Figure 2 ( 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 FISHquantification 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 ) . ( 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 = 2μmTime 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 . ( 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 =

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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 FISHquantification 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).(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 = 2μmTime 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.(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=


Figure 3 ( 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 . ( 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 . ( 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 ) . ( 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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Figure 3(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.(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.(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).(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.


Figure 4 ( 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 . ( 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 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 ) . ( 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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Figure 4(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.(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 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).(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).


Figure 5 ( 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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Figure 5(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.


Figure 6Representative 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 ) ( 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 .

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Figure 6Representative 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)(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.


Figure 1The 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 .

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Figure 1The 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.


Figure 2Ubiquitylation 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 .

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Figure 2Ubiquitylation 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.


Figure 3Reconstituted 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 .

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Figure 3Reconstituted 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.


Figure 4CMG 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 .

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Figure 4CMG 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.


Figure 5TAP - 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 .

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Figure 5TAP-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.


Figure 6Embryonic 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Embryonic 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.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%).


Figure 1 . 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 .

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Figure 1. 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.


Figure 2 . 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 .

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Figure 2. 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.


Figure 3 . 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 .

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Figure 3. 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.


Figure 4 . 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 ) .

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Figure 4. 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).


Figure 5 . 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 ) .

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Figure 5. 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).


Figure 6 . 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 ) .

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Figure 6. 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).


Figure 7 . 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 ) .

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Figure 7. 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).


Figure 8 . 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 ) .

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Figure 8. 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).


Figure 1Western 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 .

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Figure 1Western 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.


Figure 2Circos 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 . ChIP - seq ( red ) and RIP - seq ( green ) data show enrichment of DNA and RNA , respectively , from var genes in the promoter - targeted and non - targeted " Control " dCas9 immunoprecipitation from non - clonal bulk population of ring stage parasites . 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 dCas9 ChIP enrichment in the var gene promoter - targeted dCas9 strain ( red ) , dCas9 RIP enrichment normalized to IgG control in the var gene promoter - targeted dCas9 strain or non - targeted " Control " dCas9 strain ( green ) , or var gene transcript levels in the promoter - targeted dCas9 strain ( RPKM , grey ) . One replicate was performed for ChIP - seq and RNA - seq and one replicate was performed for HA and IgG control RIP - seq .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Circos 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.ChIP-seq (red) and RIP-seq (green) data show enrichment of DNA and RNA, respectively, from var genes in the promoter-targeted and non-targeted "Control" dCas9 immunoprecipitation from non-clonal bulk population of ring stage parasites. 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 dCas9 ChIP enrichment in the var gene promoter-targeted dCas9 strain (red), dCas9 RIP enrichment normalized to IgG control in the var gene promoter-targeted dCas9 strain or non-targeted "Control" dCas9 strain (green), or var gene transcript levels in the promoter-targeted dCas9 strain (RPKM, grey). One replicate was performed for ChIP-seq and RNA-seq and one replicate was performed for HA and IgG control RIP-seq.


