Figure 6A . Western blot analysis of the IFT22RNAi + GFP : : IFT22A86R cell line probed with the anti - IFT22 antibody ( bottom ) and with an anti - BiP as loading control ( top ) . B . IFA with the indicated cell lines using the mAb25 ( marker for the axoneme , central panels ) and an anti - IFT172 antibody ( marker for IFT , bottom panels ) . The top panels show the phase contrast images merged with DAPI ( cyan ) that stains nuclear and mitochondrial DNA . The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells . Scale bars correspond to 5 µm . C . Sections of IFT22RNAi + GFP : : IFT22rescue ( top panels ) or IFT22RNAi + GFP : : IFT22A86R cells ( bottom panels ) were analysed by transmission electron microscopy . Sections through the flagellar pocket , the transition zone and the flagellum are shown . Scale bars are 500 nm ( flagellar pocket sections ) or 200 nm ( transition zone and flagellum sections ) . The white arrow indicates an endocytic vesicle budding off the flagellar pocket whereas the black one points at an IFT train . ( Axo = axoneme , TZ = transition zone , BB = basal body )

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Figure 6A. Western blot analysis of the IFT22RNAi+GFP::IFT22A86R cell line probed with the anti-IFT22 antibody (bottom) and with an anti-BiP as loading control (top).B. IFA with the indicated cell lines using the mAb25 (marker for the axoneme, central panels) and an anti-IFT172 antibody (marker for IFT, bottom panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells. Scale bars correspond to 5 µm.C. Sections of IFT22RNAi+GFP::IFT22rescue (top panels) or IFT22RNAi+GFP::IFT22A86R cells (bottom panels) were analysed by transmission electron microscopy. Sections through the flagellar pocket, the transition zone and the flagellum are shown. Scale bars are 500 nm (flagellar pocket sections) or 200 nm (transition zone and flagellum sections). The white arrow indicates an endocytic vesicle budding off the flagellar pocket whereas the black one points at an IFT train. (Axo = axoneme, TZ = transition zone, BB = basal body)


Figure 1All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . A ) Immunohistochemistry analysis of small intestinal sections , using the indicated antibodies . All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . B ) Western blot analysis with protein extracts obtained from ex vivo - purified intestinal crypts and probed with the indicated antibodies . All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . C ) Haematoxylin and eosin staining of sections prepared from different intestinal tracts . All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . D ) Immunohistochemistry analysis of small intestinal sections , using specific antibodies for the proliferation marker KI67 . All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . E ) Expression analysis by qRT - PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts . Graphs show mean ± SD for three replicates .

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Figure 1All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.B) Western blot analysis with protein extracts obtained from ex vivo-purified intestinal crypts and probed with the indicated antibodies.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.


Figure 2A ) PAS and ( B ) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . C ) Quantification ( mean ± SD ) of Goblet cells in crypts and villi from the same mice sown in A and B . More than 100 crypts and villi were scored for each condition after 15 days from the first β - naphthoflavone administration . D ) Immunohistochemistry analyses in AhCre Eed + / + and AhCre Eedfl / fl mice injected with β - naphthoflavone and sacrificed after 15 days using a lysozyme - specific antibody ( upper panels : LYZ , a Paneth cell - specific marker ) . Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels ( ALPI , an enterocyte - specific marker ) . Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels ( Alcian / KI67 ) . E ) GFP expression from an Lgr5 - eGFPtransgene identifying LGR5 + ISCs in near - native agarose - embedded sections from Eed + / + and Eed - / - mice at 30 days from the first β - naphthoflavone administration . Cell nuclei were counterstained with DAPI .

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Figure 2A) PAS and (B) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Quantification (mean ± SD) of Goblet cells in crypts and villi from the same mice sown in A and B. More than 100 crypts and villi were scored for each condition after 15 days from the first β-naphthoflavone administration.D) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice injected with β-naphthoflavone and sacrificed after 15 days using a lysozyme-specific antibody (upper panels: LYZ, a Paneth cell-specific marker). Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels (ALPI, an enterocyte-specific marker). Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels (Alcian/KI67).E) GFP expression from an Lgr5-eGFPtransgene identifying LGR5+ ISCs in near-native agarose-embedded sections from Eed+/+ and Eed-/- mice at 30 days from the first β-naphthoflavone administration. Cell nuclei were counterstained with DAPI.


Figure 3A ) Radiation - induced intestinal regeneration : AhCre Eed + / + and AhCre Eedfl / fl mice were injected with β - naphthoflavone , irradiated with 10 Gy 15 days later , and sacrificed 4 and 8 days after irradiation . Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin ( n = 6 ) . B ) Immunohistochemistry analyses in AhCre Eed + / + and AhCre Eedfl / fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post - irradiation . C ) Quantification ( mean ± SD ) of KI67 positive cells per crypt ( B ) . More than 100 crypts were scored for each condition . D ) In vitro organoids formation using crypts isolated from AhCre Eed + / + and AhCre Eed - / - mice 15 days after the first β - naphthoflavone injection . Organoids pictures were taken 5 days later at low and higher magnification ( insets ) .

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Figure 3A) Radiation-induced intestinal regeneration: AhCre Eed+/+ and AhCre Eedfl/fl mice were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 4 and 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (n=6).B) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post-irradiation.C) Quantification (mean ± SD) of KI67 positive cells per crypt (B). More than 100 crypts were scored for each condition.D) In vitro organoids formation using crypts isolated from AhCre Eed+/+ and AhCre Eed-/- mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).


Figure 4A ) Volcano plot showing the expression changes with a minimal fold change of 3 ( FC = 3 ) at genome - wide levels in AhCre Eed - / - crypts relative to AhCre Eed + / + crypts 15 days after β - naphthoflavone administration . B ) Bar plot showing the percentage of H3K27me3 - positive promoters among the differentially regulated genes in Eed - / - crypts . All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls . C ) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up - regulated genes with respect to down - regulated genes in Eed - / - crypts . D ) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms . E ) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed + / + and AhCre Eed - / - crypts 15 days after β - naphthoflavone administration . F ) Expression analysis by qRT - PCR ( mean ± SD ) of both Cdkn2a isoforms ( p16Ink4A and p19Arf ) in AhCre Eed + / + and AhCre Eed - / - crypts 15 days after β - naphthoflavone administration . G ) Gene set enrichment analysis ( GSEA ) using specific secretory and enterocytic transcriptional signatures with respect to the list of up - regulated genes in Eed - / - crypts .

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Figure 4A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in AhCre Eed-/- crypts relative to AhCre Eed+/+ crypts 15 days after β-naphthoflavone administration.B) Bar plot showing the percentage of H3K27me3-positive promoters among the differentially regulated genes in Eed-/- crypts. All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls.C) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up-regulated genes with respect to down-regulated genes in Eed-/- crypts.D) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms.E) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.F) Expression analysis by qRT-PCR (mean ± SD) of both Cdkn2a isoforms (p16Ink4A and p19Arf) in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in Eed-/- crypts.


Figure 5A ) Haematoxylin and eosin ( top panels ) and PAS staining ( bottom panels ) of sections prepared from different intestinal tracts in AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice 15 days after the first β - naphthoflavone administration ( n = 5 ) . B ) Quantification ( mean ± SD ) of Goblet cells along the crypt - to - villus axis from the staining shown in A . More than 100 crypts and villi were scored for each condition . C ) Immunhistochemistry analysis using anti - KI67 antibody of intestinal sections prepared from AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice 15 days after the first β - naphthoflavone administration . D ) Radiation - induced intestinal regeneration : AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice ( n = 6 ) were injected with β - naphthoflavone , irradiated with 10 Gy 15 days later , and sacrificed 8 days after irradiation . Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin ( first panels ) and Alcian Blue ( last panels ) . Sections were also stained for immunohistochemistry analysis ( second and third panels ) using H3K27me3 and Ki67 - specific antibodies .

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Figure 5A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5).B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition.C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration.D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemistry analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.


