore currents for unmodified and m6A - modified in - vitro transcribed sams - 3 / sams - 4 RNAs at nucleotide positions - 4 through + 4 relevant to the m6A site . One hundred reads are lotted for each . Color codes are indicated . ( D ) Distribution of mean ( top ) and standard deviation ( bottom ) of normalized Nanopore currents at nucleotide positions - 4 through + 4 relevant to the m6A site for 5 , 000 of the unmodified and 2 , 680 of the m6A - modified sams - 3 / sams - 4 RNAs and 85 of an endogenous sams - 3 mRNA isoform E2 / E3L . Color codes are indicated . Central bands represent the median , boxes represent the 25th and 75th percentiles , and whiskers represent the lowest and highest values . A red line at position 0 in the top panel indicates a cut - off line ( mean current = 1 . 7557 ) that discriminates between the unmodified and m6A - modified sams - 3 / sams - 4 RNAs with accuracies of 64 . 58 % for both . (

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

C) Examples of normalized Nanopore currents for unmodified and m6A-modified in-vitro transcribed sams-3/sams-4 RNAs at nucleotide positions -4 through +4 relevant to the m6A site. One hundred reads are lotted for each. Color codes are indicated. (D) Distribution of mean (top) and standard deviation (bottom) of normalized Nanopore currents at nucleotide positions -4 through +4 relevant to the m6A site for 5,000 of the unmodified and 2,680 of the m6A-modified sams-3/sams-4 RNAs and 85 of an endogenous sams-3 mRNA isoform E2/E3L. Color codes are indicated. Central bands represent the median, boxes represent the 25th and 75th percentiles, and whiskers represent the lowest and highest values. A red line at position 0 in the top panel indicates a cut-off line (mean current = 1.7557) that discriminates between the unmodified and m6A-modified sams-3/sams-4 RNAs with accuracies of 64.58% for both. (


( A - C ) Immunoblots for indicated proteins in BAT , extensor digitorum longus ( EDL ) , epididymal white adipose tissue ( eWAT ) and heart from 10 ‐ to 12 ‐ month ( mo ) ‐ old control ( Con ) and knock out ( KO ) mice . Arrows depict LC3 ‐ I and II .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Immunoblots for indicated proteins in BAT, extensor digitorum longus (EDL), epididymal white adipose tissue (eWAT) and heart from 10‐ to 12‐month (mo)‐old control (Con) and knock out (KO) mice. Arrows depict LC3‐I and II.


( D ) ATG5 and ATG7 mRNA levels in indicated tissues ( n = 4 )

(D) ATG5 and ATG7 mRNA levels in indicated tissues (n=4)


( E ) immunoblots for indicated proteins in spleen , liver , lung , kidney , mediobasal hypothalamus ( MBH ) , inguinal white adipose tissue ( ingWAT ) and perinephric fat ( pnWAT ) from 10 ‐ to 12 ‐ mo ‐ old Con and KO mice .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) immunoblots for indicated proteins in spleen, liver, lung, kidney, mediobasal hypothalamus (MBH), inguinal white adipose tissue (ingWAT) and perinephric fat (pnWAT) from 10‐ to 12‐mo‐old Con and KO mice.


( F ) Body weights ( wt ) of chow diet ( RD ) ‐ fed ( n = 6 - 29 ) , and ( G ) high ‐ fat diet ( HFD ) ‐ fed Con and KO mice at indicated ages ( n = 4 - 17 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body weights (wt) of chow diet (RD)‐fed (n=6-29), and (G) high‐fat diet (HFD)‐fed Con and KO mice at indicated ages (n=4-17).


( H ) Total body fat and lean mass of 4 - 7 mo RD ‐ fed Con and KO mice ( n = 8 - 12 ) .

O O O O O 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) Total body fat and lean mass of 4-7 mo RD‐fed Con and KO mice (n=8-12).


( I ) BAT wt ( n = 4 - 7 ) , ( J ) gastrocnemius ( GA ) wt ( n = 5 - 7 ) , ( K ) soleus wt ( n = 4 - 7 ) , ( L ) eWAT wt ( n = 5 - 7 ) and ( M ) visceral ( Visc ) and subcutaneous ( Subc ) body fat distribution in 10 ‐ mo ‐ old Con and KO mice ( n = 4 ) . Values are mean ± s . e . , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

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

(I) BAT wt (n=4-7), (J) gastrocnemius (GA) wt (n=5-7), (K) soleus wt (n=4-7), (L) eWAT wt (n=5-7) and (M) visceral (Visc) and subcutaneous (Subc) body fat distribution in 10‐mo‐old Con and KO mice (n=4). Values are mean±s.e., *P0.05, **P0.01, ***P0.001.