Figure 3Volcano plot representation of label - free quantitative proteomic analysis of P . falciparum proteins present in intron - ( left ) and promoter - targeted ( right ) dCas9 immunoprecipitations . Each dot represents a protein , and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment . For the main volcano plot , x - axis = log2 ( fold - change ) , y - axis = - log10 ( p - value ) , horizontal yellow line indicates adjusted p - value = 0 . 05 , and vertical yellow lines indicate absolute fold - change = 1 . 5 . Side panels indicate proteins uniquely identified in either sample ( y - axis = number of peptides per 100 amino acids ) . dCas9 protein is highlighted in green for reference . Two replicates were used for the promoter - targeted dCas9 and four replicates were used for the intron - targeted dCas9 . Fold enrichment and adjusted p - values for each protein highlighted in red ( promoter ) and blue ( intron ) can be found in Tables EV5 and EV6 , respectively . Correlation plot of label - free quantitative proteomic analysis of P . falciparum proteins enriched in promoter - targeted ( top right quadrant in red ) versus non - targeted control ( top left quadrant in grey ) or intron - ( bottom right quadrant in blue ) dCas9 immunoprecipitations . Each dot represents a protein , and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment . The x - axis = log2 [ fold - change ( promoter / non - targeted control ) ] and y - axis = log2 [ fold - change ( promoter / intron ) ] . dCas9 protein is highlighted in green for reference . Two replicates were used for the promoter - targeted dCas9 and four replicates were used for the intron - targeted and non - targeted dCas9 . Fold enrichment and adjusted p - values can be found for each protein highlighted in red ( promoter ) in Tables EV5 ( intron comparison ) and EV7 ( control comparison ) and for each protein highlighted in blue ( intron ) in Tables EV6 ( promoter comparison ) and EV8 ( control comparison ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Volcano plot representation of label-free quantitative proteomic analysis of P. falciparum proteins present in intron- (left) and promoter-targeted (right) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. For the main volcano plot, x-axis = log2(fold-change), y-axis = -log10(p-value), horizontal yellow line indicates adjusted p-value = 0.05, and vertical yellow lines indicate absolute fold-change = 1.5. Side panels indicate proteins uniquely identified in either sample (y-axis = number of peptides per 100 amino acids). dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted dCas9. Fold enrichment and adjusted p-values for each protein highlighted in red (promoter) and blue (intron) can be found in Tables EV5 and EV6, respectively.Correlation plot of label-free quantitative proteomic analysis of P. falciparum proteins enriched in promoter-targeted (top right quadrant in red) versus non-targeted control (top left quadrant in grey) or intron- (bottom right quadrant in blue) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. The x-axis = log2[fold-change(promoter/non-targeted control)] and y-axis = log2[fold-change(promoter/intron)]. dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted and non-targeted dCas9. Fold enrichment and adjusted p-values can be found for each protein highlighted in red (promoter) in Tables EV5 (intron comparison) and EV7 (control comparison) and for each protein highlighted in blue (intron) in Tables EV6 (promoter comparison) and EV8 (control comparison).