Figure 6A ) Expression analysis by qRT - PCR ( mean ± SD ) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis , regionalization , and Goblet cell differentiation ( n = 3 ) . B ) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice ( n = 3 ) . C ) Expression analysis by qRT - PCR ( mean ± SD ) of the Goblet cell markers ( Atoh1 , Gfi1 , Spdef ) , early secretory precursor cells ( Dll1 ) , NOTCH targets ( Hes1 , Olfm4 ) and intestinal stem cells ( Lgr5 , Rnf43 ) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ . DMSO served as vehicle negative control ( n = 4 ) . D ) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody . The same crypts described in C were used . H3K27me3 levels were normalized on Histone H3 density ( percentage of H3 signal ) . Graphs show mean ± SD .

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Figure 6A) Expression analysis by qRT-PCR (mean ± SD) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis, regionalization, and Goblet cell differentiation (n=3).B) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice (n=3).C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (Atoh1, Gfi1, Spdef), early secretory precursor cells (Dll1), NOTCH targets (Hes1, Olfm4) and intestinal stem cells (Lgr5, Rnf43) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4).D) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody. The same crypts described in C were used. H3K27me3 levels were normalized on Histone H3 density (percentage of H3 signal). Graphs show mean ± SD.


Figure 1B Western blot analysis confirming two Mettl5 KO clones by specific METTL5 antibody . α - TUBULIN was used as a loading control . C LC - MS / MS analysis of m6A / A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO ( KO - 6B ) mESCs . D PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1832 site and A1825 site ( for input control ) in 18S rRNA of WT and Mettl5 KO ( KO - 6B ) mESCs , respectively . E LC - MS / MS analysis of d3 - m6A / A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro . No enzyme group was used as a negative control .

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Figure 1B Western blot analysis confirming two Mettl5 KO clones by specific METTL5 antibody. α-TUBULIN was used as a loading control.C LC-MS/MS analysis of m6A/A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO (KO-6B) mESCs.D PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1832 site and A1825 site (for input control) in 18S rRNA of WT and Mettl5 KO (KO-6B) mESCs, respectively.E LC-MS/MS analysis of d3-m6A/A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro. No enzyme group was used as a negative control.


Figure 2A , B Representative bright - filed ( A ) and AP staining ( B ) images of the WT and Mettl5 KO mESCs cultured in 2i / Lif medium . Scale bars represent 100 μm and 50 μm , respectively . C RT - qPCR analysis of the expression of pluripotency genes in WT and Mettl5 KO mESCs . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . ns , not significant ; * * * , P < 0 . 001 . D Western blot analysis of the expression of pluripotency markers in WT and Mettl5 KO mESCs . α - TUBULIN was used as a loading control . E Immunostaining analysis of the expression of pluripotency markers ( POU5F1 , SOX2 and NANOG ) in WT and Mettl5 KO mESCs , respectively . DAPI ( blue ) was used as a nuclear counterstain . Scale bars represent 10 μm .

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Figure 2A,B Representative bright-filed (A) and AP staining (B) images of the WT and Mettl5 KO mESCs cultured in 2i/Lif medium. Scale bars represent 100 μm and 50 μm, respectively.C RT-qPCR analysis of the expression of pluripotency genes in WT and Mettl5 KO mESCs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; ***, P<0.001.D Western blot analysis of the expression of pluripotency markers in WT and Mettl5 KO mESCs. α-TUBULIN was used as a loading control.E Immunostaining analysis of the expression of pluripotency markers (POU5F1, SOX2 and NANOG) in WT and Mettl5 KO mESCs, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 10 μm.


Figure 3A Western blot analysis showing dynamic expression patterns of POU5F1 and METTL5 during the indicated time points of EB induction . α - TUBULIN was used as a loading control . B Histogram showing the threshold cycles of qPCR ( qPCRCT ) for detecting m6A1832 site in 18S rRNA during the indicated time points of EB induction . Data are shown as mean ± SD from three biological replicates . Student ' s t test , two - tailed . ns , not significant ; * , P < 0 . 05 . C Immunostaining analysis of the expression of pluripotency marker POU5F1 ( left ) , endoderm marker FOXA2 ( middle ) and neuroectoderm marker NESTIN ( right ) in control and Mettl5 KO EBs at different time points , respectively . DAPI ( blue ) was used as a nuclear counterstain . Scale bars represent 100 μm . D Quantification of the percentages of positive cells shown in ( C ) . Quantitative analysis was based on at least three independent experiments . Data are represented as mean ± SD . Student ' s t test , two - tailed . * * , P < 0 . 01 ; * * * , P < 0 . 001 . E RT - qPCR analysis of the expressions of pluripotency and lineage - specific markers in Day7 EBs . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . ns , not significant ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . F Western blot analysis showing pluripotency ( POU5F1 ) and three germ layer markers ( FOXA2 , T and NESTIN ) expression in Day7 EBs . α - TUBULIN was used as a loading control .

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Figure 3A Western blot analysis showing dynamic expression patterns of POU5F1 and METTL5 during the indicated time points of EB induction. α-TUBULIN was used as a loading control.B Histogram showing the threshold cycles of qPCR (qPCRCT) for detecting m6A1832 site in 18S rRNA during the indicated time points of EB induction. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *, P<0.05.C Immunostaining analysis of the expression of pluripotency marker POU5F1(left), endoderm marker FOXA2 (middle) and neuroectoderm marker NESTIN (right) in control and Mettl5 KO EBs at different time points, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 100 μm. D Quantification of the percentages of positive cells shown in (C). Quantitative analysis was based on at least three independent experiments. Data are represented as mean ± SD. Student's t test, two-tailed. **, P < 0.01; ***, P<0.001. E RT-qPCR analysis of the expressions of pluripotency and lineage-specific markers in Day7 EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P<0.001.F Western blot analysis showing pluripotency (POU5F1) and three germ layer markers (FOXA2, T and NESTIN) expression in Day7 EBs. α-TUBULIN was used as a loading control.


Figure 4A Volcano plot depicting down - regulated ( left ) and up - regulated ( right ) genes in Mettl5 KO mESCs compared with WT mESCs in Ribo - seq . Significantly altered genes are defined as those ≥ 1 . 5 - fold change and P value < 0 . 05 ( shown in red dots ) . Blue dots represent genes with < 1 . 5 - fold change and P value < 0 . 05 . Green dots represent genes with ≥ 1 . 5 - fold change and P value ≥ 0 . 05 . Black dots represent genes with ＜ 1 . 5 - fold change and P value ≥ 0 . 05 . P values were inferred from two - sided Wald tests ( DESeq2 ) and P value adjustments were made for multiple comparisons . Examples of up - or down - regulated or non - significantly changed genes are annotated in the graph . B Track plot showing translational changes of Fbxw7 in Mettl5 KO mESCs compared with controls . C RT - qPCR analysis of Fbxw7 mRNA levels in WT and Mettl5 KO cells during the indicated time points course of EB induction . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . ns , not significant . D Western blot analysis showing FBXW7 expression in control and Mettl5 KO cells during the indicated time points of EB induction . GAPDH was used as a loading control . E RT - qPCR analysis showing Fbxw7 mRNA levels in Day7 KO , KO + HA - MT5WT , and KO + HA - MT5Mut EBs . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . ns , not significant . F Western blot analysis showing FBXW7 expression in Day7 KO , KO + HA - MT5WT , and KO + HA - MT5Mut EBs . GAPDH was used as a loading control .