( A - B ) mRNA for indicated genes in BAT ( n = 4 )

(A-B) mRNA for indicated genes in BAT (n=4)


( c ) mRNA for the indicated genes in epididymal white adipose tissue ( eWAT ) ( n = 4 )

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

(c) mRNA for the indicated genes in epididymal white adipose tissue (eWAT) (n=4)


( D ) immunoblots for indicated proteins in BAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice . Arrows depict protein isoforms .

O O O O O O O O O O O O O O O O O O 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) immunoblots for indicated proteins in BAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( E ) immunoblots for indicated proteins in eWAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice . Arrows depict protein isoforms .

(E) immunoblots for indicated proteins in eWAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( F ) Electron micrographs ( × 10 , 000 magnification ) of BAT depicting mitochondria from 4 ‐ mo ‐ old Con and KO mice . m , mitochondria ; LD , lipid droplet ; n , nucleus .

O O O O O O O O O O O O O O O O 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 ( × 10,000 magnification) of BAT depicting mitochondria from 4‐mo‐old Con and KO mice. m, mitochondria; LD, lipid droplet; n, nucleus.


( G ) mRNA levels ( n = 4 ) in BAT from 10 ‐ mo ‐ old Con and KO

(G) mRNA levels (n=4) in BAT from 10‐mo‐old Con and KO mice


( H ) hematoxylin and eosin ( HE ) and ( I ) Sirius Red stains in BAT from 10 ‐ mo ‐ old Con and KO mice ( n = 3 - 4 ) . Average adipocyte and LD size , and LD number in BAT are shown . Scale bar , 50 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

(H) hematoxylin and eosin (HE) and (I) Sirius Red stains in BAT from 10‐mo‐old Con and KO mice (n=3-4). Average adipocyte and LD size, and LD number in BAT are shown. Scale bar, 50 μm.


( J ) BAT mRNA levels from 4 ‐ mo ‐ old RD ‐ fed Con and KO mice cold ‐ challenged for 75 min ( n = 3 ) . Values are mean ± s . e . * * * P0 . 001 .

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

(J) BAT mRNA levels from 4‐mo‐old RD‐fed Con and KO mice cold‐challenged for 75 min (n=3). Values are mean±s.e. ***P0.001.


( A ) Body temperature before ( Basal ) and during cold exposure ( n = 7 - 8 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO )

(A) Body temperature before (Basal) and during cold exposure (n=7-8) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice


( B ) Oxygen consumption ( VO2 ) , ( C ) carbon dioxide production ( VCO2 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice ( n = 4 ) .

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

(B) Oxygen consumption (VO2), (C) carbon dioxide production (VCO2) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4).


( D ) energy expenditure in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice ( n = 4 )

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

(D) energy expenditure in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4)


( E ) VO2 , ( F ) VCO2 and ( G ) energy expenditure in 10 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con and KO mice ( n = 3 - 5 ) .

(E) VO2, (F) VCO2 and (G) energy expenditure in 10‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=3-5).


( H ) Ambulation ( AMB ) in 10 ‐ mo ‐ old RD ‐ fed ( n = 4 ) and HFD ‐ fed Con and KO mice ( n = 3 - 5 ) .

(H) Ambulation (AMB) in 10‐mo‐old RD‐fed (n=4) and HFD‐fed Con and KO mice (n=3-5).


( I ) Hematoxylin and eosin ( HE ) ‐ stained epididymal white adipose tissue ( eWAT ) from 6 ‐ mo ‐ old RD ‐ fed Con and KO mice . Quantification for cell size is shown ( n = 3 ) . Scale bar , 50 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

(I) Hematoxylin and eosin (HE)‐stained epididymal white adipose tissue (eWAT) from 6‐mo‐old RD‐fed Con and KO mice. Quantification for cell size is shown (n=3). Scale bar, 50 μm.


( J ) Adβ3 mRNA in eWAT from 10 ‐ mo ‐ old RD ‐ fed Con and KO mice ( n = 4 ) , and in ( K ) inguinal ( ingWAT ) from 4 ‐ mo ‐ oldcold ‐ challenged RD ‐ fed Con and KO mice ( n = 3 ) .

(J) Adβ3 mRNA in eWAT from 10‐mo‐old RD‐fed Con and KO mice (n=4), and in (K) inguinal (ingWAT) from 4‐mo‐oldcold‐challenged RD‐fed Con and KO mice (n=3).