Figure 4Western blot analysis of cytoplasmic ( Cty ) and nuclear extracts from a bulk population of ISWI - 3HA - ribo parasites in the absence ( - ) or presence ( + ) of glucosamine ( GlcN ) . ISWI - 3HA is detected with an anti - HA antibody . Antibodies against aldolase ( Ald . ) and histone H3 are controls for the cytoplasmic and nuclear extracts , respectively . Molecular weights are shown to the right . MA plot of log2 [ glucosamine - treated / untreated , M ] plotted over the mean abundance of each gene ( A ) at 12 hpi . Transcripts with a significantly higher ( above x - axis ) or lower ( below x - axis ) abundance in the presence of glucosamine are highlighted in red ( q ≤ 0 . 05 ) . iswi is highlighted in blue ( q = 6 . 71x10 - 10 ) and the active var gene is highlighted in green ( q = 2 . 53x10 - 5 ) . Two and three replicates were used for untreated and glucosamine - treated parasites , respectively . p - values were calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 ( Love et al , 2014 ) . q = Bonferroni corrected p - value . Frequency plot showing the time in the IDC of peak transcript level [ comparison to microarray time course in ( Bozdech et al , 2003 ; Data ref : Bozdech et al , 2003 ) ] for genes that are significantly down - regulated ( blue ) or up - regulated ( yellow ) following ISWI knockdown . Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA parasites at 12 hpi from 1 . 5 kb upstream of the translation start site ( " Start " ) to 1 . 5 kb downstream of the translation stop site ( " Stop " ) for genes that are down - ( blue ) or up - regulated ( yellow ) upon ISWI knockdown . One replicate was used for the ISWI ChIP - seq . ChIP - seq data show enrichment of ISWI ( blue ) in clonal ISWI - 3HA parasites at 12 hpi relative to regions of accessible chromatin ( " ATAC , " red ) . Chromatin accessibility ( ATAC - seq ) data were taken from the 15 hpi time point in ( Toenhake et al , 2018 ; Data ref : Toenhake et al , 2018 ) . Genome location is indicated at the top of the 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 enrichment ( ChIP / Input or ATAC - seq / genomic DNA ) . One replicate was used for the ISWI ChIP - seq . Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA parasites at 12 hpi from 1 . 5 kb upstream of the translation start site ( " Start " ) to 1 . 5 kb downstream of the translation stop site ( " Stop " ) for all genes , which are grouped into quartiles based on their transcript levels ( RPKM ) at 12 hpi . Dark red represents genes with the highest transcript levels ( " 1st " ) , light red represents genes with the second highest transcript levels ( " 2nd " ) , orange represents genes with the third highest transcript levels ( " 3rd " ) , and yellow represents genes with the lowest transcript levels ( " 4th " ) . One replicate was used for the ISWI ChIP - seq and the transcription data are an average from the two replicates of the untreated ISWI - 3HA clone used for the differential expression analysis . ChIP - seq data show enrichment of ISWI ( blue ) and H3K9ac ( green ) in clonal ISWI - 3HA parasites at 12 hpi at the active var gene ( PF3D7 _ 1240600 ) . RNA - seq data from this clone at 12 hpi show transcript levels for this gene ( grey ) . Genome location is indicated at the top of the 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 ChIP / Input for ChIP data and RPKM for RNA - seq data . One replicate was used for each ChIP - seq and the RNA - seq data is from a single replicate from the untreated ISWI - 3HA clone used for the differential expression analysis . Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA parasites at 12 hpi from 1 . 5 kb upstream of the translation start site ( " Start " ) to 1 . 5 kb downstream of the translation stop site ( " Stop " ) for the active var gene ( PF3D7 _ 1240600 , blue ) or silent var genes ( yellow ) . One replicate was used for the ISWI ChIP - seq .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Western blot analysis of cytoplasmic (Cty) and nuclear extracts from a bulk population of ISWI-3HA-ribo parasites in the absence (-) or presence (+) of glucosamine (GlcN). ISWI-3HA is detected with an anti-HA antibody. Antibodies against aldolase (Ald.) and histone H3 are controls for the cytoplasmic and nuclear extracts, respectively. Molecular weights are shown to the right.MA plot of log2[glucosamine-treated/untreated, M] plotted over the mean abundance of each gene (A) at 12 hpi. Transcripts with a significantly higher (above x-axis) or lower (below x-axis) abundance in the presence of glucosamine are highlighted in red (q ≤ 0.05). iswi is highlighted in blue (q = 6.71x10-10) and the active var gene is highlighted in green (q = 2.53x10-5). Two and three replicates were used for untreated and glucosamine-treated parasites, respectively. p-values were calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected p-value.Frequency plot showing the time in the IDC of peak transcript level [comparison to microarray time course in (Bozdech et al, 2003; Data ref: Bozdech et al, 2003)] for genes that are significantly down-regulated (blue) or up-regulated (yellow) following ISWI knockdown.Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for genes that are down- (blue) or up-regulated (yellow) upon ISWI knockdown. One replicate was used for the ISWI ChIP-seq.ChIP-seq data show enrichment of ISWI (blue) in clonal ISWI-3HA parasites at 12 hpi relative to regions of accessible chromatin ("ATAC," red). Chromatin accessibility (ATAC-seq) data were taken from the 15 hpi time point in (Toenhake et al, 2018; Data ref: Toenhake et al, 2018). Genome location is indicated at the top of the 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 enrichment (ChIP/Input or ATAC-seq/genomic DNA). One replicate was used for the ISWI ChIP-seq.Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for all genes, which are grouped into quartiles based on their transcript levels (RPKM) at 12 hpi. Dark red represents genes with the highest transcript levels ("1st"), light red represents genes with the second highest transcript levels ("2nd"), orange represents genes with the third highest transcript levels ("3rd"), and yellow represents genes with the lowest transcript levels ("4th"). One replicate was used for the ISWI ChIP-seq and the transcription data are an average from the two replicates of the untreated ISWI-3HA clone used for the differential expression analysis.ChIP-seq data show enrichment of ISWI (blue) and H3K9ac (green) in clonal ISWI-3HA parasites at 12 hpi at the active var gene (PF3D7_1240600). RNA-seq data from this clone at 12 hpi show transcript levels for this gene (grey). Genome location is indicated at the top of the 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 ChIP/Input for ChIP data and RPKM for RNA-seq data. One replicate was used for each ChIP-seq and the RNA-seq data is from a single replicate from the untreated ISWI-3HA clone used for the differential expression analysis.Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for the active var gene (PF3D7_1240600, blue) or silent var genes (yellow). One replicate was used for the ISWI ChIP-seq.