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Figure 4A Volcano plot depicting down-regulated (left) and up-regulated (right) genes in Mettl5 KO mESCs compared with WT mESCs in Ribo-seq. Significantly altered genes are defined as those ≥1.5-fold change and P value < 0.05 (shown in red dots). Blue dots represent genes with <1.5-fold change and P value < 0.05. Green dots represent genes with ≥1.5-fold change and P value ≥ 0.05. Black dots represent genes with ＜1.5-fold change and P value ≥ 0.05. P values were inferred from two-sided Wald tests (DESeq2) and P value adjustments were made for multiple comparisons. Examples of up- or down-regulated or non-significantly changed genes are annotated in the graph.B Track plot showing translational changes of Fbxw7 in Mettl5 KO mESCs compared with controls.C RT-qPCR analysis of Fbxw7 mRNA levels in WT and Mettl5 KO cells during the indicated time points course of EB induction. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant.D Western blot analysis showing FBXW7 expression in control and Mettl5 KO cells during the indicated time points of EB induction. GAPDH was used as a loading control.E RT-qPCR analysis showing Fbxw7 mRNA levels in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant.F Western blot analysis showing FBXW7 expression in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. GAPDH was used as a loading control.


Figure 5 A Western blot analysis showing dynamic expression patterns of FBXW7 and c - MYC during the indicated time points of EB induction . α - TUBULIN was used as a loading control . B Western blot analysis showing FBXW7 and c - MYC expression in control , Mettl5 KO ( KO - 6B ) and KO + HA - MT5WT Day7 EBs , respectively . α - TUBULIN was used as a loading control . D RT - qPCR analysis of the expression of pluripotency and lineage - specific markers in Day7 FBXW7 - induced EBs at the presence or absence of 10 ng / ml Dox . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . E Immunostaining analysis of the expression of pluripotency marker POU5F1 , lineage - specific markers FOXA2 , T and NESTIN in Day7 FBXW7 - induced EBs at the presence or absence of 10 ng / ml Dox . Scale bars represent 100 μm . G RT - qPCR analysis the expression of pluripotency and lineage - specific markers in Day7 c - MYC - induced EBs at the presence or absence of 10 ng / ml Dox . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . H Immunostaining analysis of the expression of pluripotency marker POU5F1 , lineage - specific markers FOXA2 , T and NESTIN in Day7 c - MYC - induced EBs at the presence or absence of 10 ng / ml Dox . Scale bars represent 100 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 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 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 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 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

Figure 5 A Western blot analysis showing dynamic expression patterns of FBXW7 and c-MYC during the indicated time points of EB induction. α-TUBULIN was used as a loading control. B Western blot analysis showing FBXW7 and c-MYC expression in control, Mettl5 KO (KO-6B) and KO+HA-MT5WT Day7 EBs, respectively. α-TUBULIN was used as a loading control.D RT-qPCR analysis of the expression of pluripotency and lineage-specific markers in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.E Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.G RT-qPCR analysis the expression of pluripotency and lineage-specific markers in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.H Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.


Figure 1E . Total RNAs from naïve CD4 + T cells , PHA - stimulated CD4 + T cells and resting memory CD4 + T cells were extracted and proceeded to RT - qPCR to quantitate the relative expression of CBX4 . The expression of CBX4 in PHA - stimulated and resting memory CD4 + T cells was normalized to naïve CD4 + T cells . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by one - way ANOVA with Tukey ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 1E. Total RNAs from naïve CD4+ T cells, PHA-stimulated CD4+ T cells and resting memory CD4+ T cells were extracted and proceeded to RT-qPCR to quantitate the relative expression of CBX4. The expression of CBX4 in PHA-stimulated and resting memory CD4+ T cells was normalized to naïve CD4+ T cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.


Figure 3B . Super - resolution SIM image of CBX4 in HEK293T cells . DAPI was used to dye DNA which was colored into blue . FITC - tagged antibodies were used to label endogenous CBX4 which was colored into green . Data information : Scale bars in ( B ) 5 μm . All the samples were imaged to obtain at least three sets of images . C . The distribution of CBX4 bodies diameters . The black line indicated the median value of diameters which was 512 nm . Data were collected from three cells of three independent samples . F - L . RFP - tagged EZH2 , RING1B , SUMO4 , SUZ12 , CHAF1A , DNMT1 and HP1α were co - overexpressed with GFP - tagged CBX4 in HEK293T cells , respectively . SIM imaging was performed for each combination . The line scan profiles of SIM images which showed co - localization and non - co - localization between CBX4 and its partners were shown on the right of each panel . The red arrows in merged images indicated the positions where the line scans profiled . M - O . GFP - tagged CBX4 was overexpressed in HEK293T cells , followed by treating with AF568 - tagged antibodies against H3K4me3 , H3K9me3 and H3K27me3 , respectively . CBX4 and each histone modification were imaged with SIM . Line scan profiles were shown on the right of each panel . Red arrows in merged images indicated the positions where line scans profiled . Data information : Scale bars 5 μm . All the samples were imaged to obtain at least three sets of images .

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 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

Figure 3B. Super-resolution SIM image of CBX4 in HEK293T cells. DAPI was used to dye DNA which was colored into blue. FITC-tagged antibodies were used to label endogenous CBX4 which was colored into green. Data information: Scale bars in (B) 5 μm. All the samples were imaged to obtain at least three sets of images.C. The distribution of CBX4 bodies diameters. The black line indicated the median value of diameters which was 512 nm. Data were collected from three cells of three independent samples.F-L. RFP-tagged EZH2, RING1B, SUMO4, SUZ12, CHAF1A, DNMT1 and HP1α were co-overexpressed with GFP-tagged CBX4 in HEK293T cells, respectively. SIM imaging was performed for each combination. The line scan profiles of SIM images which showed co-localization and non-co-localization between CBX4 and its partners were shown on the right of each panel. The red arrows in merged images indicated the positions where the line scans profiled. M-O. GFP-tagged CBX4 was overexpressed in HEK293T cells, followed by treating with AF568-tagged antibodies against H3K4me3, H3K9me3 and H3K27me3, respectively. CBX4 and each histone modification were imaged with SIM. Line scan profiles were shown on the right of each panel. Red arrows in merged images indicated the positions where line scans profiled. Data information: Scale bars 5 μm. All the samples were imaged to obtain at least three sets of images.


Figure 4A . FRAP images of CBX4 bodies to indicate the internal diffusion and diffusion across boundary properties . GFP - tagged CBX4 was overexpressed in HEK293T cells . These live cells were proceeded to time series imaging 24 hours post transfection . Six GFP - CBX4 bodies were circled and marked as region of interest 1 ( ROI 1 ) , ROI 2 , ROI 3 , ROI 4 , ROI 5 and ROI 6 . Three CBX4 bodies ( ROI 4 , ROI 5 and ROI 6 ) were bleached with strong 488 nm laser pulse . Another three unbleached CBX4 bodies ( ROI 1 , ROI 2 and ROI 3 ) were set as control . Images were captured every 4 s . The right histogram showed relative fluorescence intensities of unbleached and bleached CBX4 bodies in each time point . Data information : Data represented mean ± SEM in biological triplicate in ( A ) . Scale bars in ( A ) represented 5 μm . All the samples were imaged to obtain at least three sets of images . P . RFP - tagged EZH2 was co - overexpressed with GFP - tagged CBX4 mutant named CBX4mut which was CBX4 - LTVTmut . SIM images were captured . The line scan profile was shown on the right . The red arrow within merged image indicated the line scan position . Data information represented 5 μm . All the samples were imaged to obtain at least three sets of images .

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 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

Figure 4A. FRAP images of CBX4 bodies to indicate the internal diffusion and diffusion across boundary properties. GFP-tagged CBX4 was overexpressed in HEK293T cells. These live cells were proceeded to time series imaging 24 hours post transfection. Six GFP-CBX4 bodies were circled and marked as region of interest 1 (ROI 1), ROI 2, ROI 3, ROI 4, ROI 5 and ROI 6. Three CBX4 bodies (ROI 4, ROI 5 and ROI 6) were bleached with strong 488 nm laser pulse. Another three unbleached CBX4 bodies (ROI 1, ROI 2 and ROI 3) were set as control. Images were captured every 4 s. The right histogram showed relative fluorescence intensities of unbleached and bleached CBX4 bodies in each time point. Data information: Data represented mean ± SEM in biological triplicate in (A). Scale bars in (A) represented 5 μm. All the samples were imaged to obtain at least three sets of images.P. RFP-tagged EZH2 was co-overexpressed with GFP-tagged CBX4 mutant named CBX4mut which was CBX4-LTVTmut. SIM images were captured. The line scan profile was shown on the right. The red arrow within merged image indicated the line scan position. Data information represented 5 μm. All the samples were imaged to obtain at least three sets of images.


