( B ) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual - Luciferase miRNA Target Expression Vector with either CoV2 - miR - O7a . 2 target site or CoV2 - miR - O7a . 2 site with mutations in seed region at 24 h on co - transfection with CoV2 - miR - O7a . 2 or control miRNA mimics . Activity on transfecting control miRNA mimic was considered 100 % . Bars and error bars represent the average and standard deviation from three independent experiments .

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(B) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual-Luciferase miRNA Target Expression Vector with either CoV2-miR-O7a.2 target site or CoV2-miR-O7a.2 site with mutations in seed region at 24 h on co-transfection with CoV2-miR-O7a.2 or control miRNA mimics. Activity on transfecting control miRNA mimic was considered 100 %. Bars and error bars represent the average and standard deviation from three independent experiments.


( D ) Kinetics of BATF2 and LAMP3 mRNA levels in non - induced and interferon IFN - α - induced ( for 8 , 16 , and 24 h ) A549 - ACE2 cells transfected with either control or CoV2 - miR - O7a . 2 mimics . Normalized read abundances in transcript per million ( TPM ) are shown for two individual experiments ( n = 2 ) .

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(D) Kinetics of BATF2 and LAMP3 mRNA levels in non-induced and interferon IFN-α-induced (for 8, 16, and 24 h) A549-ACE2 cells transfected with either control or CoV2-miR-O7a.2 mimics. Normalized read abundances in transcript per million (TPM) are shown for two individual experiments (n=2).


( E ) Genomic view of the human BATF2 gene showing normalized RNA - seq reads from non - induced and IFN - α - induced ( for 8 h ) A549 - ACE2 cells transfected with either control , CoV2 - miR - O7a . 1 or CoV2 - miR - O7a . 2 mimics . The complementary site of CoV2 - miR - O7a to BATF2 3 ´ UTR is shown .

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(E) Genomic view of the human BATF2 gene showing normalized RNA-seq reads from non-induced and IFN-α-induced (for 8 h) A549-ACE2 cells transfected with either control, CoV2-miR-O7a.1 or CoV2-miR-O7a.2 mimics. The complementary site of CoV2-miR-O7a to BATF2 3´UTR is shown.


( A , B ) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN - α treatment in A549 - ACE2 cells transfected with CoV2 - miR - O7a . 1 ( A ) or CoV2 - miR - O7a . 2 ( B ) compared to control mimic . Significantly downregulated genes are marked in red , and upregulated genes in blue . The orange horizontal line indicates two - tailed P = 0 . 05 . n = 2 , dashed lines represent + 1 and - 1 log2 fold change .

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(A, B) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN-α treatment in A549-ACE2 cells transfected with CoV2-miR-O7a.1 (A) or CoV2-miR-O7a.2 (B) compared to control mimic. Significantly downregulated genes are marked in red, and upregulated genes in blue. The orange horizontal line indicates two-tailed P = 0.05. n=2, dashed lines represent +1 and -1 log2 fold change.


( C ) Log2 fold change of ISGs at 8 , 16 , 24 hours of IFN - α treatment and categorized based on CoV2 - miR - O7a . 2 target sites 8mer , 7mer - m8 , 7mer - A1 , and no seed as shown in the schematic . The mean and standard error of the mean is shown . Number of ISGs with 8mer site n = 8 , 7mer - m8 n = 32 , 7mer - A1 n = 26 and no seed n = 92 . The two - tailed p values were calculated using the Mann - Whitney - Wilcoxon test . ISGs were calculated as all the upregulated genes ( ≥ 3 - fold ; padj < 0 . 05 ) in two replicates of IFN - induced versus non - induced conditions in all time points .

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(C) Log2 fold change of ISGs at 8, 16, 24 hours of IFN-α treatment and categorized based on CoV2-miR-O7a.2 target sites 8mer, 7mer-m8, 7mer-A1, and no seed as shown in the schematic. The mean and standard error of the mean is shown. Number of ISGs with 8mer site n=8, 7mer-m8 n= 32, 7mer-A1 n=26 and no seed n= 92. The two-tailed p values were calculated using the Mann-Whitney-Wilcoxon test. ISGs were calculated as all the upregulated genes (≥ 3-fold; padj < 0.05) in two replicates of IFN-induced versus non-induced conditions in all time points.


( A ) Heat map showing log2 fold change of expression of ISGs in A549 - ACE2 cells across 8 , 16 and 24 h time points upon IFN - α treatment compared to non - treated controls . The common upregulated genes ( ≥ 3 - fold ; padj < 0 . 05 ) in IFN - induced versus non - induced conditions at all time points were categorized as ISGs .

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(A) Heat map showing log2 fold change of expression of ISGs in A549-ACE2 cells across 8, 16 and 24 h time points upon IFN-α treatment compared to non-treated controls. The common upregulated genes (≥ 3-fold; padj < 0.05) in IFN-induced versus non-induced conditions at all time points were categorized as ISGs.


( A ) Expression levels of CoV2 - miR - O7a . 1 , CoV2 - miR - O7a . 2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs ( Control ORF6 ) analyzed by stem - loop RT - qPCR from nasopharyngeal swabs of patients tested positive for COVID - 19 or another seasonal human coronavirus ( HCoV ) . Relative expression to hsa - miR - let - 7a is shown . Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples .

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(A) Expression levels of CoV2-miR-O7a.1, CoV2-miR-O7a.2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs (Control ORF6) analyzed by stem-loop RT-qPCR from nasopharyngeal swabs of patients tested positive for COVID-19 or another seasonal human coronavirus (HCoV). Relative expression to hsa-miR-let-7a is shown. Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples.


C - F Division pattern of seam cells at the postembryonic stage in hermaphrodites . Seam cells V1 - V4 and V6 divide symmetrically at the L2 stage ( highlighted in red ) , thus doubling in number .

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C-F Division pattern of seam cells at the postembryonic stage in hermaphrodites. Seam cells V1-V4 and V6 divide symmetrically at the L2 stage (highlighted in red), thus doubling in number.


( G , H ) dcr ‐ 1 young adults have an increased number of seam cells ( shown in brackets ) and gaps in the alae ( between arrows ) .

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(G,H) dcr‐1 young adults have an increased number of seam cells (shown in brackets) and gaps in the alae (between arrows).


( I , J ) atg ‐ 2 mutations suppress the retarded heterochronic defect in dcr ‐ 1mutants . Complete alae ( arrow ) are formed in dcr ‐ 1 ; atg ‐ 2 double mutants .

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(I,J) atg‐2 mutations suppress the retarded heterochronic defect in dcr‐1mutants. Complete alae (arrow) are formed in dcr‐1; atg‐2 double mutants.


K - M Confocal images showing expression of hbl ‐ 1 : : gfp : : hbl ‐ 1 reporter in VNC in wild type ( K ) , atg ‐ 2 ( L ) and epg ‐ 6 ( M ) L3 larvae .

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K-M Confocal images showing expression of hbl‐1::gfp::hbl‐1 reporter in VNC in wild type (K), atg‐2 (L) and epg‐6 (M) L3 larvae.


N Distribution of relative fluorescence intensity of HBL ‐ 1 : : GFP in 50 unit areas in VNC in the indicated strains .

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N Distribution of relative fluorescence intensity of HBL‐1::GFP in 50 unit areas in VNC in the indicated strains.


O - Q Confocal images showing the expression of the col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) reporter in wild type ( O ) , atg ‐ 2 ( P ) and atg ‐ 3 ( Q ) late L4 larvae .

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O-Q Confocal images showing the expression of the col‐10::gfp::lin‐41(3′UTR) reporter in wild type (O), atg‐2 (P) and atg‐3 (Q) late L4 larvae.


R Distribution of relative fluorescence intensity of col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in 50 unit areas of hypodermal cells in the indicated strains .

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R Distribution of relative fluorescence intensity of col‐10::gfp::lin‐41(3′UTR) in 50 unit areas of hypodermal cells in the indicated strains.