( L ) Serum ‐ free fatty acid ( FFA ) and glycerol ( Gly ) from 4 ‐ to 6 ‐ mo ‐ old Con and KO mice untreated ( basal ) or treated with isoproterenol ( Iso ) i . p . for 20 min ( n = 4 - 6 ) , and ( M ) from 4 ‐ mo ‐ old RD ‐ fed cold ‐ challenged ( 75 min ) mice ( n = 3 - 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

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

(L) Serum‐free fatty acid (FFA) and glycerol (Gly) from 4‐ to 6‐mo‐old Con and KO mice untreated (basal) or treated with isoproterenol (Iso) i.p. for 20 min (n=4-6), and (M) from 4‐mo‐old RD‐fed cold‐challenged (75 min) mice (n=3-4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A , B ) Tmem26 and tbx1 mRNA levels in epididymal eWAT , and ( C , D ) ingWAT from Atg7Flox / Floxmice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses and cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

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

(A,B) Tmem26 and tbx1 mRNA levels in epididymal eWAT, and (C,D) ingWAT from Atg7Flox/Floxmice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( E , F ) hematoxylin and eosin ( HE ) ‐ stained ingWAT section from Atg7Flox / Flox mice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses and cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

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

(E,F) hematoxylin and eosin (HE)‐stained ingWAT section from Atg7Flox/Flox mice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( G ) mRNA for indicated genes in ingWAT from 4 ‐ mo ‐ old cold ‐ exposed Con and KO mice ( n = 4 ) .

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

(G) mRNA for indicated genes in ingWAT from 4‐mo‐old cold‐exposed Con and KO mice (n=4).


( H , I ) mRNA levels in eWAT from 10 ‐ mo ‐ old Con and KO mice ( n = 4 ) .

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

(H,I) mRNA levels in eWAT from 10‐mo‐old Con and KO mice (n=4).


( J ) β ‐ oxidation in eWAT and ingWAT from 4 ‐ mo ‐ old Con and KO mice ( n = 4 ) .

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

(J) β‐oxidation in eWAT and ingWAT from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA levels in soleus from 4 ‐ mo ‐ old cold ‐ exposed Con and KO mice ( n = 3 - 6 ) .

(K) mRNA levels in soleus from 4‐mo‐old cold‐exposed Con and KO mice (n=3-6).


( M ) BAT from 4 ‐ mo ‐ old Con and KO mice ( n = 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

(M) BAT from 4‐mo‐old Con and KO mice (n=4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A ) Blood glucose in 6 ‐ to 10 ‐ month ( mo ) ‐ old mice ( n = 12 - 15 ) , and ( B ) serum insulin levels in 10 ‐ mo ‐ old fed control ( Con ) and KO mice ( n = 6 ) .

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

(A) Blood glucose in 6‐ to 10‐month (mo)‐old mice (n=12-15), and (B) serum insulin levels in 10‐mo‐old fed control (Con) and KO mice (n=6).


( C ) Glucose tolerance tests in 10 ‐ mo ‐ old chow diet ( RD ) ‐ fed ( n = 5 ) , and ( D ) in 10 ‐ to 12 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con and KO mice ( n = 4 - 9 ) .

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

(C) Glucose tolerance tests in 10‐mo‐old chow diet (RD)‐fed (n=5), and (D) in 10‐ to 12‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=4-9).


( E ) Insulin tolerance test in 10 ‐ mo ‐ old RD ‐ fed Con and KO mice ( n = 5 ) .

(E) Insulin tolerance test in 10‐mo‐old RD‐fed Con and KO mice (n=5).


( F ) Immunoblots for indicated proteins in skeletal muscle from 10 ‐ mo ‐ old Con and KO mice .

(F) Immunoblots for indicated proteins in skeletal muscle from 10‐mo‐old Con and KO mice.


( G ) Hematoxylin and eosin ( HE ) ‐ stained gastrocnemius ( GA ) sections from 6 ‐ mo ‐ old Con and KO mice . Myofiber cross ‐ sectional area ( μm2 ) is shown ( n = 3 ) .

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

(G) Hematoxylin and eosin (HE)‐stained gastrocnemius (GA) sections from 6‐mo‐old Con and KO mice. Myofiber cross‐sectional area (μm2) is shown (n=3).


( H ) Expression of MuRF ‐ 1 and Atrogin ‐ 1 in GA and TA from 10 ‐ mo ‐ old Con and KO mice ( n = 4 )

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

(H) Expression of MuRF‐1 and Atrogin‐1 in GA and TA from 10‐mo‐old Con and KO mice (n=4)


( I ) myogenic genes in GA from 10 ‐ mo ‐ old Con and KO mice ( n = 6 ) .

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

(I) myogenic genes in GA from 10‐mo‐old Con and KO mice (n=6).


( J ) Immunoblots for indicated proteins in tissues from 4 ‐ mo ‐ old Con and KO mice ( n = 4 ) .

(J) Immunoblots for indicated proteins in tissues from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA for IRS1 , ( L ) IRS2 in epididymal white adipose tissue ( eWAT ) and GA from 10 ‐ mo ‐ old Con and KO mice ( n = 6 ) .

(K) mRNA for IRS1, (L) IRS2 in epididymal white adipose tissue (eWAT) and GA from 10‐mo‐old Con and KO mice (n=6).