Figure 5G . HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO1 , Flag - tagged UBC9 or Flag - tagged CBX4 , and siNC . In the last group , HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO1 , Flag - tagged UBC9 and siCBX4 . EZH2 was IP with anti - HA beads . Both total and IP samples were IB with antibodies against HA , Flag and GAPDH . The expression ratios of SUMO1 - EZH2 within each group were showed below the figure . H . HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO4 , Flag - tagged UBC9 or Flag - tagged CBX4 , and siNC . In the last group , HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO4 , Flag - tagged UBC9 and siCBX4 . EZH2 was IP with anti - HA beads . Both total and IP samples were IB with antibodies against HA , Flag and GAPDH . The expression ratios of SUMO4 - EZH2 within each group were showed below the figure .

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Figure 5G. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO1-EZH2 within each group were showed below the figure. H. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO4-EZH2 within each group were showed below the figure.


Figure 6B . Two micrograms ( 2 μg ) of HA - tagged EZH2 were co - overexpressed with 4 μg of Flag - tagged SUMO4 , 250 ng of Flag - tagged UBC9 , 250 ng of Flag - tagged CBX4 or CBX4mut within 6 cm dishes . The CBX4mut was the LLPS - deficient CBX4 . EZH2 was IP with anti - HA beads . Both total and IP samples were IB with antibodies against HA , Flag and GAPDH . The expression ratios of SUMO4 - EZH2 were marked below the panel . D . The endogenous EZH2 in TZM - bl cells within 6 cm dishes was knocked down by siRNAs targeting 3 ' UTR of EZH2 mRNA , followed by the overexpression of 2 μg of EZH2 , 4 μg of Flag - tagged SUMO4 , 250 ng of Flag - tagged UBC9 or 250 ng of Flag - tagged CBX4 . EZH2 was IP with anti - EZH2 antibodies . IP samples were IB with anti - EZH2 antibodies . Total samples were IB with anti - EZH2 , anti - H3K27me3 , anti - Histone H3 and anti - GAPDH antibodies . The expression ratios of SUMO4 - EZH2 and H3K27me3 were marked below the panel . E . In the first group , HEK293T cells within 6 cm dishes were transfected with empty vectors . In the second group , 3 μg of EZH2 was overexpressed . In the third group , 3 μg of EZH2 was co - overexpressed with 2 μg of EED and 2 μg of SUZ12 . In the fourth and sixth group , 3 μg of EZH2 was co - overexpressed with 2 μg of EED , 2 μg of SUZ12 , 2 μg of SUMO4 , 500 ng of UBC9 and 2 μg of CBX4 . In the fifth group , 3 μg of EZH2mut ( Y731D ) was co - overexpressed with 2 μg of EED , 2 μg of SUZ12 , 2 μg of SUMO4 , 500 ng of UBC9 and 2 μg of CBX4 . Forty - eight hours post transfection , EZH2 and EZH2mut were IP with anti - EZH2 , followed by the incubation with 2 μg of mononucleosomes ( H3 . 1 ) and 20 μM of the cofactor S - adenosyl - L - methionine ( SAM ) to measure the methyltransferase activity of EZH2 . The IP reaction in the sixth group was incubated with 2 μg of mononucleosomes ( H3 . 1 K27M ) and 20 μM of SAM . Both total and IP samples were IB with anti - EZH2 , GAPDH , H3K27me3 and H3 . 1 antibodies . The purities of recombinant mononucleosomes which included H3 . 1 , H2B , H2A and H4 were verified by Coomassie blue staining . The expression ratios of SUMO4 - EZH2 and H3K27me3 were marked below the panel .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6B. Two micrograms (2 μg) of HA-tagged EZH2 were co-overexpressed with 4 μg of Flag-tagged SUMO4, 250 ng of Flag-tagged UBC9, 250 ng of Flag-tagged CBX4 or CBX4mut within 6 cm dishes. The CBX4mut was the LLPS-deficient CBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO4-EZH2 were marked below the panel.D. The endogenous EZH2 in TZM-bl cells within 6 cm dishes was knocked down by siRNAs targeting 3'UTR of EZH2 mRNA, followed by the overexpression of 2 μg of EZH2, 4 μg of Flag-tagged SUMO4, 250 ng of Flag-tagged UBC9 or 250 ng of Flag-tagged CBX4. EZH2 was IP with anti-EZH2 antibodies. IP samples were IB with anti-EZH2 antibodies. Total samples were IB with anti-EZH2, anti-H3K27me3, anti-Histone H3 and anti-GAPDH antibodies. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.E. In the first group, HEK293T cells within 6 cm dishes were transfected with empty vectors. In the second group, 3 μg of EZH2 was overexpressed. In the third group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED and 2 μg of SUZ12. In the fourth and sixth group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. In the fifth group, 3 μg of EZH2mut (Y731D) was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. Forty-eight hours post transfection, EZH2 and EZH2mut were IP with anti-EZH2, followed by the incubation with 2 μg of mononucleosomes (H3.1) and 20 μM of the cofactor S-adenosyl-L-methionine (SAM) to measure the methyltransferase activity of EZH2. The IP reaction in the sixth group was incubated with 2 μg of mononucleosomes (H3.1 K27M) and 20 μM of SAM. Both total and IP samples were IB with anti-EZH2, GAPDH, H3K27me3 and H3.1 antibodies. The purities of recombinant mononucleosomes which included H3.1, H2B, H2A and H4 were verified by Coomassie blue staining. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.


Figure 7A . PHA - activated primary CD4 + T cells were infected with wildtype HIV - 1 viruses which harbored a GFP ORF after Nef gene , followed by infecting with shluc and shCBX4 lentiviruses respectively . The percentages of GFP - positive cells , which were HIV - 1 - expressing cells , were monitored every three days . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B , C . Primary CD4 + T cells were treated as in ( A ) . On Day 6 post infection with HIV - 1 viruses and shRNA lentiviruses , ChIP assays with antibodies against H3K27me3 and H2AK119Ub were conducted in both shluc and shCBX4 groups . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . D . PHA - activated CD4 + T cells were infected with wildtype HIV - 1 viruses as in ( A ) . One group of HIV - 1 - infected CD4 + T cells were left untreated . One group of HIV - 1 - infected cells were infected with shluc . Another group of HIV - 1 - infected cells were infected with shCBX4 . Two weeks later , one part of HIV - 1 - infected CD4 + T cells were reactivated by αCD3 / αCD28 / IL - 2 . One part of shluc - and shCBX4 - infected cells were co - stimulated with LRA JQ - 1 . GFP - positive cells in each group were measured by flow cytometry to indicate reactivated HIV - 1 - infected cells . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by one - way ANOVA with Tukey ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E - G . Primary CD4 + T cells , which contained latent HIV - 1 - infected cells , were isolated from three HIV - 1 - infected individuals . One group of cells were untreated . One group of cells were activated with αCD3 / αCD28 / IL - 2 . Another two groups of cells were transfected with siNC and siCBX4 utilizing 4D - Nucleofector System respectively . Three days post transfection , the amounts of intracellular HIV - 1 RNAs within each group were quantitated by RT - qPCR and represented as 103 copies per million CD4 + T cells . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by one - way ANOVA with Tukey ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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 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

Figure 7A. PHA-activated primary CD4+ T cells were infected with wildtype HIV-1 viruses which harbored a GFP ORF after Nef gene, followed by infecting with shluc and shCBX4 lentiviruses respectively. The percentages of GFP-positive cells, which were HIV-1-expressing cells, were monitored every three days. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.B, C. Primary CD4+ T cells were treated as in (A). On Day 6 post infection with HIV-1 viruses and shRNA lentiviruses, ChIP assays with antibodies against H3K27me3 and H2AK119Ub were conducted in both shluc and shCBX4 groups. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.D. PHA-activated CD4+ T cells were infected with wildtype HIV-1 viruses as in (A). One group of HIV-1-infected CD4+ T cells were left untreated. One group of HIV-1-infected cells were infected with shluc. Another group of HIV-1-infected cells were infected with shCBX4. Two weeks later, one part of HIV-1-infected CD4+ T cells were reactivated by αCD3/αCD28/IL-2. One part of shluc- and shCBX4-infected cells were co-stimulated with LRA JQ-1. GFP-positive cells in each group were measured by flow cytometry to indicate reactivated HIV-1-infected cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.E-G. Primary CD4+ T cells, which contained latent HIV-1-infected cells, were isolated from three HIV-1-infected individuals. One group of cells were untreated. One group of cells were activated with αCD3/αCD28/IL-2. Another two groups of cells were transfected with siNC and siCBX4 utilizing 4D-Nucleofector System respectively. Three days post transfection, the amounts of intracellular HIV-1 RNAs within each group were quantitated by RT-qPCR and represented as 103 copies per million CD4+ T cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.