S , T Distribution of relative fluorescence intensity of hbl ‐ 1 : : gfp in VNC and col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in hypodermal cells in 50 unit areas in wild type , let ‐ 363 ( RNAi ) or starvation ‐ treated animals . Black lines in ( N , R - T ) represent the average fluorescence intensity of the reporter in each strain . P values are determined by Student ' s two ‐ tailed unpaired t ‐ test in ( N , R - T ) . ( C , E , G , I ) Scale bar , 20 μm ; ( D , F , H , J - M , O - Q ) scale bar , 10 μm . GFP , green fluorescent protein ; miRISC , miRNA ‐ induced silencing complex ; RNAi , RNA ‐ mediated interference ; UTR , untranslated region ; VNC , ventral nerve cord .

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S,T Distribution of relative fluorescence intensity of hbl‐1::gfp in VNC and col‐10::gfp::lin‐41(3′UTR) in hypodermal cells in 50 unit areas in wild type, let‐363(RNAi) or starvation‐treated animals. Black lines in (N,R-T) represent the average fluorescence intensity of the reporter in each strain. P values are determined by Student's two‐tailed unpaired t‐test in (N,R-T). (C,E,G,I) Scale bar, 20 μm; (D,F,H,J-M,O-Q) scale bar, 10  μm. GFP, green fluorescent protein; miRISC, miRNA‐induced silencing complex; RNAi, RNA‐mediated interference; UTR, untranslated region; VNC, ventral nerve cord.


A - C Wild ‐ type and atg ‐ 2 hermaphrodites have a single vulva ( arrow ) . ( B ) let ‐ 60 ( n1046gf ) mutants show a Muv phenotype . Black arrows indicate pseudovulvae .

A-C Wild‐type and atg‐2 hermaphrodites have a single vulva (arrow). (B) let‐60(n1046gf) mutants show a Muv phenotype. Black arrows indicate pseudovulvae.


D Loss of autophagy activity suppresses the Muv phenotype in let ‐ 60 ( n1046 ) mutants . The number of adult animals examined was : wild type ( n = 450 ) , let ‐ 60 ( n = 406 ) , epg ‐ 6 ; let ‐ 60 ( n = 444 ) , let ‐ 60 ; atg ‐ 2 ( n = 803 ) , epg ‐ 5 ; let ‐ 60 ( n = 672 ) , epg ‐ 5 ; let ‐ 60 ; ain ‐ 1 ( ku322 ) ( n = 635 ) . P values are determined by a two ‐ tailed χ2 test .

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D Loss of autophagy activity suppresses the Muv phenotype in let‐60(n1046) mutants. The number of adult animals examined was: wild type (n=450), let‐60 (n=406), epg‐6; let‐60 (n=444), let‐60; atg‐2 (n=803), epg‐5; let‐60 (n=672), epg‐5; let‐60; ain‐1(ku322) (n=635). P values are determined by a two‐tailed χ2 test.


E , F lsy ‐ 6 mutants show defects in ASEL fate specification and do not express the ASEL ‐ specific reporter lim ‐ 6pro : : GFP .

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E,F lsy‐6 mutants show defects in ASEL fate specification and do not express the ASEL‐specific reporter lim‐6pro::GFP.


G Expression of lim ‐ 6pro : : GFP in various strains . aCompared with lsy ‐ 6 ( ot150 ) . bCompared with lsy ‐ 6 ( ot150 ) ; ain ‐ 1 ( ku322 ) . P values are determined by a two ‐ tailed χ2 test .

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G Expression of lim‐6pro::GFP in various strains. aCompared with lsy‐6(ot150).bCompared with lsy‐6(ot150); ain‐1(ku322). P values are determined by a two‐tailed χ2 test.


AIN ‐ 1 : : GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild ‐ type animals . ( A ) Nomarski image of the embryo shown in ( B ) . Scale bar , 10 μm for whole embryo . C . elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size . Thus , scale bars for embryos are only shown once in each figure . ( C ) AIN ‐ 1 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos . ( A - C ) are confocal images .

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AIN‐1::GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild‐type animals. (A) Nomarski image of the embryo shown in (B). Scale bar, 10 μm for whole embryo. C. elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size. Thus, scale bars for embryos are only shown once in each figure. (C) AIN‐1::GFP forms a large number of aggregates in epg‐6 mutant embryos. (A-C) are confocal images.


( D ) Western blotting shows that endogenous AIN ‐ 1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type .

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(D) Western blotting shows that endogenous AIN‐1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type.


( E ) No AIN ‐ 1 : : GFP aggregates are formed in sqst ‐ 1 mutant embryos .

(E) No AIN‐1::GFP aggregates are formed in sqst‐1 mutant embryos.


( F ) AIN ‐ 1 : : GFP accumulates into numerous aggregates in epg ‐ 7 mutant embryos .

(F) AIN‐1::GFP accumulates into numerous aggregates in epg‐7 mutant embryos.


( G , H ) Number of AIN ‐ 1 : : GFP ( G ) and ALG ‐ 2 : : GFP ( H ) aggregates per focal plane in the indicated strains . Error bars indicate the s . d . of four examined embryos .

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(G,H) Number of AIN‐1::GFP (G) and ALG‐2::GFP (H) aggregates per focal plane in the indicated strains. Error bars indicate the s.d. of four examined embryos.


( I , J ) ALG ‐ 2 : : GFP is diffusely localized in the cytoplasm in wild ‐ type embryos . ( I ) Nomarski image of the embryo shown in ( J ) .

(I,J) ALG‐2::GFP is diffusely localized in the cytoplasm in wild‐type embryos. (I) Nomarski image of the embryo shown in (J).


( K ) ALG ‐ 2 : : GFP forms a few aggregates in epg ‐ 6 mutant embryos . ( L ) ALG ‐ 2 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos under stress conditions . GFP , green fluorescent protein ; s . d . , standard deviation .

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(K) ALG‐2::GFP forms a few aggregates in epg‐6 mutant embryos. (L) ALG‐2::GFP forms a large number of aggregates in epg‐6 mutant embryos under stress conditions. GFP, green fluorescent protein; s.d., standard deviation.


( A - D ) Both AIN ‐ 1 : : GFP and SQST ‐ 1 , detected by anti ‐ SQST ‐ 1 antibody , are very weakly expressed and diffusely localized in wild ‐ type embryos .

(A-D) Both AIN‐1::GFP and SQST‐1, detected by anti‐SQST‐1 antibody, are very weakly expressed and diffusely localized in wild‐type embryos.


( E - H ) AIN ‐ 1 : : GFP aggregates colocalize with SQST ‐ 1 aggregates in atg ‐ 3 mutant embryos . Scale bars , 10 μm for insets that show a magnified view .

(E-H) AIN‐1::GFP aggregates colocalize with SQST‐1 aggregates in atg‐3 mutant embryos. Scale bars, 10 μm for insets that show a magnified view.


( I - L ) AIN ‐ 1 : : GFP aggregates are separable from PGL granules , detected by anti ‐ SEPA ‐ 1 , in atg ‐ 3 mutant embryos .

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(I-L) AIN‐1::GFP aggregates are separable from PGL granules, detected by anti‐SEPA‐1, in atg‐3 mutant embryos.


( M - P ) Colocalization of AIN ‐ 1 : : GFP aggregates with LGG ‐ 1 puncta in epg ‐ 6 mutant embryos .

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(M-P) Colocalization of AIN‐1::GFP aggregates with LGG‐1 puncta in epg‐6 mutant embryos.


( Q ) % of AIN ‐ 1 : : GFP aggregates colocalized with SQST ‐ 1 aggregates , SEPA ‐ 1 aggregates and LGG ‐ 1 puncta in autophagy mutants . Error bars indicate the s . d . of four examined embryos .

(Q) % of AIN‐1::GFP aggregates colocalized with SQST‐1 aggregates, SEPA‐1 aggregates and LGG‐1 puncta in autophagy mutants. Error bars indicate the s.d. of four examined embryos.


( R ) A co ‐ IP assay shows that AIN ‐ 1 associates with LGG ‐ 1 in vivo .

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(R) A co‐IP assay shows that AIN‐1 associates with LGG‐1 in vivo.


( S ) Direct interaction between AIN ‐ 1 and LGG ‐ 1 in an in vitro pull ‐ down assay . 5 % of the His fusion proteins used for pull ‐ down are shown as input . Corresponding Ponceau ‐ stained blots show the amount of proteins used for pull ‐ down .