A - F Indirect calorimetry was performed using the Oxymax / CLAMS system in Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 5 ) mice . Whole - body oxygen consumption rate ( VO2 ) ( A - B ) , energy expenditure ( EE ) ( C - D ) and respiratory exchange ratio ( RER ) ( E - F ) were measured at 24 ° C and during a cold challenge at 6 ° C during both the light ( white rectangle ) and dark ( black rectangle ) phases over a 2 - day period .

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

A-F Indirect calorimetry was performed using the Oxymax/CLAMS system in Cdk4+/+ (n=6) and Cdk4-/- (n=5) mice. Whole-body oxygen consumption rate (VO2) (A-B), energy expenditure (EE) (C-D) and respiratory exchange ratio (RER) (E-F) were measured at 24°C and during a cold challenge at 6°C during both the light (white rectangle) and dark (black rectangle) phases over a 2-day period.


CalR was used to GLM - regression plot for each group corresponding to the association between energy balance and total body mass at 24 ° C ( G ) and the association between energy balance and total body mass at 6 ° C ( H ) .

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

CalR was used to GLM-regression plot for each group corresponding to the association between energy balance and total body mass at 24°C (G) and the association between energy balance and total body mass at 6°C (H).


J - K Basal rectal temperature was measured at 24 ° C ( J ) , and rectal temperature was monitored during acute cold exposure at 4 ° C for 6 hours ( K ) in Cdk4 + / + ( n = 10 ) and Cdk4 - / - ( n = 8 ) mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K Basal rectal temperature was measured at 24°C (J), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (K) in Cdk4+/+ (n=10) and Cdk4-/- (n=8) mice.


A Gross morphology of iBAT from Cdk4 + / + and Cdk4 - / - mice .

A Gross morphology of iBAT from Cdk4+/+ and Cdk4-/- mice.


C Hematoxylin - eosin staining and UCP1 immunohistochemical ( IHC ) staining of iBAT sections ( scale bar 100 μm ) from Cdk4 + / + and Cdk4 - / - mice .

O O O O O O O O O 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 Hematoxylin-eosin staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm) from Cdk4+/+ and Cdk4-/- mice.


D - E Western blot analysis of UCP1 and CDK4 protein expression ( D ) and quantified protein levels of UCP1 ( E ) ( n = 4 biological replicates ) in iBAT . HSP90 was used as loading 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

D-E Western blot analysis of UCP1 and CDK4 protein expression (D) and quantified protein levels of UCP1 (E) (n=4 biological replicates) in iBAT. HSP90 was used as loading control.


F Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 6 ) mice as assessed by RT - PCR .

O O O O O O O O O O O O O O O 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 Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4+/+ (n=6) and Cdk4-/- (n=6) mice as assessed by RT-PCR.


G - H Transmission electron microscopy ( TEM ) images of iBAT ( G ) ( scale bar 5 μm ( upper panel ) and 1 μm ( lower panel , LD indicates lipid droplet and M mitochondria ) ) and quantification of lipid droplet number ( LD ) and mitochondrial volume ( Mito ) ( H ) ( Cdk4 + / + ( n = 4 ) and Cdk4 - / - ( n = 4 ) ) .

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

G-H Transmission electron microscopy (TEM) images of iBAT (G) (scale bar 5 μm (upper panel) and 1 μm (lower panel, LD indicates lipid droplet and M mitochondria)) and quantification of lipid droplet number (LD) and mitochondrial volume (Mito) (H) (Cdk4+/+ (n=4) and Cdk4-/- (n=4)).


I Respirometry analysis of iBAT as measured by the Oroboros system ( Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 6 ) ) .

I Respirometry analysis of iBAT as measured by the Oroboros system (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).


A - B Rectal temperature was measured at 24 ° C ( A ) , and rectal temperature was monitored for 6 hours during acute cold exposure at 4 ° C ( B ) in Cdk4flox / flox ( n = 9 ) and Cdk4flox / flox Ucp1 - Cre mice ( n = 9 ) .

A-B Rectal temperature was measured at 24°C (A), and rectal temperature was monitored for 6 hours during acute cold exposure at 4°C (B) in Cdk4flox/flox (n=9) and Cdk4flox/flox Ucp1-Cre mice (n=9).


C Gross morphology of iBAT from Cdk4flox / flox and Cdk4flox / flox Ucp1 - Cre mice D iBAT mass compared to body weight of Cdk4flox / flox ( n = 8 ) and Cdk4flox / flox Ucp1 - Cre ( n = 7 ) mice .

C Gross morphology of iBAT from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice D iBAT mass compared to body weight of Cdk4flox/flox (n=8) and Cdk4flox/flox Ucp1-Cre (n=7) mice.


E Hematoxylin - eosin ( H & E ) staining and UCP1 immunohistochemical ( IHC ) staining of iBAT sections ( scale bar 100 μm ( upper panel ) and 50 μm ( lower panel ) ) from Cdk4flox / flox and Cdk4flox / flox Ucp1 - Cre mice .