Fig . 1 . The emerging timeline for highly pathogenic viruses and the proposed Disease X . b . Schematic representation of SARS - CoV - 2 S protein . Its S1 subunit contains NTD ( 14 - 305 aa ) , RBD ( 319 - 541 aa ) , and RBM ( 437 - 508 aa ) . Its S2 subunit contains FP ( 788 - 806 aa ) , HR1 ( 912 - 984 aa ) , HR2 ( 1163 - 1213 aa ) , TM ( 1214 - 1237 aa ) and CP ( 1238 - 1273 aa ) . c . The formation of syncytium in Huh - 7 cells 24 h after SARS - CoV - 2 infection , with scale bar of 200 μm . d . Images of SARS - CoV - and SARS - CoV - 2 S - mediated cell - cell fusion on 293T / ACE2 cells at 2 h ( left ) and 24 h ( right ) . e . SARS - CoV ( I - II ) and SARS - CoV - 2 ( III - IV ) S - mediated syncytium formation on 293T / ACE2 cells at 48 h . f . SARS - CoV ( I - II ) and SARS - CoV - 2 ( III - IV ) S - mediated syncytium formation on Huh - 7 cells at 48 h . Scale bar equals 400 μm in d - f .

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Fig. 1.The emerging timeline for highly pathogenic viruses and the proposed Disease X. b. Schematic representation of SARS-CoV-2 S protein. Its S1 subunit contains NTD (14-305 aa), RBD (319-541 aa), and RBM (437-508 aa). Its S2 subunit contains FP (788-806 aa), HR1 (912-984 aa), HR2 (1163-1213 aa), TM (1214-1237 aa) and CP (1238-1273 aa). c. The formation of syncytium in Huh-7 cells 24 h after SARS-CoV-2 infection, with scale bar of 200 μm.d. Images of SARS-CoV- and SARS-CoV-2 S-mediated cell-cell fusion on 293T/ACE2 cells at 2 h (left) and 24 h (right).e. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on 293T/ACE2 cells at 48 h. f. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on Huh-7 cells at 48 h. Scale bar equals 400 μm in d-f.


Fig . 4 . b . SARS - CoV - 2 S protein - mediated cell - cell fusion in the presence of EK1 - scramble ( I ) , EK1 ( II ) , EK1C ( III ) , and EK1P ( IV ) at 2 . 5 μM ( scale bar : 400 μm ) . c . Inhibitory activity of EK1 - scramble , EK1 , EK1C and EK1P against SARS - CoV - 2 S - mediated cell - cell fusion . e . Inhibitory activity of EK1 - lipopeptides on SARS - CoV - 2 S - mediated cell - cell fusion . f . Inhibitory activity of EK1 - lipopeptides on SARS - CoV - 2 PsV infection . Experiments were repeated twice , and the data are expressed as means ± SD ( error bar ) .

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Fig. 4.b. SARS-CoV-2 S protein-mediated cell-cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 μm).c. Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell-cell fusion.e. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell-cell fusion.f. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar).


Fig . 5 . HCoVs . a to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of SARS - CoV ( a ) HCoVs . a to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of MERS - CoV ( b ) HCoVs . a to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of HCoV - OC43 ( c ) HCoVs . a to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of HCoV - 229E ( d ) to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of HCoV - NL63 ( e ) to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of WIV1 ( f ) to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of Rs3367 ( g ) to h . Inhibitory activity of EK1C4 in cell - cell fusion mediated by the S proteins of SHC014 ( h ) . i to o . Inhibitory activity of EK1C4 in PsV infection assays against SARS - CoV ( i ) to o . Inhibitory activity of EK1C4 in PsV infection assays against MERS - CoV ( j ) Inhibitory activity of EK1C4 in PsV infection assays against HCoV - OC43 ( k ) Inhibitory activity of EK1C4 in PsV infection assays against HCoV - 229E ( l ) Inhibitory activity of EK1C4 in PsV infection assays against , NL63 ( m ) Inhibitory activity of EK1C4 in PsV infection assays against WIV1 ( n ) Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 ( o ) .

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Fig. 5.HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SARS-CoV (a)HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of MERS-CoV (b)HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-OC43 (c)HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-229E (d)to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-NL63 (e)to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of WIV1 (f)to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of Rs3367 (g)to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SHC014 (h).i to o. Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i)to o. Inhibitory activity of EK1C4 in PsV infection assays against MERS-CoV (j)Inhibitory activity of EK1C4 in PsV infection assays against HCoV-OC43(k)Inhibitory activity of EK1C4 in PsV infection assays against HCoV-229E (l)Inhibitory activity of EK1C4 in PsV infection assays against , NL63 (m)Inhibitory activity of EK1C4 in PsV infection assays against WIV1 (n)Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 (o).


Fig . 6 . Inhibitory activity of EK1 on live HCoV replication for SARS - CoV - 2 ( a ) , MERS - CoV ( b ) , HCoV - OC43 ( c ) , HCoV - 229E ( d ) , and HCoV - NL63 ( e ) . In vivo prophylactic efficacy of EK1C4 against HCoV - OC43 infection in mice . Body weight change ( f ) and survival curves ( g ) of mice challenged with HCoV - OC43 . h - i . In vivo therapeutic efficacy of EK1C4 against HCoV - OC43 infection in mice . Body weight change ( h ) and survival curves ( i ) of mice challenged with HCoV - OC43 . Experiments were repeated twice , and the data are expressed as means ± SD .

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Fig. 6.Inhibitory activity of EK1 on live HCoV replication for SARS-CoV-2 (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), and HCoV-NL63 (e).In vivo prophylactic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (f) and survival curves (g) of mice challenged with HCoV-OC43. h-i. In vivo therapeutic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (h) and survival curves (i) of mice challenged with HCoV-OC43. Experiments were repeated twice, and the data are expressed as means ± SD.


Figure 1 ( A - D ) Color fundus photographs and SW - AF examinations on four patients of β - thalassemia subject to chelation therapy by DFO for at least 16 years . Fundus phenotypes , including RPE mottling and depigmentation in the macula ( Case Ⅰ ) ( Gelman et al , 2014 ) , choroidal sclerosis in the perimacular areas ( Case Ⅱ ) , peripapillary ( Case Ⅲ and Ⅳ ) , and subretinal pigmentation ( Case Ⅳ ) were evaluated . RPE lesions including concentric distribution of stippled hyper - autofluorescence at the macula ( Case Ⅰ ) , large areas of hypo - autofluorescent regions at parapapillary or perimacular area ( Case Ⅱ and III ) , and extensive RPE loss in both eyes ( Case Ⅳ ) were observed by SW - AF .