Figure 1Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL / 6 mouse lines . Tissues were isolated from indicated C57BL / 6 or iNKT TN mouse lines and stained with anti - CD3 and CD1d ( PBS57 ) tetramer . Spleen , mLN , sdLN , liver , adipose , and lung lymphocytes from Jα18 - / - or Vα14 mice were stimulated in vitro with RAW - CD1d cells and 1μg α - GalCer . An additional sample of Vα14 lymphocytes from each organ was plated with RAW - CD1d cells but no α - GalCer . Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array . Error bars are SD of mean values from three different mice per group . Results shown are representative of two independent experiments where n = 3 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d (PBS57) tetramer.Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18-/- or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n=3 biological replicates.


Figure 2Lymphocytes from the indicated tissues of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer , before they were fixed , permeabilized , and stained with antibodies to T - bet , RORγt , and PLZF . Results shown are gated on CD3 + CD1d - tetramer + cells . The percentage of CD3 + CD1d - tetramer + iNKT cells in each organ that stained positively for PLZF , T - bet , and RORγt are shown . * * p < 0 . 01 , Mann - Whitney test . Error bars are SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells.The percentage of CD3+ CD1d-tetramer+ iNKT cells in each organ that stained positively for PLZF, T-bet, and RORγt are shown. **p<0.01, Mann-Whitney test. Error bars are SD.


Figure 3Lymphocytes from the Peyer ' s patches of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer . Percentage of lymphocytes that were CD3 + CD1d - tetramer + iNKT cells among the Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 22 ; Vα14 n = 16 ; Jα18 - / - n = 8 . ( C - E ) Mice were analyzed for total fecal IgA by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 . ( C - E ) Mice were analyzed for total fecal IgG1 ( D ) or total serum IgG1 ( E ) by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 . Percentages of total B cells that were IgG1 + in the spleen , mLN , and Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 15 ; Vα14 n = 15 ; Jα18 - / - n =

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Lymphocytes from the Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer. Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among the Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=22; Vα14 n=16; Jα18-/- n=8.(C-E)Mice were analyzed for total fecal IgA by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.(C-E)Mice were analyzed for total fecal IgG1 (D) or total serum IgG1 (E) by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.Percentages of total B cells that were IgG1+ in the spleen, mLN, and Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=15; Vα14 n=15; Jα18-/- n=5