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(S) Direct interaction between AIN‐1 and LGG‐1 in an in vitro pull‐down assay. 5% of the His fusion proteins used for pull‐down are shown as input. Corresponding Ponceau‐stained blots show the amount of proteins used for pull‐down.


( T ) A fragment of AIN ‐ 1 directly binds to LGG ‐ 1 in an in vitro pull ‐ down assay .

(T) A fragment of AIN‐1 directly binds to LGG‐1 in an in vitro pull‐down assay.


( B ) Representative images of GFAP + Sox2 + BrdU + RGLs , GFAP - Sox2 + BrdU + progenitors , Tbr2 + BrdU + IPCs in the DG . Scale bar = 20 μm . ( C ) Quantification of GFAP + Sox2 + BrdU + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 022 ) , GFAP - Sox2 + BrdU + progenitors ( WT vs . KO , Student ' s t - test , p = 0 . 0046 ) , Tbr2 + BrdU + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 4 mice ; KO , n = 6 mice . ( D ) Representative images of GFAP + Mcm2 + RGLs , Tbr2 + Mcm2 + IPCs , DCX + Mcm2 + NBs in the DG . Scale bar = 20 μm . ( E ) Quantification of GFAP + Mcm2 + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 016 ) , Tbr2 + Mcm2 + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) , DCX + Mcm2 + NBs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 3 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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(B) Representative images of GFAP+Sox2+BrdU+ RGLs, GFAP-Sox2+BrdU+ progenitors, Tbr2+BrdU+ IPCs in the DG. Scale bar = 20 μm. (C) Quantification of GFAP+Sox2+BrdU+ RGLs (WT vs. KO, Student's t-test, p = 0.022), GFAP-Sox2+BrdU+ progenitors (WT vs. KO, Student's t-test, p = 0.0046), Tbr2+BrdU+ IPCs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 4 mice; KO, n = 6 mice. (D) Representative images of GFAP+Mcm2+ RGLs, Tbr2+Mcm2+ IPCs, DCX+Mcm2+ NBs in the DG. Scale bar = 20 μm. (E) Quantification of GFAP+Mcm2+ RGLs (WT vs. KO, Student's t-test, p = 0.016), Tbr2+Mcm2+ IPCs (WT vs. KO, Student's t-test, p = 0.003), DCX+Mcm2+ NBs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 3 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( B ) Representative images used for fate mapping of tdT + cells in the DG by co - stained with GFAP ( RGL ) , Tbr2 ( IPC ) , DCX ( IN ) , NeuN ( MN ) . Scale bar = 20 μm .

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(B) Representative images used for fate mapping of tdT+ cells in the DG by co-stained with GFAP (RGL), Tbr2 (IPC), DCX (IN), NeuN (MN). Scale bar = 20 μm.


( C ) Quantification of composition of tdT + cells ( RGL : WT vs . KO , Student ' s t - test , p = 0 . 026 ; IPC : WT vs . KO , Student ' s t - test , p = 0 . 028 ; IN : WT vs . KO , Student ' s t - test , p = 0 . 049 ; N : WT vs . KO , Student ' s t - test , p = 0 . 048 ) in the DG of WT and cKO mice . WT , n = 3 mice ; cKO , n = 3 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 .

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(C) Quantification of composition of tdT+ cells (RGL: WT vs. KO, Student's t-test, p = 0.026; IPC: WT vs. KO, Student's t-test, p = 0.028; IN: WT vs. KO, Student's t-test, p = 0.049; N: WT vs. KO, Student's t-test, p = 0.048) in the DG of WT and cKO mice. WT, n = 3 mice; cKO, n = 3 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01


( H ) Representative images of BrdU - retaining RGLs ( tdT + BrdU + ) in the DG . Scale bar = 20 μm . ( I ) Quantification of BrdU - retaining RGLs ( BrdU + tdT + GFAP + , WT vs . cKO , Student ' s t - test , p = 0 . 009 ) in the DG of WT and cKO mice . WT , n = 4 mice ; cKO , n = 3 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 O O O 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) Representative images of BrdU-retaining RGLs (tdT+BrdU+) in the DG. Scale bar = 20 μm. (I) Quantification of BrdU-retaining RGLs (BrdU+tdT+GFAP+, WT vs. cKO, Student's t-test, p = 0.009) in the DG of WT and cKO mice. WT, n = 4 mice; cKO, n = 3 mice.


( B ) Representative images of Nestin - GFP + GFAP + Mcm2 + RGLs in the DG . Scale bar = 50 μm . ( C ) Quantification of GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 001 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . ( D ) Percentage of GFAP + Nestin - GFP + Mcm2 + activated RGLs among total GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 0013 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of Nestin-GFP+GFAP+Mcm2+ RGLs in the DG. Scale bar = 50 μm. (C) Quantification of GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.001) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. (D) Percentage of GFAP+Nestin-GFP+Mcm2+ activated RGLs among total GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.0013) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.


( I ) Representative images of NeuN + BrdU + adult - born neurons in the DG of 6 month - old mice . Scale bar = 50 μm . ( J ) Quantification of NeuN + BrdU + adult - born neurons ( WT vs . KO , Student ' s t - test , p = 0 . 0052 ) in the DG of WT and KO mice at 7 months of age . WT , n = 4 mice ; KO , n = 3 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(I) Representative images of NeuN+BrdU+ adult-born neurons in the DG of 6 month-old mice. Scale bar = 50 μm. (J) Quantification of NeuN+BrdU+ adult-born neurons (WT vs. KO, Student's t-test, p = 0.0052) in the DG of WT and KO mice at 7 months of age. WT, n = 4 mice; KO, n = 3 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.


( A ) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling . Scale bar = 50 μm . ( B ) Percentage of BrdU + aNPCs among total aNPCs isolated from WT or KO mice ( WT vs . KO , Student ' s t - test , p = 0 . 0075 ) . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * * , p < 0 . 01 , * * * , p < 0 . 001 .

(A) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling. Scale bar = 50 μm. (B) Percentage of BrdU+ aNPCs among total aNPCs isolated from WT or KO mice (WT vs. KO, Student's t-test, p = 0.0075). WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; **, p < 0.01, ***, p < 0.001.


( A ) DARTS analysis indicated HK1 as a molecular target of L - arginine in aNPCs . The aNPC lysates treated with L - arginine , digested with pronase and then analyzed by Western blot with anti - HK1 and GAPDH antibodies . ( B ) Quantification of HK1 expression level in aNPCs lysates treated with L - arginine and followed by digesting with pronase ( One - way ANOVA , F ( 3 , 8 ) = 3 . 61 , p = 0 . 043 ) . n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(A) DARTS analysis indicated HK1 as a molecular target of L-arginine in aNPCs. The aNPC lysates treated with L-arginine, digested with pronase and then analyzed by Western blot with anti-HK1 and GAPDH antibodies. (B) Quantification of HK1 expression level in aNPCs lysates treated with L-arginine and followed by digesting with pronase (One-way ANOVA, F(3, 8)= 3.61, p = 0.043). n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( I ) Co - IP of HK1 with VDAC1 in vehicle or L - arginine - treated cells . ( J ) Quantification of the co - IP efficacy of HK1 and VDAC interaction in vehicle or L - arginine - treated cells ( IP : HK1 , vehicle vs . L - arginine , Student ' s t - test , p < 0 . 001 ; IP : VDAC1 , vehicle vs . L - arginine , Student ' s t - test , p = 0 . 0078 ) . Vehicle , n = 3 independent experiments ; L - arginine , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(I) Co-IP of HK1 with VDAC1 in vehicle or L-arginine-treated cells. (J) Quantification of the co-IP efficacy of HK1 and VDAC interaction in vehicle or L-arginine-treated cells (IP: HK1, vehicle vs. L-arginine, Student's t-test, p < 0.001; IP: VDAC1, vehicle vs. L-arginine, Student's t-test, p = 0.0078). Vehicle, n = 3 independent experiments; L-arginine, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( C - E ) Quantification of glucose ( C , WT vs . KO , Student ' s t - test , p < 0 . 001 ) and glutamine ( E , WT vs . KO , Student ' s t - test , p = 0 . 94 ) consumption and lactate production ( D , WT vs . KO , Student ' s t - test , p = 0 . 019 ) in WT - and KO - aNPCs . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 * * * , p < 0 . 001 .