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Figure 1(A - D) Color fundus photographs and SW-AF examinations on four patients of β-thalassemia subject to chelation therapy by DFO for at least 16 years. Fundus phenotypes, including RPE mottling and depigmentation in the macula (Case Ⅰ) (Gelman et al, 2014), choroidal sclerosis in the perimacular areas (Case Ⅱ), peripapillary (Case Ⅲ and Ⅳ), and subretinal pigmentation (Case Ⅳ) were evaluated. RPE lesions including concentric distribution of stippled hyper-autofluorescence at the macula (Case Ⅰ), large areas of hypo-autofluorescent regions at parapapillary or perimacular area (Case Ⅱ and III), and extensive RPE loss in both eyes (Case Ⅳ) were observed by SW-AF.


Figure 2 ( A - C ) Adult C57BL / 6J mice ( four months old ) were intraperitoneally injected with DFO at 100 mg / kg three times a week for three months in a row . SW - AF images were acquired longitudinally at one , two and three months post injection . ( I , J ) Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death . Hoechst was used for nuclear staining . Untreated mice of similar age were used as the healthy controls . Flat mount images were captured by 10 × confocal microscopy and stitched together . ( K - N ) The RPE was dissected from the mice injected with DFO for three and nine months respectively and stained with phalloidin . Age - matched untreated mice were included as the controls . The images were taken at 40 × magnification .

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Figure 2(A - C) Adult C57BL/6J mice (four months old) were intraperitoneally injected with DFO at 100 mg/kg three times a week for three months in a row. SW-AF images were acquired longitudinally at one, two and three months post injection.(I, J) Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death. Hoechst was used for nuclear staining. Untreated mice of similar age were used as the healthy controls. Flat mount images were captured by 10× confocal microscopy and stitched together.(K - N) The RPE was dissected from the mice injected with DFO for three and nine months respectively and stained with phalloidin. Age-matched untreated mice were included as the controls. The images were taken at 40× magnification.


Figure 3 ( A - D ) Light microscopic examination of the iRPE cells 24 and 48 hours post treatment . Scale bar : 50 µm . ( E - H ) iRPE cells were stained with ZO - 1 24 and 48 hours post treatment . Untreated iRPE cells were used as the control . ( I - L ) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment . Untreated iRPE cells were included as the control . The cells were counterstained with Hoechst .

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Figure 3(A - D) Light microscopic examination of the iRPE cells 24 and 48 hours post treatment. Scale bar: 50 µm.(E - H) iRPE cells were stained with ZO-1 24 and 48 hours post treatment. Untreated iRPE cells were used as the control.(I - L) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment. Untreated iRPE cells were included as the control. The cells were counterstained with Hoechst.


Figure 4 ( A ) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment . The iRPE cells supplemented with 0 . 1 % DMSO in the culture media were used as the control . Actin was used as the loading control . HIF1α ( post - translationally modified ) was predicted to be ~ 100 kDa ; HIF2α ( post translationally modified ) was predicted to be ~ 120 kDa . ( B ) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment . Untreated iRPE cells were used as the normal control . Histone H3 was used as the loading control . (

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Figure 4(A) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the control. Actin was used as the loading control. HIF1α (post-translationally modified) was predicted to be ~100 kDa; HIF2α (post translationally modified) was predicted to be ~ 120 kDa. (B) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment. Untreated iRPE cells were used as the normal control. Histone H3 was used as the loading control. (


Figure 5 ( A ) Ferrous iron was measured . The statistics are analyzed by one - way ANOVA with the Tukey test . The results are presented as mean ± S . E . M . , n = 3 iRPE lines for each group . * p < 0 . 05 . ( B ) The transcripts of ferroptosis - associated markers were determined by qPCR using iRPE cells as the template . The RNA extract from untreated iRPE was included as the control . The level of each transcript was normalized to ACTB . The statistics are analyzed by ratio paired student ' s t - test for each target gene . The results are presented as mean ± S . E . M . , n = 3 iRPE lines for each group . * p < 0 . 05 ; * * p < 0 . 01 . Round dots : untreated iRPE ; square dots : DFO - treated iRPE . ( C ) The level of ROS was measured . The statistics are analyzed by one - way ANOVA with the Tukey test . The results are presented as mean ± S . E . M . , n = 6 iRPE lines for each group . * p < 0 . 05 , * * p < 0 . 01 . DHE : Dihydroethidium . ( D ) Mitochondrial respiration of the iRPE cells with DFO treatment was determined by seahorse extracellular flux assay . OCR was determined over a course of time . Each data point is shown as mean ± S . E . M . , n = 4 iRPE lines for each group . OCR : oxygen consumption rate ; FCCP : carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ; Rot : rotenone ; AA : antimycin A .

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Figure 5(A) Ferrous iron was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05.(B) The transcripts of ferroptosis-associated markers were determined by qPCR using iRPE cells as the template. The RNA extract from untreated iRPE was included as the control. The level of each transcript was normalized to ACTB. The statistics are analyzed by ratio paired student's t-test for each target gene. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05; ** p < 0.01. Round dots: untreated iRPE; square dots: DFO-treated iRPE.(C) The level of ROS was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 6 iRPE lines for each group. * p < 0.05, ** p < 0.01. DHE: Dihydroethidium.(D) Mitochondrial respiration of the iRPE cells with DFO treatment was determined by seahorse extracellular flux assay. OCR was determined over a course of time. Each data point is shown as mean ± S.E.M., n = 4 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.


Figure 6 ( A - C ) Light microscopy of morphological protection of AKG against DFO toxicity on iRPE . The cells were treated with DFO at 100 mM for 48 hours ( B ) . AKG was supplemented at 10 mM simultaneously ( C ) . The control group was supplemented with 0 . 1 % DMSO ( A ) . Scale bar : 100 µm . ( D - F ) The DFO - treated iRPE cells with or without AKG supplementation were subject to phalloidin staining ( E and F ) . The iRPE cells treated with 0 . 1 % DMSO - containing media were included as the controls ( D ) . The images were captured at 40 × magnification . Scale bar : 30 µm . ( G - I ) Detection of cell death in the DFO - treated iRPE cells with or without AKG supplementation by TUNEL staining . Hoechst was used for nuclear staining . The images were captured at 40 × magnification . Scale bar : 30 µm . ( J ( J - L ) SW - AF was performed longitudinally on the mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months .

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Figure 6(A - C) Light microscopy of morphological protection of AKG against DFO toxicity on iRPE. The cells were treated with DFO at 100 mM for 48 hours (B). AKG was supplemented at 10 mM simultaneously (C). The control group was supplemented with 0.1% DMSO (A). Scale bar: 100 µm. (D - F) The DFO-treated iRPE cells with or without AKG supplementation were subject to phalloidin staining (E and F). The iRPE cells treated with 0.1% DMSO-containing media were included as the controls (D). The images were captured at 40× magnification. Scale bar: 30 µm. (G - I) Detection of cell death in the DFO-treated iRPE cells with or without AKG supplementation by TUNEL staining. Hoechst was used for nuclear staining. The images were captured at 40× magnification. Scale bar: 30 µm. (J(J - L) SW-AF was performed longitudinally on the mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months.


Figure 7 ( A ) Mitochondrial respiration of the DFO - treated ( 100 mM ) iRPE cells with AKG supplementation ( 10 mM ) for 24 hours was determined by seahorse extracellular flux assay . OCR was measured over a course of time . Each dot is shown as mean ± S . E . M . , n = 5 iRPE lines for each group . OCR : oxygen consumption rate ; FCCP : carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ; Rot : rotenone ; AA : antimycin A . ( G ) The DFO - treated ( 100 mM ) iRPE cells supplemented with AKG ( 10 mM ) for 48 hours were lysed for immunoblotting against HIF2α . The iRPE cells supplemented with 0 . 1 % DMSO in the culture media were used as the controls . HIF2α ( post translationally modified ) was predicted to be ~ 120 kDa . Actin was included as the loading control . ( H ) RPE lysate from the one - year - old mice subject to 10 - month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α . The RPE of the age - matched untreated mice was included as the control . HIF2α ( degraded ) was predicted to be 40 - 80 kDa . Actin was used as the loading control .