Figure 4CD1d ( PBS57 ) tetramer + CD3 + cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4 + CD3 + CD1d tetramer - cells from PP ( PP CD4 ) . RNAseq was performed . Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample . Spleen , mLN , and PP lymphocytes from Vα14 and C57BL / 6 mice were stimulated with PMA and ionomycin . Lymphocytes were stained with anti - CD3 , CD1d ( PBS57 ) tetramer , anti - IL - 4 , and anti - IFNγ . Percentages of iNKT cells and non - iNKT T cells within the population of CD3 + IL - 4 + cells and CD3 + IFNγ + cells , n = 5 mice for C57BL / 6 group and n = 4 mice for Vα14 group . Error bars are SD . Vα14 TN mice were administered α - GalCer either 2μg intravenously or 5μg by oral gavage . Mice were given Brefeldin A intraperitoneally after 30 min , and tissues were harvested 3 hours later . Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with antibodies to IL - 4 , IFNγ , and IL - 17 . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells . Quantification of data from C , n = 2 mice per group . Mann - Whitney test . Error bars are SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample.Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD.Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.Quantification of data from C, n=2 mice per group. Mann-Whitney test. Error bars are SEM.


Figure 5Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α - GalCer and with or without agnostic anti - CD40 . IgM was measured by ELISA of culture supernatants 4 days later . Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti - CD40 activated wild type B cells with or without 1μg α - GalCer and with blocking antibody to CD1d ( 1μg / mL , clone 1B1 ) or isotype control as indicated . IgM , IgG1 , and IgA were measured by ELISA of 4 day culture supernatants . Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . IL - 4 were measured by ELISA of culture supernatants . Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 ( D - E ) were measured by ELISA of culture supernatants . Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + and IgA + class switched B cells were enumerated by flow cytometry after 4 days of cocultureSpleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + class switched B cells were enumerated by flow cytometry after 4 days of

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants.Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. IL-4 were measured by ELISA of culture supernatants.Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1 (D-E) were measured by ELISA of culture supernatants.Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of cocultureSpleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


Figure 6OVA - specific antibody titers from feces were determined by ELISA from the mice in ( A ) . Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD . OVA - specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD . Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with the indicated antibodies . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells . Quantification of data from E , n = 3 mice per group . Mann - Whitney test . Error bars are SEM . * p < 0 . 05 .

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Figure 6OVA-specific antibody titers from feces were determined by ELISA from the mice in (A). Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.OVA-specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with the indicated antibodies. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. Quantification of data from E, n=3 mice per group. Mann-Whitney test. Error bars are SEM. *p<0.05.


Figure 7Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP - iNKT cells . Heatmap of FPKM values from genes uniquely upregulated in PP - iNKT cells as compared to both spleen iNKT and PP CD4 cells . Red indicates higher expression and blue indicates lower expression . Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies , analyzed by flow cytometry and gated on CD1d ( PBS57 ) tetramer + CD3 + cells . Quantification of data from D , n = 11 mice per group . Mann - Whitney test . Error bars are SEM .

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Figure 7Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP-iNKT cells.Heatmap of FPKM values from genes uniquely upregulated in PP-iNKT cells as compared to both spleen iNKT and PP CD4 cells. Red indicates higher expression and blue indicates lower expression.Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies, analyzed by flow cytometry and gated on CD1d (PBS57) tetramer+CD3+ cells.Quantification of data from D, n=11 mice per group. Mann-Whitney test. Error bars are SEM.


Figure 2Determination of maximum growth rate ( Vmax ) using a label - free cell count - based proliferation assay . Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi - log scale displaying total numbers of cells over time . Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints . Western blot for NUP58 protein expression in mutant clones ( B - E ) and control cells ( clone A and WT ) . Whole - cell lysate hybridized with customized antibody 1 ( Ab1 , top panel ) and antibody 2 ( Ab2 , bottom panel ) . Actin was used as loading control .

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Figure 2Determination of maximum growth rate (Vmax) using a label-free cell count-based proliferation assay. Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi-log scale displaying total numbers of cells over time. Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints.Western blot for NUP58 protein expression in mutant clones (B-E) and control cells (clone A and WT). Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Actin was used as loading control.