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 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-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

(C-E) Quantification of glucose (C, WT vs. KO, Student's t-test, p < 0.001) and glutamine (E, WT vs. KO, Student's t-test, p = 0.94) consumption and lactate production (D, WT vs. KO, Student's t-test, p = 0.019) in WT- and KO-aNPCs. WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05 ***, p < 0.001.


( J ) Representative profile of mitochondria stress test showing the OCR of WT - or KO - aNPCs . WT , n = 4 independent experiments , KO , n = 4 independent experiments .

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

(J) Representative profile of mitochondria stress test showing the OCR of WT- or KO-aNPCs. WT, n = 4 independent experiments, KO, n = 4 independent experiments.


( C ) Quantification of Nestin - GFP + GFAP + RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 99 ) . WT + Vehicle , n = 4 mice ; WT + IACS , n = 4 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 3 mice . ( D ) Quantification of Nestin - GFP + GFAP + BrdU + activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( E ) Quantification of Nestin - GFP + GFAP - BrdU + progenitors ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 028 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( F ) Quantification of Nestin - GFP + Tbr2 + BrdU + IPCs ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 02 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 4 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(C) Quantification of Nestin-GFP+GFAP+ RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.99). WT + Vehicle, n = 4 mice; WT + IACS, n = 4 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 3 mice. (D) Quantification of Nestin-GFP+GFAP+BrdU+ activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (E) Quantification of Nestin-GFP+GFAP-BrdU+ progenitors (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.028) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (F) Quantification of Nestin-GFP+Tbr2+BrdU+ IPCs (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.02) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 4 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( H ) Representative images of NeuN + BrdU + adult - born neurons in the DG . Scale bar = 20 μm . ( I ) Quantification of NeuN + BrdU + adult - born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 5 mice ; WT + IACS , n = 5 mice ; KO + Vehicle , n = 5 mice , KO + IACS , n = 5 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(H) Representative images of NeuN+BrdU+ adult-born neurons in the DG. Scale bar = 20 μm. (I) Quantification of NeuN+BrdU+ adult-born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 5 mice; WT + IACS, n = 5 mice; KO + Vehicle, n = 5 mice, KO + IACS, n = 5 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( a ) Species relative abundance as a function of time for all pairwise communities . Experimental measurements and model fits based on T3 are represented as data points and lines , respectively . In each subplot , time and species relative abundance are displayed on the x and y - axis , respectively . Stars denote datasets with a sum of mean squared errors greater than 0 . 15 . Error bars represent 1 s . d . from the mean of at least three biological replicates . ( b ) Temporal changes in species relative abundance of a selected set of pairwise assemblages inoculated at 5 % species A , 95 % species B or 95 % species A , 5 % species B based on OD600 values . Time and relative abundance are represented on the x and y - axis , respectively . Data points and lines represent experimental measurements and model fits to T3 , respectively . Error bars represent 1 s . d . from the mean of at least three biological replicates . Stars denote datasets with a sum of mean squared errors greater than 0 . 15 . ( c ) Inferred inter - species interaction coefficients for the gLV model trained on T3 . Gray and green edges denote negative ( αij < 0 ) and positive ( αij > 0 ) interaction coefficients . The edge width and node size represent the magnitude of the inter - species interaction coefficient and steady - state monospecies abundance ( xe = - μiαii - 1 ) , respectively . To highlight significant interactions , inter - species interaction coefficients with a magnitude less than 1e - 5 were not displayed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Species relative abundance as a function of time for all pairwise communities. Experimental measurements and model fits based on T3 are represented as data points and lines, respectively. In each subplot, time and species relative abundance are displayed on the x and y-axis, respectively. Stars denote datasets with a sum of mean squared errors greater than 0.15. Error bars represent 1 s.d. from the mean of at least three biological replicates. (b) Temporal changes in species relative abundance of a selected set of pairwise assemblages inoculated at 5% species A, 95% species B or 95% species A, 5% species B based on OD600 values. Time and relative abundance are represented on the x and y-axis, respectively. Data points and lines represent experimental measurements and model fits to T3, respectively. Error bars represent 1 s.d. from the mean of at least three biological replicates. Stars denote datasets with a sum of mean squared errors greater than 0.15. (c) Inferred inter-species interaction coefficients for the gLV model trained on T3. Gray and green edges denote negative (αij < 0) and positive (αij > 0) interaction coefficients. The edge width and node size represent the magnitude of the inter-species interaction coefficient and steady-state monospecies abundance (xe = -μiαii-1), respectively. To highlight significant interactions, inter-species interaction coefficients with a magnitude less than 1e-5 were not displayed.


( a ) Stacked bar plots of all 11 and 12 - member ( full ) multi - species communities . The text above each subplot denotes the absent organism in the community . Time and relative abundance are represented on the x and y - axis of each subplot , respectively . Colors represent different organisms in the community . ( b ) Difference in community assembly for single - species dropout communities . Difference in community assembly was computed as $ \ sum _ { i = 1 , \ } ^ { 11 } { \ frac { 1 } { 7 } \ sum _ { j = 1 } ^ { 7 } { ( { \ widehat { v } } _ { ji , FULL } - v _ { ji , X } ) } } ^ { 2 } $ where i and j represent species and time points , respectively . X represents a single - species dropout community lacking the organism X . v ̂ denotes the renormalized relative abundance of the shared set of species in the full ( 12 - member ) and single - species dropout community . Here $ { \ widehat { v } } _ { i } = \ \ frac { v _ { i } } { \ sum _ { i \ \ in C } ^ { } v _ { i } } $ where C denotes the set of 11 shared organisms in the full and single - species dropout community . Data points represent biological replicates ( n = 6 , circles ) and the mean of biological replicates ( blue squares ) , respectively . ( c ) Box plot of the Pearson correlation coefficient for three model training sets ( left ) including T1 : monospecies ( M ) ; T2 : M and pairwise 1 ( PW1 ) ; and T3 : M , PW1 and pairwise 2 ( PW2 ) . On each box , the red line represents the median , the edges of the box are the 25th and 75th percentiles , the whiskers extend to the most extreme data points and the outliers are plotted as red crosses . ( d ) Box plot of the sum of mean squared errors for the model predictions of the time - resolved measurements of multi - species communities trained on T1 , T2 or T3 training sets . On each box , the red line represents the median , the edges of the box are the 25th and 75th percentiles , the whiskers extend to the most extreme data points and the outliers are plotted as red crosses . ( e ) Comparison between model prediction based on T1 - T4 and experimental data for each organism in the full community as a function of time . On each subplot , the x and y - axis represent time and relative abundance of each species , respectively . Data points ( red ) and lines denote experimental data and model predictions based on T1 - T4 . Species names are displayed in the upper left corner of each subplot . Error bars represent 1 s . d . from the mean of six biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stacked bar plots of all 11 and 12-member (full) multi-species communities. The text above each subplot denotes the absent organism in the community. Time and relative abundance are represented on the x and y-axis of each subplot, respectively. Colors represent different organisms in the community. (b) Difference in community assembly for single-species dropout communities. Difference in community assembly was computed as $\sum_{i = 1,\ }^{11}{\frac{1}{7}\sum_{j = 1}^{7}{({\widehat{v}}_{ji,FULL} - v_{ji,X})}}^{2}$ where i and j represent species and time points, respectively. X represents a single-species dropout community lacking the organism X. v̂ denotes the renormalized relative abundance of the shared set of species in the full (12-member) and single-species dropout community. Here ${\widehat{v}}_{i} = \ \frac{v_{i}}{\sum_{i\ \in C}^{}v_{i}}$ where C denotes the set of 11 shared organisms in the full and single-species dropout community. Data points represent biological replicates (n = 6, circles) and the mean of biological replicates (blue squares), respectively. (c) Box plot of the Pearson correlation coefficient for three model training sets (left) including T1: monospecies (M); T2: M and pairwise 1 (PW1); and T3: M, PW1 and pairwise 2 (PW2). On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (d) Box plot of the sum of mean squared errors for the model predictions of the time-resolved measurements of multi-species communities trained on T1, T2 or T3 training sets. On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (e) Comparison between model prediction based on T1-T4 and experimental data for each organism in the full community as a function of time. On each subplot, the x and y-axis represent time and relative abundance of each species, respectively. Data points (red) and lines denote experimental data and model predictions based on T1-T4. Species names are displayed in the upper left corner of each subplot. Error bars represent 1 s.d. from the mean of six biological replicates.