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E Hematoxylin-eosin (H&E) staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm (upper panel) and 50 μm (lower panel)) from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice.


F - G Western blot analysis of UCP1 and CDK4 protein expression ( F ) and quantified protein levels of UCP1 ( G ) ( n = 4 biological replicates ) in iBAT . HSP90 was used as a loading 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

F-G Western blot analysis of UCP1 and CDK4 protein expression (F) and quantified protein levels of UCP1(G) (n=4 biological replicates) in iBAT. HSP90 was used as a loading control.


H Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4flox / flox ( n = 5 ) and Cdk4flox / flox Ucp1 - Cre ( n = 6 ) mice as assessed by RT - PCR .

O O O O O O O O O O O O O O O O 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 Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4flox/flox (n=5) and Cdk4flox/flox Ucp1-Cre (n=6) mice as assessed by RT-PCR.


A - B TH immunohistochemical staining in iBAT sections ( A ) ( scale bar 20 μm , arrows indicate TH parenchymal fibers ) and corresponding quantification of the number of TH fibers relative to 100 adipocytes ( B ) ( Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 6 ) ) .

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

A-B TH immunohistochemical staining in iBAT sections (A) (scale bar 20 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (B) (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).


C Hematoxylin - eosin staining in scWAT showing multilocular adipocytes in Cdk4 - / - ( scale bar 50 μm ) .

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C Hematoxylin-eosin staining in scWAT showing multilocular adipocytes in Cdk4-/- (scale bar 50 μm).


D Expression of thermogenic genes and brown adipocyte markers in scWAT of Cdk4 + / + ( n = 5 ) and Cdk4 - / - ( n = 6 ) mice as assessed by RT - PCR .

D Expression of thermogenic genes and brown adipocyte markers in scWAT of Cdk4+/+ (n=5) and Cdk4-/- (n=6) mice as assessed by RT-PCR.


E - F Acute cold test ( 4 ° C ) after treatment with the β - adrenergic antagonist propranolol ( Prop ) in Cdk4 + / + ( n = 7 ) and Cdk4 - / - ( n = 6 ) animals or with vehicle ( NaCl ) ( Cdk4 + / + ( n = 7 ) and Cdk4 - / - ( n = 6 ) ) ( E ) and corresponding quantification of the area under the curve ( AUC ) .

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E-F Acute cold test (4°C) after treatment with the β-adrenergic antagonist propranolol (Prop) in Cdk4+/+ (n=7) and Cdk4-/- (n=6) animals or with vehicle (NaCl) (Cdk4+/+ (n=7) and Cdk4-/- (n=6)) (E) and corresponding quantification of the area under the curve (AUC).


A Cdk4 mRNA expression in the dissected VMH of Cdk4flox / flox ( n = 5 ) and Cdk4flox / flox Sf1 - Cre ( n = 6 )

O O O O O 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 Cdk4 mRNA expression in the dissected VMH of Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre (n=6) mice


B - C Rectal temperature was measured at 24 ° C ( B ) , and rectal temperature was monitored during acute cold exposure at 4 ° C for 6 hours ( C ) in Cdk4flox / flox ( n = 15 ) and Cdk4flox / flox Sf1 - Cre mice ( n = 15 ) .

B-C Rectal temperature was measured at 24°C (B), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (C) in Cdk4flox/flox (n=15) and Cdk4flox/flox Sf1-Cre mice (n=15).


D Hematoxylin - eosin ( H & E ) staining of iBAT sections ( scale bar 100 μm ) from Cdk4flox / flox and Cdk4flox / flox Sf1 - Cre mice .

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D Hematoxylin-eosin (H&E) staining of iBAT sections (scale bar 100 μm) from Cdk4flox/flox and Cdk4flox/flox Sf1-Cre mice.


E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox / flox ( n = 9 ) and Cdk4flox / flox Sf1 - Cre mice ( n = 9 ) mice as assessed by RT - PCR .

E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox/flox (n=9) and Cdk4flox/flox Sf1-Cre mice (n=9) mice as assessed by RT-PCR.


F - G TH immunohistochemical ( IHC ) staining in iBAT sections ( F ) ( scale bar 40 μm , arrows indicate TH parenchymal fibers ) and corresponding quantification of the number of TH fibers relative to 100 adipocytes ( G ) ( Cdk4flox / flox ( n = 5 ) and Cdk4flox / flox Sf1 - Cre mice ( n = 5 ) ) .

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F-G TH immunohistochemical (IHC) staining in iBAT sections (F) (scale bar 40 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (G) (Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre mice (n=5)).


H - I Western blot analysis ( H ) and quantification of p - CREBS133 protein expression following 6 hours of cold exposure ( 4 ° C ) ( I ) ( n = 3 biological replicates ) . HSP90 was used as a loading control .