Figure 3Representation of the highest growth rates ( Vmax ) for each technical replicate for each indicated cell line . Each dot represents a technical replicate ( WT n = 35 , C1 n = 27 , C2 n = 28 , C3 n = 34 , C4 n = 32 , C5 n = 35 , C6 n = 38 ) of one independent experimental replicate performed .

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Figure 3Representation of the highest growth rates (Vmax) for each technical replicate for each indicated cell line. Each dot represents a technical replicate (WT n=35, C1 n=27, C2 n=28, C3 n=34, C4 n=32, C5 n=35, C6 n=38) of one independent experimental replicate performed.


Figure 4Western blot for NUP58 protein expression in mutant clones and control cells . Whole - cell lysate hybridized with customized antibody 1 ( Ab1 , top panel ) and antibody 2 ( Ab2 , bottom panel ) . Blots were loaded with wild - type HAP1 cell line ( WT ) , empty vector ( EV ) , non - targeting siRNA ( siNT ) and NUP58 targeting siRNA ( siNUP58 ) , followed by NUP58 mutant clones ( C1 / C2 / C3 / C4 in left panels , C5 / C6 in right panels ) . Actin was used as loading control . Asterisks indicate possible NUP58 alternative isoforms .

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Figure 4Western blot for NUP58 protein expression in mutant clones and control cells. Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Blots were loaded with wild-type HAP1 cell line (WT), empty vector (EV), non-targeting siRNA (siNT) and NUP58 targeting siRNA (siNUP58), followed by NUP58 mutant clones (C1 / C2 / C3 / C4 in left panels, C5 / C6 in right panels). Actin was used as loading control. Asterisks indicate possible NUP58 alternative isoforms.


Figure 5Cellular viability expressed as number of viable colonies following Cas9 - induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1 - KPNA4 - eGFP ( KAPs ) .

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Figure 5Cellular viability expressed as number of viable colonies following Cas9-induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1-KPNA4-eGFP (KAPs).


Figure 6Improvement in active nuclear import upon cell passaging . Dots represent analysed cells ( WT P3 n = 16 , WT P19 n = 25 , C5 P1 n = 24 , C5 P3 n = 27 , C5 P19 n = 23 ) of one independent experimental replicate performed . NUP58 protein expression in C5 at P1 and P19 . Blots were performed using Antibody 1 ( Ab1 , top ) or Antibody 2 ( Ab2 , bottom ) . Actin was used as loading control . The band corresponding to the full - length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk . Cellular viability following Cas9 - induced gene editing of native NUP58 ( left ) or NUP85 ( right ) in WT cell line ( black circles ) and in a cell line overexpressing NUP58 mutant isoform ( grey circles ) .

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Figure 6Improvement in active nuclear import upon cell passaging. Dots represent analysed cells (WT P3 n=16, WT P19 n=25, C5 P1 n=24, C5 P3 n=27, C5 P19 n=23) of one independent experimental replicate performed.NUP58 protein expression in C5 at P1 and P19. Blots were performed using Antibody 1 (Ab1, top) or Antibody 2 (Ab2, bottom). Actin was used as loading control. The band corresponding to the full-length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk.Cellular viability following Cas9-induced gene editing of native NUP58 (left) or NUP85 (right) in WT cell line (black circles) and in a cell line overexpressing NUP58 mutant isoform (grey circles).


Figure 7Diagram at the top represents G - banding pattern of chromosome 13 and highlights chromosome locations of tested genes ( NUP58 , GRK1 , ADPRHL1 ) . Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number . Representative images of NUP58 gene - specific FISH hybridization for control HAP1 cell lines ( WT P3 - WT P19 ) and mutant C5 at P19 . Each dot represents a NUP58 locus of cluster thereof . Scale bar , 31 . 3

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Figure 7Diagram at the top represents G-banding pattern of chromosome 13 and highlights chromosome locations of tested genes (NUP58, GRK1, ADPRHL1). Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number.Representative images of NUP58 gene-specific FISH hybridization for control HAP1 cell lines (WT P3-WT P19) and mutant C5 at P19. Each dot represents a NUP58 locus of cluster thereof. Scale bar, 31.3 μm