( a ) Bipartite network of species ( left ) and metabolites ( right ) for metabolites that decreased by at least two - fold compared to the abundance of each metabolite at the beginning of the experiment . Colors represent modules containing many overlapping interactions in the network . Network partitioning into modules was performed using BiMat ( Flores Metabolites in bold were depleted and secreted by distinct organisms . ( b ) Bipartite network of species ( left ) and metabolites ( right ) for metabolites that increased in abundance by at least two - fold compared to the beginning of the experiment . Metabolites highlighted in bold were depleted or secreted by different species . Colors represent modules containing many overlapping interactions in the network . Network partitioning into modules was performed using BiMat (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bipartite network of species (left) and metabolites (right) for metabolites that decreased by at least two-fold compared to the abundance of each metabolite at the beginning of the experiment. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores Metabolites in bold were depleted and secreted by distinct organisms. (b) Bipartite network of species (left) and metabolites (right) for metabolites that increased in abundance by at least two-fold compared to the beginning of the experiment. Metabolites highlighted in bold were depleted or secreted by different species. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores


( c ) Scatter plot of the number of consumed metabolites that decreased by at least two - fold compared to the beginning of the experiment vs . the OD600 value of the monospecies culture at the corresponding time point . Error bars represent 1 s . d . from the mean of three biological replicates .

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(c) Scatter plot of the number of consumed metabolites that decreased by at least two-fold compared to the beginning of the experiment vs. the OD600 value of the monospecies culture at the corresponding time point. Error bars represent 1 s.d. from the mean of three biological replicates.


RNA - Seq analysis of : different time points of primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock - infected controls For ( A ) data were analyzed by Fisher test ( number of donors = 3 biological replicates ) . Boxplots in ( A ) depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . For each time point , dots illustrate the pathway enrichment analysis of genes up - regulated in infected vs matched mock - infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of differentially expressed genes ( DEGs ) in the pathway ; grey dots are not statistically

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RNA-Seq analysis of: different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock-infected controls For (A) data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in (A) depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes (DEGs) in the pathway; grey dots are not statistically significant


RNA - Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART - treated vs ART - naïve animals ( B ) . ( B ) data were analyzed by Gene Set Enrichment Analysis ( GSEA , number of animals = 8 ) . Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART - naïve and ART - treated time points . Red color indicates leading edge genes . All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress ( 184 genes ; GO : 0034599 ) .

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RNA-Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART-treated vs ART-naïve animals (B). (B) data were analyzed by Gene Set Enrichment Analysis (GSEA, number of animals =8). Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART-naïve and ART-treated time points. Red color indicates leading edge genes. All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress (184 genes; GO:0034599).


( A ) Total to oxidized glutathione ratio ( GSH / GSSG ) in mock infected or HIV - 1 infected CD4 + T - cells ( 7 dpi , n = 4 biological replicates ; mean ± SEM ) .

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(A) Total to oxidized glutathione ratio (GSH/GSSG) in mock infected or HIV-1 infected CD4+ T-cells (7 dpi, n=4 biological replicates; mean±SEM).


representative picture ( B ) of ROS content in HIV - 1 infected or mock infected primary CD4 + T - cells at 7dpi ( productive infection ) and 14 dpi ( latent infection ) . ROS content was measured by immunofluorescence , HIV - 1 DNA + cells were identified by

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representative picture (B) of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH


quantification of ROS content in HIV - 1 infected or mock infected primary CD4 + T - cells at 7dpi ( productive infection ) and 14 dpi ( latent infection ) . ROS content was measured by immunofluorescence , HIV - 1 DNA + cells were identified by FISH ( D ) . The black dash in ( C ) indicates the median . Box plots in ( D ) depict median and 25 - 75 percentile , while whiskers extend from min to max . n = number of cells . Scale bar = 2μm .

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quantification of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH (D). The black dash in (C) indicates the median. Box plots in (D) depict median and 25-75 percentile, while whiskers extend from min to max. n= number of cells. Scale bar = 2μm.


( E ) Subcellular localization of Nrf2 in CD4 + T - cells infected with HIV - 1 or mock infected ( 3dpi ) as analyzed by biochemical fractionation and western blot ; Whole Cell Extract ( WCE ) , Cytoplasm ( Cyt ) , Nucleoplasm ( Nuc ) .

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(E) Subcellular localization of Nrf2 in CD4+ T-cells infected with HIV-1 or mock infected (3dpi) as analyzed by biochemical fractionation and western blot; Whole Cell Extract (WCE), Cytoplasm (Cyt), Nucleoplasm (Nuc).


( F ) Nuclear localization ( left ) and content ( right ) of Nrf2 in HIV - 1 RNA + and HIV - 1 RNA - cells ( 7dpi ) as measured by combining immunofluorescence and HIV - 1 RNA FISH . Black dash indicates median , n = number of cells . Scale bar =

(F) Nuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA+ and HIV-1 RNA- cells (7dpi) as measured by combining immunofluorescence and HIV-1 RNA FISH. Black dash indicates median, n= number of cells. Scale bar = 2μm


Time course of the mRNA levels of main downstream antioxidant targets of Nrf2 during the transition from productive ( 3 - 9 dpi ) to latent ( 14 dpi ) infection , as measured by qPCR Data in ( G ) are depicted as mean ± SEM of 3 biological replicates . raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in infected vs mock infected cells Trx = thioredoxin ; NQO1 = NAD ( P ) H [ quinone ] dehydrogenase 1 ; HMOX - 1 = heme oxygenase 1 ; G6PD = glucose - 6 - phosphate dehydrogenase ; GCLC = glutamate - cysteine ligase ; TrxR1 = thioredoxin reductase 1 .

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Time course of the mRNA levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection, as measured by qPCR Data in (G) are depicted as mean±SEM of 3 biological replicates. raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in infected vs mock infected cells Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC= glutamate-cysteine ligase ; TrxR1= thioredoxin reductase 1.


Time course of the protein levels of main downstream antioxidant targets of Nrf2 during the transition from productive ( 3 - 9 dpi ) to latent ( 14 dpi ) infection , as measured by western blot GCLC and HMOX - 1 in ( H ) were probed upon membrane stripping .

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Time course of the protein levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection, as measured by western blot GCLC and HMOX-1 in (H) were probed upon membrane stripping.


( I ) Relative mRNA expression of Nrf2 downstream antioxidant targets in J - Lat 9 . 2 cells left untreated ( latent ) or treated for 24h with 10µM TPA ( reactivated ) . Data are depicted as mean ± SEM of 3 biological replicates . raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in TPA - reactivated vs latent cells Trx = thioredoxin ; NQO1 = NAD ( P ) H [ quinone ] dehydrogenase 1 ; HMOX - 1 = heme oxygenase 1 ; G6PD = glucose - 6 - phosphate dehydrogenase ; GCLC =

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(I) Relative mRNA expression of Nrf2 downstream antioxidant targets in J-Lat 9.2 cells left untreated (latent) or treated for 24h with 10µM TPA (reactivated). Data are depicted as mean±SEM of 3 biological replicates. raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in TPA-reactivated vs latent cells Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC=


( A ) gag mRNA expression ( left ) and relative viability ( right ) of HIV - 1 infected Jurkat - TAg cells transfected with non targeting siRNA ( NC ) or siRNAs targeting the HMOX - 1 gene . Cells were infected 24hr post - transfection and assayed for mRNA expression ( by qPCR ) or viability ( by MTT assay ) 48hr post - infection . Data ( mean ± SEM ; n = 3 technical replicates ) were normalized using the NC control and analyzed by unpaired t - test . P < 0 . 05 ; * * P < 0 . 01 .