H-I Western blot analysis (H) and quantification of p-CREBS133 protein expression following 6 hours of cold exposure (4°C) (I) (n=3 biological replicates). HSP90 was used as a loading control.


A - D Confocal illustrations of frontal hypothalamic sections from Cdk4 + / + mice ( A , C , N = 3 ) and Cdk4 - / - mice ( B , D , N = 3 ) . Low magnification showing nuclei ( Hoechst , left pannel ) and c - Fos activated - cells ( white , right pannel ) in the mPOA ( A1 - 2 , B1 - 2 ) and the ARC , the VMH , the DMH , the LH ( C1 - 2 , D1 - 2 ) . High magnifications corresponding to the frame area in A1 - 2 , B1 - 2 , C2 , D2 and showing c - Fos - IR cells ( A3 - 4 , B3 - 4 , C3 - 6 , D3 - 6 ) . Scale bars ( A1 - 2 , B1 - 2 , C1 - 2 , D1 - 2 ) : 200μm , ( A3 - 4 , B3 - 4 , C3 - 4 , D3 - 4 ) : 50μm . E Quantification of c - Fos IR cells in brain sections of Cdk4 + / + and Cdk4 - / - mice ( n = 15 - 24 slices according hypothalamic nucleus and genotype ) .

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A-D Confocal illustrations of frontal hypothalamic sections from Cdk4+/+ mice (A, C, N=3) and Cdk4-/- mice (B, D, N=3). Low magnification showing nuclei (Hoechst, left pannel) and c-Fos activated-cells (white, right pannel) in the mPOA (A1-2, B1-2) and the ARC, the VMH, the DMH, the LH (C1-2, D1-2). High magnifications corresponding to the frame area in A1-2, B1-2, C2, D2 and showing c-Fos-IR cells (A3-4, B3-4, C3-6, D3-6). Scale bars (A1-2, B1-2, C1-2, D1-2): 200μm, (A3-4, B3-4, C3-4, D3-4): 50μm. E Quantification of c-Fos IR cells in brain sections of Cdk4+/+ and Cdk4-/- mice (n=15-24 slices according hypothalamic nucleus and genotype).


( A ) Survival curves of C57BL / 6J naïve and adrenalectomized ( Adx ) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged , i . p . , with 30 µg ( naïve ) or 2 µg ( Adx ) TNF , solved in sterile PBS , per 20 g body weight . No deaths occurred later than 72 h after TNF injection . Results represent combined data of 3 experiments .

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(A) Survival curves of C57BL/6J naïve and adrenalectomized (Adx) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 30 µg (naïve) or 2 µg (Adx) TNF, solved in sterile PBS, per 20 g body weight. No deaths occurred later than 72 h after TNF injection. Results represent combined data of 3 experiments.


( B ) Corticosterone levels in the serum of C57BL / 6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water ( N = 6 per group ) .

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(B) Corticosterone levels in the serum of C57BL/6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water (N=6 per group).


( C ) Survival curves of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged , i . p . , with 50 µg ( GRWT ) or 12 . 5 µg ( GRDim ) TNF , solved in sterile PBS , per 20 g bodyweight . No deaths occurred later than 50 h after TNF injection . Results represent combined data of 2 experiments .

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(C) Survival curves of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 50 µg (GRWT) or 12.5 µg (GRDim) TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 50 h after TNF injection. Results represent combined data of 2 experiments.


Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of C57BL / 6J naïve and Adx mice ( D , N = 3 - 6 ) ,

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

Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of C57BL/6J naïve and Adx mice (D, N=3-6),


Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRWT and GRDim mice ( E , N = 6 - 9 ) .

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

Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRWT and GRDim mice (E, N=6-9).


( F ) Agtpbp1 gene - expression measured with RT - qPCR in the ileum of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water ( N = 3 per group ) .

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(F) Agtpbp1 gene-expression measured with RT-qPCR in the ileum of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water (N=3 per group).


( G ) Survival curves of GRfl / fl and GRVillKO mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged , i . p . , with 35 µg TNF , solved in sterile PBS , per 20 g bodyweight . No deaths occurred later than 150 h after TNF injection . Results represent combined data of 6 experiments .

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(G) Survival curves of GRfl/fl and GRVillKO mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 35 µg TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 150 h after TNF injection. Results represent combined data of 6 experiments.


( H ) Mouse Nr3c1 gene - expression measured with RT - qPCR in the intestinal epithelial cells ( IECs ) of GRfl / fl and GRVillKO mice ( N = 3 - 6 ) . Hprt and Villin were used as house - keeping genes .

O O O O O O O O O O O O O O O O O O O O O O O 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) Mouse Nr3c1 gene-expression measured with RT-qPCR in the intestinal epithelial cells (IECs) of GRfl/fl and GRVillKO mice (N=3-6). Hprt and Villin were used as house-keeping genes.


( I ) Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRfl / fl and GRVillKO mice ( N = 9 per group ) .