Figure 1A . qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected ( CA : Control , n = 6 ; cKO , n = 6 . DG : Control , n = 6 ; cKO , n = 6 . Cortex : Control , n = 7 ; cKO , n = 7 . Cerebellum : Control , n = 4 ; cKO , n = 4 ) . * p - value < 0 . 05 ( Student t - test ) . B . Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice ( Control , n = 4 ; cKO , n = 4 ) . * * p - value < 0 . 01 ( Student t - test ) . C . Immunohistochemical staining ( upper level ) for marker proteins of neuronal integrity and quantification ( lower panel ) show no difference in control and Setd1b cKO mice ( NeuN : Control , n = 6 ; cKO , n = 6 ; Student t - test p - value = 0 . 57 . MAP2 : Control , n = 4 ; cKO , n = 4 ; Student t - test p - value = 0 . 72 . Iba1 : Control , n = 5 ; cKO , n = 5 ; Student t - test p - value = 0 . 8 . Gfap : Control , n = 5 ; cKO , n = 5 ; Student t - test p - value = 0 . 09 . ) . Scale bar : 100 µm . D . Average speed ( left panel ) and percent time spent in the center ( right panel ) during exposure to the open field test was similar in control and Setd1b cKO mice ( Average speed : Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 075 . Time spent in center : Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 96 ) . E . Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y - maze test ( Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 3 ) . F . Depressive - like behavior was measured in the Porsolt forced swim test . There was no significant difference in the % time spent immobile between groups ( Control , n = 8 ; cKO , n = 8 ; Student t - test p - value = 0 . 5 ) . G . Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice ( Control : n = 15 , cKO : n = 15 . Repeated measures ANOVA , genotype effect : F ( 1 , 28 ) = 82 . 34 , * * * * p - value < 0 . 0001 ) . H . Plots showing the specific search strategies during water maze training . Note the failure of Setd1b cKO mice to adopt hippocampus - dependent search strategies . I . The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice ( Student t - test : * * * p - value < 0 . 001 ) .

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Figure 1A. qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected (CA: Control, n = 6; cKO, n = 6. DG: Control, n = 6; cKO, n = 6. Cortex: Control, n = 7; cKO, n = 7. Cerebellum: Control, n = 4; cKO, n = 4). * p-value < 0.05 (Student t-test).B. Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice (Control, n = 4; cKO, n = 4). ** p-value < 0.01 (Student t-test).C. Immunohistochemical staining (upper level) for marker proteins of neuronal integrity and quantification (lower panel) show no difference in control and Setd1b cKO mice (NeuN: Control, n = 6; cKO, n = 6; Student t-test p-value = 0.57. MAP2: Control, n = 4; cKO, n = 4; Student t-test p-value = 0.72. Iba1: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.8. Gfap: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.09.). Scale bar: 100 µm.D. Average speed (left panel) and percent time spent in the center (right panel) during exposure to the open field test was similar in control and Setd1b cKO mice (Average speed: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.075. Time spent in center: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.96).E. Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y-maze test (Control, n = 15; cKO, n = 15; Student t-test p-value = 0.3).F. Depressive-like behavior was measured in the Porsolt forced swim test. There was no significant difference in the % time spent immobile between groups (Control, n = 8; cKO, n = 8; Student t-test p-value = 0.5).G. Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice (Control: n = 15, cKO: n = 15. Repeated measures ANOVA, genotype effect: F (1,28) = 82.34, **** p-value < 0.0001).H. Plots showing the specific search strategies during water maze training. Note the failure of Setd1b cKO mice to adopt hippocampus-dependent search strategies. I. The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice (Student t-test: *** p-value < 0.001).