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(A) gag mRNA expression (left) and relative viability (right) of HIV-1 infected Jurkat-TAg cells transfected with non targeting siRNA (NC) or siRNAs targeting the HMOX-1 gene. Cells were infected 24hr post-transfection and assayed for mRNA expression (by qPCR) or viability (by MTT assay) 48hr post-infection. Data (mean±SEM; n=3 technical replicates) were normalized using the NC control and analyzed by unpaired t-test. P<0.05; **P<0.01.


( B RNA - Seq analysis of different time points of primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected . The gene set used for the analyses is GO iron ion import ( 12 genes , GO : 0097286 ) . Data in ( B ) were analyzed by Fisher test . Boxplots depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . Dots below boxplots illustrate , for each time point , the pathway enrichment analysis of genes up - regulated in infected vs matched mock - infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of DEGs in the pathway ; grey dots are not statistically significant .

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(B RNA-Seq analysis of different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. The gene set used for the analyses is GO iron ion import (12 genes, GO:0097286). Data in (B) were analyzed by Fisher test. Boxplots depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. Dots below boxplots illustrate, for each time point, the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of DEGs in the pathway; grey dots are not statistically significant.


( C ) Heatmaps of the standardized expression of the GO iron ion import pathway genes in HIV - 1 infected and mock infected samples over time . Expression levels were standardized [ ( mean gene expression - SD ) / SD ] for each gene in each time point . The TFRC ( TfR1 ) gene , which was considered for further analysis , is highlighted .

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(C) Heatmaps of the standardized expression of the GO iron ion import pathway genes in HIV-1 infected and mock infected samples over time. Expression levels were standardized [(mean gene expression - SD)/SD] for each gene in each time point. The TFRC (TfR1) gene, which was considered for further analysis, is highlighted.


( D ) TfR1 expression in HIV - 1 infected or mock infected CD4 + T - cells as visualized by confocal microscopy . Fluorescent values shown were calculated as described in ( McCloy et al , 2014 ) . Data were analyzed by one - way ANOVA followed by Sidak ´ s post - test to compare corresponding mock infected vs infected pairs . Black dash indicates median , n = number of cells . Scale bar = 2μm .

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(D) TfR1 expression in HIV-1 infected or mock infected CD4+ T-cells as visualized by confocal microscopy. Fluorescent values shown were calculated as described in (McCloy et al, 2014). Data were analyzed by one-way ANOVA followed by Sidak´s post-test to compare corresponding mock infected vs infected pairs. Black dash indicates median, n= number of cells. Scale bar = 2μm.


( E ) Western blot ( upper panel ) and relative expression ( lower panel ) of FTH - 1 protein in HIV - 1 infected vs mock infected CD4 + T - cells over time . Western blot quantification was performed with Fiji - Image J ( Schindelin et al , 2012 ) , normalized to the housekeeping protein beta - actin and expressed as Log2 fold change in HIV - 1 infected vs mock infected cells ( mean ± SEM ; n = 3 biological replicates ) . P < 0 . 05 ;

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(E) Western blot (upper panel) and relative expression (lower panel) of FTH-1 protein in HIV-1 infected vs mock infected CD4+ T-cells over time. Western blot quantification was performed with Fiji-Image J (Schindelin et al, 2012), normalized to the housekeeping protein beta-actin and expressed as Log2 fold change in HIV-1 infected vs mock infected cells (mean±SEM; n=3 biological replicates). P<0.05;


( F ) Percentage of intracellular p24 + gag CD4 + T - cells infected with HIV - 1 and left untreated or treated for 48hr with the iron donor Fe - NTA at 150μM concentration ( n = 3 ; mean ± range of three biological replicates ) .

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(F) Percentage of intracellular p24+ gag CD4+ T-cells infected with HIV-1 and left untreated or treated for 48hr with the iron donor Fe-NTA at 150μM concentration (n=3; mean ± range of three biological replicates).


( G ) Percentage of GFP + J - Lat cells ( clones 9 . 2 and 15 . 4 ) left untreated or treated for 48hr with TPA ( 10μM ) , the iron donor FeNT ( 150μM ) or a combination of the two . Data are expressed as mean ± SEM of four technical replicates for each cell line and were analyzed by one - way ANOVA followed by Tukey ´ s post - test . * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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(G) Percentage of GFP+ J-Lat cells (clones 9.2 and 15.4) left untreated or treated for 48hr with TPA (10μM), the iron donor FeNT (150μM) or a combination of the two. Data are expressed as mean±SEM of four technical replicates for each cell line and were analyzed by one-way ANOVA followed by Tukey´s post-test. *P<0.05; ***P<0.001; ****P<0.0001.


( A RNA - Seq analyses of different time points in primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected . Data were analyzed by Fisher test ( number of donors = 3 biological replicates ) . Boxplots in depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . For each time point , dots below boxplots illustrate the pathway enrichment analysis of genes up - regulated in infected vs matched mock infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of differentially expressed genes in the pathway ; grey dots are not statistically significant . The gene set considered for the analyses is Biocarta _ PML _ pathway ( 17 genes , M4891 ) .

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(A RNA-Seq analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes in the pathway; grey dots are not statistically significant. The gene set considered for the analyses is Biocarta_PML_pathway (17 genes, M4891).


B ) proteomic analyses of different time points in primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected . Data were analyzed by Fisher test ( number of donors = 3 biological replicates ) . Boxplots in depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . For each time point , dots below boxplots illustrate the pathway enrichment analysis of proteins up - regulated in infected vs matched mock infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of differentially expressed proteins in the pathway ; grey dots are not statistically significant .

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B) proteomic analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of proteins up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed proteins in the pathway; grey dots are not statistically significant.


( C Western blot of PML protein in HIV - 1 infected vs mock infected CD4 + T - cells over time . Raw data were normalized using actin ( protein ) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells .

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(C Western blot of PML protein in HIV-1 infected vs mock infected CD4+ T-cells over time. Raw data were normalized using actin (protein) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells.


D ) relative expression PML mRNA in HIV - 1 infected vs mock infected CD4 + T - cells over time . Raw data were normalized using GAPDH or 18S ( mRNA ) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells . Data were analyzed by two - way ANOVA followed by Tukey ´ s post - test ( mean ± SEM of 3 and 4 biological replicates for PML

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D) relative expression PML mRNA in HIV-1 infected vs mock infected CD4+ T-cells over time. Raw data were normalized using GAPDH or 18S (mRNA) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells. Data were analyzed by two-way ANOVA followed by Tukey´s post-test (mean±SEM of 3 and 4 biological replicates for PML mRNA


( E ) Western blot and quantification of PML protein in two different clones of J - Lat cells left untreated or treated with TPA for 24h . Data were analyzed by unpaired t - test ( mean ± SEM ; n = 3 technical replicates for each cell line ) .

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(E) Western blot and quantification of PML protein in two different clones of J-Lat cells left untreated or treated with TPA for 24h. Data were analyzed by unpaired t-test (mean±SEM; n=3 technical replicates for each cell line).


( F PML protein expression in CD4 + T - cells infected with HIV - 1 or mock infected at 7dpi as measured by western blot . Cells were left untreated or treated with the antioxidant compound NAC ( 10mM , F )

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(F PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the antioxidant compound NAC (10mM, F)


G ) PML protein expression in CD4 + T - cells infected with HIV - 1 or mock infected at 7dpi as measured by western blot . Cells were left untreated or treated with the iron chelator deferiprone ( L1 , 50μM , G ) .

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G) PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the iron chelator deferiprone (L1, 50μM, G).


( A Representative image and quantification of the number of PML NBs in HIV - 1 DNA + vs mock infected CD4 + T - cells . Scale bars = 2μm . Infected cells were identified with DNA FISH and PML NBs were stained by IF .

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(A Representative image and quantification of the number of PML NBs in HIV-1 DNA+ vs mock infected CD4+ T-cells. Scale bars = 2μm. Infected cells were identified with DNA FISH and PML NBs were stained by IF.


B ) Representative image and quantification of the number of PML NBs in HIV - 1 RNA + vs HIV - 1 RNA - CD4 + T - cells . Scale bars = 2μm . Infected cells were identified with RNA FISH and PML NBs were stained by IF .

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B) Representative image and quantification of the number of PML NBs in HIV-1 RNA+ vs HIV-1 RNA- CD4+ T-cells. Scale bars = 2μm. Infected cells were identified with RNA FISH and PML NBs were stained by IF.