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Figure 7(A) Mitochondrial respiration of the DFO-treated (100 mM) iRPE cells with AKG supplementation (10 mM) for 24 hours was determined by seahorse extracellular flux assay. OCR was measured over a course of time. Each dot is shown as mean ± S.E.M., n = 5 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.(G) The DFO-treated (100 mM) iRPE cells supplemented with AKG (10 mM) for 48 hours were lysed for immunoblotting against HIF2α. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the controls. HIF2α (post translationally modified) was predicted to be ~ 120 kDa. Actin was included as the loading control. (H) RPE lysate from the one-year-old mice subject to 10-month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α. The RPE of the age-matched untreated mice was included as the control. HIF2α (degraded) was predicted to be 40 - 80 kDa. Actin was used as the loading control.


Figure 2A - L ) Serum starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F - domains for 15 minutes and analyzed by immunoblotting for pAKT ( S473 ) , pERK1 / 2 ( T202 / Y204 ) , and pFAK ( Y397 ) phosphorylation . A , E , I ) pAKT , B , F , J ) pERK1 / 2 , and C , G , K ) pFAK phosphorylation levels are quantified and normalized to Icos - cage signaling levels . Appendix Figure S1B shows the detection of two bands expected for pERK1 / 2 . Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F - domain : D ) helical bundles , H ) trimeric and tetrameric homo - oligomers and tetrahedral nanocages , L ) Icosahedral nanocages and trimeric subunit . M - P ) Dose - response curves , EC50 , and EMAX for pAKT and pERK1 / 2 activation . The curves , EC50 and EMAX values are calculated using Prism , GraphPad . N , P ) Darker red color indicate lower EC50 and higher EMAX values .

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Figure 2A-L) Serum starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F-domains for 15 minutes and analyzed by immunoblotting for pAKT (S473), pERK1/2 (T202/Y204), and pFAK (Y397) phosphorylation. A, E, I) pAKT, B, F, J) pERK1/2, and C, G, K) pFAK phosphorylation levels are quantified and normalized to Icos-cage signaling levels. Appendix Figure S1B shows the detection of two bands expected for pERK1/2. Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F-domain: D) helical bundles, H) trimeric and tetrameric homo-oligomers and tetrahedral nanocages, L) Icosahedral nanocages and trimeric subunit.M-P) Dose-response curves, EC50, and EMAX for pAKT and pERK1/2 activation. The curves, EC50 and EMAX values are calculated using Prism, GraphPad. N, P) Darker red color indicate lower EC50 and higher EMAX values.


Figure 3C ) Comparison of cell migration induced by Ang1 , Ang2 , and designed F - domain scaffolds after 12 hours of treatment . The change in wound area is normalized to vehicle control . All experiments have at least 3 biological replicates . ( * ) is the comparison between F - domain scaffolds vs Ang1 , and ( # ) is the comparison between F - domain scaffolds vs Vehicle . E ) Representative increase in vascular stability by Icos1 . The number of nodes ( blue ) , meshes ( red ) , and tubes ( green ) were quantified using angiogenesis analyzer plugin in ImageJ . Scale bar is100 µm .

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Figure 3C) Comparison of cell migration induced by Ang1, Ang2, and designed F-domain scaffolds after 12 hours of treatment. The change in wound area is normalized to vehicle control. All experiments have at least 3 biological replicates. (*) is the comparison between F-domain scaffolds vs Ang1, and (#) is the comparison between F-domain scaffolds vs Vehicle.E) Representative increase in vascular stability by Icos1. The number of nodes (blue), meshes (red), and tubes (green) were quantified using angiogenesis analyzer plugin in ImageJ. Scale bar is100 µm.


Figure 4A - C ) Representative images of FOXO1 localization upon scaffolds administration . Scale bars are 10 µm D ) Quantifications of nuclear FOXO1 shown as percent of cells with nuclear FOXO1 stain , 100 cells were counted per sample . E ) Cells were stimulated with scaffolds or angiopoietins and stained with Tie2 antibodies for analyzing plasma membrane Tie2 clusters . Dark gray bars : Quantification shows the percent % of total cells that exhibit large Tie2 clusters on the plasma membrane . K , L ) Representatives immunoblot gel ( K ) and quantifications ( L ) for α5 integrin knockdown experiments using 20 nM of siRNA .

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Figure 4A-C) Representative images of FOXO1 localization upon scaffolds administration. Scale bars are 10 µm D) Quantifications of nuclear FOXO1 shown as percent of cells with nuclear FOXO1 stain, 100 cells were counted per sample. E) Cells were stimulated with scaffolds or angiopoietins and stained with Tie2 antibodies for analyzing plasma membrane Tie2 clusters. Dark gray bars: Quantification shows the percent % of total cells that exhibit large Tie2 clusters on the plasma membrane.K, L) Representatives immunoblot gel (K) and quantifications (L) for α5 integrin knockdown experiments using 20 nM of siRNA.


Figure 5C , D ) CCI - TBI brain sections were stained for reactive astrocytes ( GFAP , blue ) , microglia ( Iba1 , green ) and serum albumin ( magenta ) to highlight tissue margins ( dotted white line ) and quantify secondary damage as a function of lesion area ( E ; * p = 0 . 04 , n = 4 , the mean ± SEM is plotted ) . Bar , 1 mm . E ) Cumulative blood extravasation into brain tissue ( white outline ) was imaged by albumin immunofluorescence to establish an unbiased intensity - threshold mask ( red ) and quantify the area of blood serum extravasation ( mm2 ) in Icos1ø vs . Icos1 treated animals ( * * p = 0 . 01 , n = 4 , the mean ± SEM is plotted ) . Scale bar is 500 µm . F - H ) Brain tissues stained by GFAP - ( magenta ) and CD31 - immunofluorescence ( green ) were imaged by confocal microscopy to measure angiogenesis as a function of vessel diameter ( G ; * * p = 0 . 003 , n = 4 , the boxes show 25th to 75th percentiles and whiskers represent the min and max values . ) and quantify the number of CD31 - vessels ( H ; * * p = 0 . 007 , n = 4 , the mean ± SEM is plotted ) at the site of injury in Icos1ø and Icos1 treated animals 4 days post - injury . Scale bars in G and H is 100 µm . I ) Activation of pAKT ( Magenta ) was examined in Icos1ø and Icos1 treated - animals within or around vascular ( CD31 + , green ) and pericytes ( NG2 + cells , yellow ) of the BBB . High - resolution confocal images ( insets ) reveal the prevalence of activated pAKT ( magenta ) co - stain ( cyan arrows ) in pericytes ( yellow ) of the BBB along vasculature ( green , or dotted outline ) versus reactive neuroglia ( NG2 + pAKT - ) in the parenchyma ( red arrows ) in Icos1ø vs . Icos1 treated animals . Scale bar is 10 µm .

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Figure 5C, D) CCI-TBI brain sections were stained for reactive astrocytes (GFAP, blue), microglia (Iba1, green) and serum albumin (magenta) to highlight tissue margins (dotted white line) and quantify secondary damage as a function of lesion area (E; *p = 0.04, n = 4, the mean ± SEM is plotted ). Bar, 1 mm. E) Cumulative blood extravasation into brain tissue (white outline) was imaged by albumin immunofluorescence to establish an unbiased intensity-threshold mask (red) and quantify the area of blood serum extravasation (mm2) in Icos1ø vs. Icos1 treated animals (**p = 0.01, n = 4, the mean ± SEM is plotted). Scale bar is 500 µm. F-H) Brain tissues stained by GFAP- (magenta) and CD31-immunofluorescence (green) were imaged by confocal microscopy to measure angiogenesis as a function of vessel diameter (G; **p = 0.003, n = 4, the boxes show 25th to 75th percentiles and whiskers represent the min and max values.) and quantify the number of CD31-vessels (H; **p = 0.007, n = 4, the mean ± SEM is plotted) at the site of injury in Icos1ø and Icos1 treated animals 4 days post-injury. Scale bars in G and H is 100 µm.I) Activation of pAKT (Magenta) was examined in Icos1ø and Icos1 treated-animals within or around vascular (CD31+, green) and pericytes (NG2+ cells, yellow) of the BBB. High-resolution confocal images (insets) reveal the prevalence of activated pAKT (magenta) co-stain (cyan arrows) in pericytes (yellow) of the BBB along vasculature (green, or dotted outline) versus reactive neuroglia (NG2+pAKT-) in the parenchyma (red arrows) in Icos1ø vs. Icos1 treated animals. Scale bar is 10 µm.