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(I) Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRfl/fl and GRVillKO mice (N=9 per group).


( D ) Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT and GRDim mice , as measured by RNA - seq in ileum samples . All genes , induced in ileum by DEX in GRWT with LFC > 0 . 8 , p < 0 . 05 are considered , while these genes with p < 0 . 05 in GRDim were considered . The slope of the correlation curve as well as Pearson correlation coefficient were calculated by Graphpad Prism .

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(D) Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT and GRDim mice, as measured by RNA-seq in ileum samples. All genes, induced in ileum by DEX in GRWT with LFC>0.8, p<0.05 are considered, while these genes with p<0.05 in GRDim were considered. The slope of the correlation curve as well as Pearson correlation coefficient were calculated by Graphpad Prism.


( E ) C57BL / 6J mice received antibiotics ( AB ) in the drinking water for 3 weeks , followed by 1 week 25 mM ZnSO4 in the drinking water . During this week AB administration was continued by oral gavage . Mice were challenged i . v . with 12 . 5 µg TNF solved in sterile PBS and survival recorded . Combined data of 2 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 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

(E) C57BL/6J mice received antibiotics (AB) in the drinking water for 3 weeks, followed by 1 week 25 mM ZnSO4 in the drinking water. During this week AB administration was continued by oral gavage. Mice were challenged i.v. with 12.5 µg TNF solved in sterile PBS and survival recorded. Combined data of 2 experiments.


( F ) Zinc levels measured in serum after the antibiotics and ZnSO4 protocol ( N = 5 - 8 ) .

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

(F) Zinc levels measured in serum after the antibiotics and ZnSO4 protocol (N=5-8).


Mice housed in germ - free ( GF ) or specific pathogen free ( SPF ) conditions received 25 mM ZnSO4 in the drinking water for 1 week and were then challenged i . p . with 35 µg TNF , dissolved in sterile PBS , per 20 g bodyweight . Combined data of 2 experiments .

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

Mice housed in germ-free (GF) or specific pathogen free (SPF) conditions received 25 mM ZnSO4 in the drinking water for 1 week and were then challenged i.p. with 35 µg TNF, dissolved in sterile PBS, per 20 g bodyweight. Combined data of 2 experiments.


( H ) displays the blood zinc levels in SPF and GF mice treated for 1 week with water or ZnSO4 ( N = 3 / 4 per group ) .

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

(H) displays the blood zinc levels in SPF and GF mice treated for 1 week with water or ZnSO4 (N=3/4 per group).


( I ) Ileum Slurry ( IS ) was isolated from C57BL / 6J mice put on 25 mM ZnSO4 or control water for 1 week and 750 µL of this IS was injected i . p . in C57BL / 6J GF mice .

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(I) Ileum Slurry (IS) was isolated from C57BL/6J mice put on 25 mM ZnSO4 or control water for 1 week and 750 µL of this IS was injected i.p. in C57BL/6J GF mice.


Transcriptional downregulation of ISRE / IRF genes in the intestinal epithelium by zinc part 1 . RNA - seq experiment of GRWT and GRDim mice which received normal drinking water or drinking water containing 25mM ZnSO4 for 1 week . Ileum was sampled and RNA - seq performed ( N = 5 - 6 per group ) . Genes downregulated by zinc compared to water control animals ( LFC < 0 . 8 and p < 0 . 05 ) were considered . ( C ) Heat map of a selection of the 48 genes of Fig 3B .

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Transcriptional downregulation of ISRE/IRF genes in the intestinal epithelium by zinc part 1. RNA-seq experiment of GRWT and GRDim mice which received normal drinking water or drinking water containing 25mM ZnSO4 for 1 week. Ileum was sampled and RNA-seq performed (N=5-6 per group). Genes downregulated by zinc compared to water control animals (LFC<0.8 and p<0.05) were considered. (C) Heat map of a selection of the 48 genes of Fig 3B.


( A - B ) Expression levels ( RT - qPCR data ) of Mlkl and Zbp1 , from the collection of 48 , in ileum biopsy samples , showing the reducing effects of zinc in both GRWT and GRDim mice , and the higher expression in GRDim compared to GRWT ( N = 4 - 6 / group ) .

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(A-B) Expression levels (RT-qPCR data) of Mlkl and Zbp1, from the collection of 48, in ileum biopsy samples, showing the reducing effects of zinc in both GRWT and GRDim mice, and the higher expression in GRDim compared to GRWT (N=4-6/group).