Figure 6F . Left panel shows representative images of RNAscope performed for Kmt2a , Kmt2b and Setd1b . Right panel shows a bar chart quantifying the dots / cell ( n = 1500 cells from 2 mice ) indicative of the corresponding expression level . Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b ( * * * p - value < 0 , 0001 , Student t - test ) .

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Figure 6F. Left panel shows representative images of RNAscope performed for Kmt2a, Kmt2b and Setd1b. Right panel shows a bar chart quantifying the dots/cell (n = 1500 cells from 2 mice) indicative of the corresponding expression level. Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b (*** p-value < 0,0001, Student t-test).


Figure 1B Following treatment with 10 mM metformin overnight , NIH3T3 reporter cells were heat - shocked at 43 ° C for 30 min and recovered at 37 ° C for 5 h . GFP induction was quantitated by flow cytometry . HS : heat shock . C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting . D , E Following heat shock and recovery for 4 h , mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . β - actin was used as the internal control . F Reporter cells were treated with 10 mM metformin overnight , and viability was measured using Guava ViaCount ® reagents ( mean ± SD , n = 5 , n . s . , not significant , Student ' s t - test ) . G Left panel , graphic depiction of the ELISA - based DNA binding assay . HSE : heat shock element . Right panel : Immediately following heat shock at 43 ° C for 30 min , nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . Experimental details are described in Materials and Methods . I HEK293T cells stably expressing a scramble or HSF1 - targeting shRNA were treated with 10 mM metformin overnight and heat - shocked at 43 ° C for 30 min . Cells were fixed for PLA to detect HSF1 - DNA interactions . Experimental details are described in Materials and Methods . Scale bars : 50 μm for low magnification and 10 μm for high magnification . J Immediately following heat shock , cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting . LDH and Lamin A / C were used as cytoplasmic and nuclear markers , respectively . C : cytoplasm ; N : nucleus . K Reporter mice ( ventral side shown ) were pre - treated with metformin for 3 days , 2 mg / mouse / day , via i . p . injection . Following a single i . p . injection of velcade ( 5 mg / kg ) and recovery for 6 h , whole - body luciferase activities were quantitated by bioluminescence imaging . The photon flux of each mouse was expressed as photon / s / cm2 / steradian ( right panel , mean ± SD , n = 3 , one - way ANOVA , * P < 0 . 05 ; * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B Following treatment with 10 mM metformin overnight, NIH3T3 reporter cells were heat-shocked at 43°C for 30 min and recovered at 37°C for 5 h. GFP induction was quantitated by flow cytometry. HS: heat shock.C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting.D, E Following heat shock and recovery for 4 h, mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). β-actin was used as the internal control.F Reporter cells were treated with 10 mM metformin overnight, and viability was measured using Guava ViaCount® reagents (mean ± SD, n = 5, n.s., not significant, Student's t-test).G Left panel, graphic depiction of the ELISA-based DNA binding assay. HSE: heat shock element. Right panel: Immediately following heat shock at 43°C for 30 min, nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). Experimental details are described in Materials and Methods.I HEK293T cells stably expressing a scramble or HSF1-targeting shRNA were treated with 10 mM metformin overnight and heat-shocked at 43°C for 30 min. Cells were fixed for PLA to detect HSF1-DNA interactions. Experimental details are described in Materials and Methods. Scale bars: 50 μm for low magnification and 10 μm for high magnification.J Immediately following heat shock, cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting. LDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. C: cytoplasm; N: nucleus.K Reporter mice (ventral side shown) were pre-treated with metformin for 3 days, 2 mg/mouse/day, via i.p. injection. Following a single i.p. injection of velcade (5 mg/kg) and recovery for 6 h, whole-body luciferase activities were quantitated by bioluminescence imaging. The photon flux of each mouse was expressed as photon/s/cm2/steradian (right panel, mean ± SD, n = 3, one-way ANOVA, *P < 0.05; **P < 0.01).