Figure 1 ( A , B ) Flies form well - organized compound eyes when they carry GMR - GAL4 without a target gene ( A ) but develop rough eyes when GMR - GAL4 drives expression of UAS - Foxn1 ( B ) . Panel B shows the typical severity of the rough - eye phenotype . Data information : Representative phenotypes are shown in all panels . Scale bars , 100 μm . ( C , D ) Bristles are pigmented and properly shaped when flies carry hs - GAL4 without a target gene ( C ) but are often white , bent , and shortened when hs - GAL4 drives expression of UAS - Foxn1 ( D ) . Flies in panels C and D were raised at 25 ° C . Arrows denote examples of white bristles . Data information : Representative phenotypes are shown in all panels . Scale bars , 100 μm .

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Figure 1(A, B) Flies form well-organized compound eyes when they carry GMR-GAL4 without a target gene (A) but develop rough eyes when GMR-GAL4 drives expression of UAS-Foxn1 (B). Panel B shows the typical severity of the rough-eye phenotype. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.(C, D) Bristles are pigmented and properly shaped when flies carry hs-GAL4 without a target gene (C) but are often white, bent, and shortened when hs-GAL4 drives expression of UAS-Foxn1 (D). Flies in panels C and D were raised at 25°C. Arrows denote examples of white bristles. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.


Figure 3AFF4 ( green ) and FOXN1 ( red ) are shown by immunofluorescence in the following wild - type tissue samples : ( B ) murine hair follicles , P9 ; DNA is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 and FOXN1 generates yellow color , visible in cell nuclei of all samples . Arrowheads mark the CH , cortex of the hair ; Scale bars , 20 µm . AFF4 ( green ) and FOXN1 ( red ) are shown by immunofluorescence in the following wild - type tissue samples : ( C ) developing murine epidermis , E16 . 5 ; ( D ) human epidermis , adult ; ( E ) murine thymus , P0 , outer region . DNA is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 and FOXN1 generates yellow color , visible in cell nuclei of all samples . In panel C , the fluorescent signal in the periderm ( P ) is nonspecific , as it is present when primary antibodies are omitted from the staining . Inner regions of the murine thymus ( not shown ) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E . Arrowheads mark the dermal / epidermal border ( C , D ) . CT , cortex of the thymus . Scale bars , 20 µm .

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Figure 3AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (B) murine hair follicles, P9; DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. Arrowheads mark the CH, cortex of the hair; Scale bars, 20 µm.AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (C) developing murine epidermis, E16.5; (D) human epidermis, adult; (E) murine thymus, P0, outer region. DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. In panel C, the fluorescent signal in the periderm (P) is nonspecific, as it is present when primary antibodies are omitted from the staining. Inner regions of the murine thymus (not shown) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E. Arrowheads mark the dermal/epidermal border (C, D). CT, cortex of the thymus. Scale bars, 20 µm.


Figure 4Representative skin phenotypes of wild - type ( WT ) mice shown macroscopically The phenotypes compared are as follows : hair coats , 4 - month - old adultsRepresentative skin phenotypes of Foxn1nu / nu ( Nude ) , or KRT14 - cre ; Aff4flox / flox ( Aff4 cko ) mice are shown macroscopically or histologically after hematoxylin and eosin staining ( H - N ) . The phenotypes compared are as follows : hair coats , 4 - month - old adults ; ( E - G ) vibrissae , P7 ; ( H ) hair follicles with growing hairs , juveniles ; ( I - K ) regressing hair follicles , juveniles ; ( L - N ) epidermis , P9 . In panel H , micrographs from left to right are WT , Nude , and Aff4 cko . In panels I - K , HF marks examples of hair follicles . In panels L - M , arrowheads indicate the dermal / epidermal border ; arrows mark examples of bent hair shafts . Scale bars : H , L - N , 20 µm ; I - K , 40 µm .

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Figure 4Representative skin phenotypes of wild-type (WT) mice shown macroscopically The phenotypes compared are as follows: hair coats, 4-month-old adultsRepresentative skin phenotypes of Foxn1nu/nu (Nude), or KRT14-cre; Aff4flox/flox (Aff4 cko) mice are shown macroscopically or histologically after hematoxylin and eosin staining (H-N). The phenotypes compared are as follows: hair coats, 4-month-old adults; (E-G) vibrissae, P7; (H) hair follicles with growing hairs, juveniles; (I-K) regressing hair follicles, juveniles; (L-N) epidermis, P9. In panel H, micrographs from left to right are WT, Nude, and Aff4 cko. In panels I-K, HF marks examples of hair follicles. In panels L-M, arrowheads indicate the dermal/epidermal border; arrows mark examples of bent hair shafts. Scale bars: H, L-N, 20 µm; I-K, 40 µm.


Figure 5 ( A - C ) Thymi from E18 . 5 embryos are shown : ( A ) intact with genotypes indicated , or ( B , C ) after sectioning , immunofluorescent staining for DLL4 ( red ) , and counterstaining of DNA ( blue ) . Sectioned samples are wild - type ( B ) or Aff4ko / ko ; Foxn1 + / - ( C ) . Scale bars : A , 1 mm ; B - C , 50 µm .

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Figure 5(A-C) Thymi from E18.5 embryos are shown: (A) intact with genotypes indicated, or (B, C) after sectioning, immunofluorescent staining for DLL4 (red), and counterstaining of DNA (blue). Sectioned samples are wild-type (B) or Aff4ko/ko; Foxn1+/- (C). Scale bars: A, 1 mm; B-C, 50 µm.


Figure 6 ( C - E ) KRT86 ( red ) is shown by immunofluorescence in skin sections from wild - type ( WT ) , Foxn1 null , or Aff4 cko mice at P9 . DNA is stained blue . HS marks examples of hair shafts . Scale bar , 40 µm .

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Figure 6(C-E) KRT86 (red) is shown by immunofluorescence in skin sections from wild-type (WT), Foxn1 null, or Aff4 cko mice at P9. DNA is stained blue. HS marks examples of hair shafts. Scale bar, 40 µm.


Figure 7Graphs present pre - mRNA ( B , C ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P6 ( B , C ) All values are derived from 3 independent experiments and presented as mean ± S . D . Pre - mRNA measurements were analyzed statistically using two - way ANOVA with post - hoc Sidak ' s multiple comparison test at a 95 % confidence interval . Graphs present ChIP ( D - G ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 ( D - G ) . ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated . All values are derived from 3 independent experiments and presented as mean ± S . D . ChIP assays were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison test at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

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Figure 7Graphs present pre-mRNA (B, C) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P6 (B, C) All values are derived from 3 independent experiments and presented as mean ± S.D. Pre-mRNA measurements were analyzed statistically using two-way ANOVA with post-hoc Sidak's multiple comparison test at a 95% confidence interval.Graphs present ChIP (D-G) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7 (D-G). ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated. All values are derived from 3 independent experiments and presented as mean ± S.D. ChIP assays were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.


Figure 8 ( B - G ) Graphs present ChIP assays of Krt86 using antibodies to total ( B , C ) , P - Ser5 ( D , E ) , or P - Ser2 ( F , G ) Pol II and skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 . All values are derived from at least 3 independent experiments and presented as mean ± S . D . Assay results were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison test at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

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Figure 8(B-G) Graphs present ChIP assays of Krt86 using antibodies to total (B, C), P-Ser5 (D, E), or P-Ser2 (F, G) Pol II and skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7. All values are derived from at least 3 independent experiments and presented as mean ± S.D. Assay results were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.