Representative immunofluorescence images and quantifications of PML NBs ( green , maximal projection ) and mab414 / nuclear pore ( red , one Z stack ) in J - Lat 9 . 2 cells . Cells were left latent or reactivated for 24 hrs with 10μM TPA . Proteasome function was blocked for 4 hrs in both latent and TPA reactivated cells using MG132 ( 10μM ) or Bortezomib ( 100nM )

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Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Proteasome function was blocked for 4 hrs in both latent and TPA reactivated cells using MG132 (10μM) or Bortezomib (100nM)


Representative immunofluorescence images and quantifications of PML NBs ( green , maximal projection ) and mab414 / nuclear pore ( red , one Z stack ) in J - Lat 9 . 2 cells . Cells were left latent or reactivated for 24 hrs with 10μM TPA . Alternatively , ubiquitination was blocked for 4 hrs using TAK243 ( 10μM )

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

Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Alternatively, ubiquitination was blocked for 4 hrs using TAK243 (10μM)


( E , F ) Representative immunofluorescence images ( E ) and quantification ( F ) of PML NBs ( green ) and SUMO 2 / 3 ( red ) in J - Lat 9 . 2 cells left latent or reactivated for 24 hours with 10μM TPA . Cells were imaged by STED microscopy , scale bar 2 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 B-CONTROLLED_VAR O O O O O O O O O O O O O

(E,F) Representative immunofluorescence images (E) and quantification (F) of PML NBs (green) and SUMO 2/3 (red) in J-Lat 9.2 cells left latent or reactivated for 24 hours with 10μM TPA. Cells were imaged by STED microscopy, scale bar 2 μm.


( G , H ) Representative immunofluorescence images ( G ) and quantification ( H ) of PML NBs ( green , maximal projection ) and SUMO 2 / 3 ( red , maximal projection ) during mock and productive HIV - 1 infection of primary CD4 + T - cells at 7dpi . Scale bar 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence images (G) and quantification (H) of PML NBs (green, maximal projection) and SUMO 2/3 (red, maximal projection) during mock and productive HIV-1 infection of primary CD4+ T-cells at 7dpi. Scale bar 10μm.


The presence of GFP - PSF - 1 on mitotic chromatin ( indicated by white arrows ) was monitored by spinning disk confocal microscopy ( see Materials and Methods ) , in GFP - psf - 1 mCherry - Histone H2B worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments ( ' Control ' = empty vector ) . NEB = nuclear envelope breakdown . The scalebars correspond to 5 µm .

The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in GFP-psf-1 mCherry-Histone H2B worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments ('Control' = empty vector). NEB = nuclear envelope breakdown. The scalebars correspond to 5 µm.


Ubiquitylation of CMG - MCM - 7 was reconstituted with the indicated factors as described in Materials and Methods . ' Neddylation ' indicates addition of the C . elegans ULA - 1 _ RFL - 1 E1 enzyme , the UBC - 12 E2 enzyme , the DCN - 1 E3 enzyme and NED - 8 .

Ubiquitylation of CMG-MCM-7 was reconstituted with the indicated factors as described in Materials and Methods. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.


Reactions containing CMG and either CUL - 2LRR - 1 or CUL - 2VHL - 1 were performed in the presence or absence of other replisome factors as indicated . CMG was then isolated by immunoprecipitation of SLD - 5 and the indicated factors were monitored by immunoblotting .

Reactions containing CMG and either CUL-2LRR-1 or CUL-2VHL-1 were performed in the presence or absence of other replisome factors as indicated. CMG was then isolated by immunoprecipitation of SLD-5 and the indicated factors were monitored by immunoblotting.


Reconstituted CMG - MCM - 7 ubiquitylation reactions were performed as above in the presence of the indicated factors . ' Neddylation ' indicates addition of the C . elegans ULA - 1 _ RFL - 1 E1 enzyme , the UBC - 12 E2 enzyme , the DCN - 1 E3 enzyme and NED - 8 .

Reconstituted CMG-MCM-7 ubiquitylation reactions were performed as above in the presence of the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.


CMG was isolated by immunoprecipitation of SLD - 5 , after ubiquitylation in the presence of TIM - 1 _ TIPN - 1 , before incubation with the indicated factors . ' Neddylation ' indicates addition of the C . elegans ULA - 1 _ RFL - 1 E1 enzyme , the UBC - 12 E2 enzyme , the DCN - 1 E3 enzyme and NED - 8 . CMG disassembly was monitored by release of CDC - 45 or MCM - 2 - 7 proteins into the supernatant , whilst SLD - 5 and other GINS subunits remained on the beads . Asterisks indicate non - specific bands in the anti - MCM - 7 and CDC - 45 immunoblots .

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

CMG was isolated by immunoprecipitation of SLD-5, after ubiquitylation in the presence of TIM-1_TIPN-1, before incubation with the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. CMG disassembly was monitored by release of CDC-45 or MCM-2-7 proteins into the supernatant, whilst SLD-5 and other GINS subunits remained on the beads. Asterisks indicate non-specific bands in the anti-MCM-7 and CDC-45 immunoblots.


TAP - psf - 1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments , before preparation of embryonic cell extracts and isolation of TAP - PSF - 1 by immunoprecipitation . The bound material was released from beads by cleavage of the TAP tag with TEV protease , before detection of the indicated factors 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 O O O O O O O O O O O O O O

TAP-psf-1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments, before preparation of embryonic cell extracts and isolation of TAP-PSF-1 by immunoprecipitation. The bound material was released from beads by cleavage of the TAP tag with TEV protease, before detection of the indicated factors by immunoblotting.


Similar experiment comparing the impact of RNAi inactivation of lrr - 1 , tim - 1 and tipn - 1 upon CMG - MCM - 7 ubiquitylation and the association of CUL - 2LRR - 1 with the worm replisome . Asterisks indicate non - specific bands in the immunoblots .

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

Similar experiment comparing the impact of RNAi inactivation of lrr-1, tim-1 and tipn-1 upon CMG-MCM-7 ubiquitylation and the association of CUL-2LRR-1 with the worm replisome. Asterisks indicate non-specific bands in the immunoblots.


Embryonic viability was monitored as described in Materials and Methods , after feeding worms on the indicated proportions of bacteria expressing RNAi ( ' % RNAi ' ) to the denoted genes , mixed with bacteria containing empty vector ( to make a total of 100 % ) .

Embryonic viability was monitored as described in Materials and Methods, after feeding worms on the indicated proportions of bacteria expressing RNAi ('% RNAi') to the denoted genes, mixed with bacteria containing empty vector (to make a total of 100%).


The presence of GFP - PSF - 1 on mitotic chromatin ( indicated by white arrows ) was monitored by spinning disk confocal microscopy ( see Materials and Methods ) , in embryos derived from worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi ( ' Control ' = empty vector ) . The scalebars correspond to 5 µm .

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

The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in embryos derived from worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi ('Control' = empty vector). The scalebars correspond to 5 µm.


Analogous experiment to that in ( A ) , but with worms fed on the indicated proportions of bacteria expressing ubxn - 3 RNAi , in combination with 20 % bacteria expressing tim - 1 RNAi specific for tim - 1 , plus the required remainder of bacteria containing empty vector ( to make a total of 100 % ) .

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

Analogous experiment to that in (A), but with worms fed on the indicated proportions of bacteria expressing ubxn-3 RNAi, in combination with 20% bacteria expressing tim-1 RNAi specific for tim-1, plus the required remainder of bacteria containing empty vector (to make a total of 100%).


Transfection of poly ( I : C ) induces ACE2 expression . HEK293 cells were cultured in six - well plates and transfected with poly ( I : C ) as indicated above using Lipofectamine 2000 . HEK293 cells were then collected for RNA isolation and RT - qPCR analysis at 9 hrs post - transfection . Results in each panel are representative of three independent experiments .

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

Transfection of poly (I:C) induces ACE2 expression.HEK293 cells were cultured in six-well plates and transfected with poly (I:C) as indicated above using Lipofectamine 2000. HEK293 cells were then collected for RNA isolation and RT-qPCR analysis at 9 hrs post-transfection. Results in each panel are representative of three independent experiments.