RT - qPCR data of Mlkl , Zbp1 in ileum biopsy samples of C57BL / 6J mice or different TLR full knockout mice . ( N = 4 / 23 per group )

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RT-qPCR data of Mlkl, Zbp1 in ileum biopsy samples of C57BL/6J mice or different TLR full knockout mice. (N=4/23 per group)


RT - qPCR data of Ripk3 , in ileum biopsy samples of C57BL / 6J mice or different TLR full knockout mice . ( N = 4 / 23 per group )

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RT-qPCR data of Ripk3, in ileum biopsy samples of C57BL/6J mice or different TLR full knockout mice. (N=4/23 per group)


RT - qPCR data of Mlkl , in ileum biopsy samples of C57BL / 6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week , either or not pretreated with antibiotics ( AB ) for three weeks . ( N = 5 per group )

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

RT-qPCR data of Mlkl , in ileum biopsy samples of C57BL/6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week, either or not pretreated with antibiotics (AB) for three weeks. (N=5 per group)


RT - qPCR data of Zbp1 , in ileum biopsy samples of C57BL / 6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week , either or not pretreated with antibiotics ( AB ) for three weeks . ( N = 5 per group )

RT-qPCR data of Zbp1, in ileum biopsy samples of C57BL/6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week, either or not pretreated with antibiotics (AB) for three weeks. (N=5 per group)


( A - B ) C57BL / 6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i . p . with 50 µg TNF , solved in 200 µL sterile PBS , per 20 g bodyweight or with PBS only . 6h later , sections through ileum were made and stained with ( A ) Hematoxylin & Eosin and MMP7 , a Paneth cell marker . Pictures at magnification of 20x were taken and one representative picture of each condition is shown . Arrows point to crypts and their ( ab ) normal occurrence ( see text ) . Scale bars : 50

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(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only. 6h later, sections through ileum were made and stained with (A) Hematoxylin & Eosin and MMP7, a Paneth cell marker. Pictures at magnification of 20x were taken and one representative picture of each condition is shown. Arrows point to crypts and their (ab)normal occurrence (see text). Scale bars: 50 μm


( C ) Transmission electron microscopy ( TEM ) images of ileum crypts show protective effects of zinc therapy on the level of TNF induced effects on Paneth cells . C57BL / 6J mice ( N = 2 / group ) were put on normal drinking water or on 25 mM ZnSO4 water for 7 days . Mice were given 50 µG TNF or PBS and 8h later , ileum samples were taken and prepared for TEM . Figure shows ileal crypts at a magnification of 1000x . In each of the 4 conditions , 2 Paneth cells are selected in red

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(C) Transmission electron microscopy (TEM) images of ileum crypts show protective effects of zinc therapy on the level of TNF induced effects on Paneth cells. C57BL/6J mice (N=2 /group) were put on normal drinking water or on 25 mM ZnSO4 water for 7 days. Mice were given 50 µG TNF or PBS and 8h later, ileum samples were taken and prepared for TEM. Figure shows ileal crypts at a magnification of 1000x. In each of the 4 conditions, 2 Paneth cells are selected in red area


( D - F ) RT - qPCR measurement of mRNA levels of Defa6 and Lyz2 , two Paneth cell markers , and Ripk3 , a TNF - induced cell - death gene , in ileum of the experiment in A .

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(D-F) RT-qPCR measurement of mRNA levels of Defa6 and Lyz2, two Paneth cell markers, and Ripk3, a TNF-induced cell-death gene, in ileum of the experiment in A.


( A - B ) C57BL / 6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i . p . with 50 µg TNF , solved in 200 µL sterile PBS , per 20 g bodyweight or with PBS only ( N = 5 ) . 18h after TNF challenge , spleen and MLNs were isolated and lysates prepared and plated on TSA plates . Colonies were counted and expressed as bacterial colonies per ml of lysate . 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure . Data are shown as means + / - SD . p - value was analyzed by means of a Student ' s t - test ( unpaired , two - tailed ) .

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

(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only (N=5). 18h after TNF challenge, spleen and MLNs were isolated and lysates prepared and plated on TSA plates. Colonies were counted and expressed as bacterial colonies per ml of lysate. 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure. Data are shown as means +/- SD. p-value was analyzed by means of a Student's t-test (unpaired, two-tailed).


( E ) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF ( see A - B ) . Pure colonies of Staphylococcus nepalensis ( orange ) , Staphylococcus sciuri ( red ) , Eschericchia coli ( purple ) and Enterococcus faecalis ( blue ) were grown in LB medium , then diluted and plated on TSA plates containing increasing concentrations of ZnCL2 . 24h later , the amounts of colonies were counted , and the amounts found in the 0 mM ZnCl2 condition expressed as 100 % . Data are shown as mean + / - SD pooled from three independent experiments .

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

(E) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF (see A-B). Pure colonies of Staphylococcus nepalensis (orange), Staphylococcus sciuri (red), Eschericchia coli (purple) and Enterococcus faecalis (blue) were grown in LB medium, then diluted and plated on TSA plates containing increasing concentrations of ZnCL2. 24h later, the amounts of colonies were counted, and the amounts found in the 0 mM ZnCl2 condition expressed as 100%. Data are shown as mean +/- SD pooled from three independent experiments.