VSV - eGFP infection induces ACE2 expression . HEK293 cells were cultured in six - well plates and infected with VSV - eGFP as indicated above . 6 hrs later , HEK293 cells were collected for RNA isolation and RT - qPCR analysis . Results in each panel are representative of three independent experiments .

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

VSV-eGFP infection induces ACE2 expression.HEK293 cells were cultured in six-well plates and infected with VSV-eGFP as indicated above. 6 hrs later, HEK293 cells were collected for RNA isolation and RT-qPCR analysis. Results in each panel are representative of three independent experiments.


Overexpression of TBK1 or TRIF induces ACE2 expression . TBK1 is an essential activator of RNA - sensing pathway . HEK293 cells were cultured in 24 - well plates . 24 hrs later , plasmids as shown above were transfected into HEK293 cells . 36 hrs after transfection , total RNA was isolation for subsequent RT - qPCR analysis . Results in each panel are representative of three independent experiments .

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

Overexpression of TBK1 or TRIF induces ACE2 expression.TBK1 is an essential activator of RNA-sensing pathway. HEK293 cells were cultured in 24-well plates. 24 hrs later, plasmids as shown above were transfected into HEK293 cells. 36 hrs after transfection, total RNA was isolation for subsequent RT-qPCR analysis. Results in each panel are representative of three independent experiments.


ACE2 expression is upregulated in the lungs from SARS - CoV MA15 infected C57Bl / 6 mouse . Intranasal instillation of 102 , 103 PFU of SARS - CoV MA15 in 50 µl of PBS or mock - infected with PBS alone , lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis . ACE2 expression data was retrieved from Gene Expression Omnibus ( GEO ) microarray database ( NCBI Accession : PRJNA149057 ; GEO : GSE33266 ) .

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

ACE2 expression is upregulated in the lungs from SARS-CoV MA15 infected C57Bl/6 mouse.Intranasal instillation of 102, 103 PFU of SARS-CoV MA15 in 50 µl of PBS or mock-infected with PBS alone, lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis. ACE2 expression data was retrieved from Gene Expression Omnibus (GEO) microarray database (NCBI Accession: PRJNA149057; GEO: GSE33266).


ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS - CoV ( icMERS - CoV EMC2012 ) . Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell . Infected samples were collected as indicated above . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA391962 ; GEO : GSE100504 ) .

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

ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS-CoV (icMERS-CoV EMC2012).Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell. Infected samples were collected as indicated above. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA391962; GEO: GSE100504).


ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection . Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only ( used as control ) . 24 hrs post - infection , gene expression was assessed using Affymetrix U133 plus 2 . 0 human GeneChips . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA137767 ; GEO : GSE27973 ) .

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

ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection.Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only (used as control). 24 hrs post-infection, gene expression was assessed using Affymetrix U133 plus 2.0 human GeneChips. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA137767; GEO: GSE27973).


ACE2 expression is upregulated in human bronchial epithelial cells ( HBECs ) after IFNβ and H1N1 influenza stimulation . HBECs were treated with IFNβ ( 1000U / ml ) or wild type H1N1 influenza ( A / PR / 8 / 34 ) . Control samples were incubated with fresh medium under the same conditions . HBECs were harvested at indicated time points ( 0 . 25 , 0 . 5 , 1 , 1 . 5 , 2 , 4 , 6 , 8 , 12 , and 18 hours post - treatment ) . Total RNA was extracted and analyzed using the Affymetrix High Throughput Array . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA121751 ; GEO : GSE19392 ) .

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

ACE2 expression is upregulated in human bronchial epithelial cells (HBECs) after IFNβ and H1N1 influenza stimulation.HBECs were treated with IFNβ (1000U/ml) or wild type H1N1 influenza (A/PR/8/34). Control samples were incubated with fresh medium under the same conditions. HBECs were harvested at indicated time points (0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). Total RNA was extracted and analyzed using the Affymetrix High Throughput Array. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA121751; GEO: GSE19392).


ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ . Primary keratinocytes from 3 donors were either untreated ( control ) or exposed to cytokines IFNα , IFNγ . 24 hrs after treatment , total RNA was extracted for subsequent microarray analysis . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA153023 ; GEO : GSE36287 ) .

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

ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ.Primary keratinocytes from 3 donors were either untreated (control) or exposed to cytokines IFNα, IFNγ. 24 hrs after treatment, total RNA was extracted for subsequent microarray analysis. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA153023; GEO: GSE36287).


Western blot analysis of a dCas9 immunoprecipitation experiment in the promoter - targeted strain at ring stage . Levels of dCas9 and histone H3 in the cytoplasmic ( Cyt ) , nuclear ( excluding chromatin , Nuc ) , and chromatin ( Ch ) fractions are revealed with anti - HA and anti - H3 antibodies , respectively . dCas9 is enriched in the immunoprecipitated fraction ( IP ) compared to the unbound supernatant ( SN ) and input ( i . e . chromatin fraction ) of the IP . Molecular weights are shown to the right .

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

Western blot analysis of a dCas9 immunoprecipitation experiment in the promoter-targeted strain at ring stage. Levels of dCas9 and histone H3 in the cytoplasmic (Cyt), nuclear (excluding chromatin, Nuc), and chromatin (Ch) fractions are revealed with anti-HA and anti-H3 antibodies, respectively. dCas9 is enriched in the immunoprecipitated fraction (IP) compared to the unbound supernatant (SN) and input (i.e. chromatin fraction) of the IP. Molecular weights are shown to the right.


Circos plots of ChIP - seq data showing genome - wide enrichment of dCas9 in ring stage parasites . The 14 chromosomes are represented circularly by the outer grey bars , with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals ( Mbp ) . Enrichment for intron - or promoter - targeted dCas9 ( normalized to non - targeted dCas9 ) is shown as average reads per million ( RPM ) over bins of 1 , 000 nt . The maximum y - axis value is 1 , 200 RPM for the promoter - targeted dCas9 ( rings represent increments of 200 ) and 400 RPM for the intron - targeted dCas9 ( rings represent increments of 66 . 7 ) . var genes are represented by red bars . An asterisk indicates an unintended binding event .

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

Circos plots of ChIP-seq data showing genome-wide enrichment of dCas9 in ring stage parasites. The 14 chromosomes are represented circularly by the outer grey bars, with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals (Mbp). Enrichment for intron- or promoter-targeted dCas9 (normalized to non-targeted dCas9) is shown as average reads per million (RPM) over bins of 1,000 nt. The maximum y-axis value is 1,200 RPM for the promoter-targeted dCas9 (rings represent increments of 200) and 400 RPM for the intron-targeted dCas9 (rings represent increments of 66.7). var genes are represented by red bars. An asterisk indicates an unintended binding event.


ChIP - seq data show enrichment of dCas9 in strains at ring stage expressing non - targeted " Control " ( grey ) , var intron - targeted ( blue ) , or var promoter - targeted ( red ) sgRNAs . Genome location is indicated at the top of each panel . The x - axis is DNA sequence , with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction . The y - axis is input - subtracted ChIP enrichment . q - values for promoter - targeted dCas9 peaks shown are 2 . 22x10 - 289 for PF3D7 _ 0413100 and 7 . 07x10 - 257 for PF3D7 _ 1240400 . q - values for intron - targeted dCas9 peaks shown are 2 . 03x10 - 76 for PF3D7 _ 412700 , 6 . 81x10 - 46 for PF3D7 _ 0412900 , 1 . 53x10 - 62 for PF3D7 _ 0413100 , and 8 . 37x10 - 4 for PF3D7 _ 1240900 .

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

ChIP-seq data show enrichment of dCas9 in strains at ring stage expressing non-targeted "Control" (grey), var intron-targeted (blue), or var promoter-targeted (red) sgRNAs. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted ChIP enrichment. q-values for promoter-targeted dCas9 peaks shown are 2.22x10-289 for PF3D7_0413100 and 7.07x10-257 for PF3D7_1240400. q-values for intron-targeted dCas9 peaks shown are 2.03x10-76 for PF3D7_412700, 6.81x10-46 for PF3D7_0412900, 1.53x10-62 for PF3D7_0413100, and 8.37x10-4 for PF3D7_1240900.