( a , b ) Differential interaction of UVRAG with the RINT - 1 - ZW10 - NAG tethering complex and the beclin - 1 - Bif - 1 - PI ( 3 ) KC3 autophagy complex after treatment with rapamycin ( a ) and HBSS ( b ) . Cells ( HEK293T ) treated with rapamycin ( 50 nM ) in a or with HBSS in b were subjected to immunoprecipitation with anti - UVRAG ( first panel ) , anti - RINT - 1 ( second panel ) or anti - PI ( 3 ) KC3 ( third panel ) , followed by immunoblotting with the indicated antibodies . Input ( 10 % ) shows endogenous protein expression . See Supplementary Fig . S9 for uncropped data .

O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

(a,b) Differential interaction of UVRAG with the RINT-1-ZW10-NAG tethering complex and the beclin-1-Bif-1-PI(3)KC3 autophagy complex after treatment with rapamycin (a) and HBSS (b). Cells (HEK293T) treated with rapamycin (50 nM) in a or with HBSS in b were subjected to immunoprecipitation with anti-UVRAG (first panel), anti-RINT-1 (second panel) or anti-PI(3)KC3 (third panel), followed by immunoblotting with the indicated antibodies. Input (10%) shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.


( c ) Gel filtration analysis of UVRAG complex formation under normal conditions ( untreated , UT ) and after rapamycin - induced autophagy ( 50 nM ) . Affinity - purified UVRAG complexes from HCT116 cells stably expressing Flag - UVRAG were fractionated by Superose - 6 gel filtration column and the eluates were analysed by western blotting for UVRAG , RINT - 1 , ZW10 , beclin 1 , PI ( 3 ) KC3 and Bif - 1 proteins . Whole - cell lysates ( 2 . 5 % ) were used as the input ( lane - 1 ) . The elution profile of each protein was quantified by densitometry analysis and normalized . Relative densitometry units were plotted against fraction number . Black arrows indicate the positions of the molecular weight size markers . Red arrows indicate the peak shift of the UVRAG eluates .

O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O

(c) Gel filtration analysis of UVRAG complex formation under normal conditions (untreated, UT) and after rapamycin-induced autophagy (50 nM). Affinity-purified UVRAG complexes from HCT116 cells stably expressing Flag-UVRAG were fractionated by Superose-6 gel filtration column and the eluates were analysed by western blotting for UVRAG, RINT-1, ZW10, beclin 1, PI(3)KC3 and Bif-1 proteins. Whole-cell lysates (2.5%) were used as the input (lane-1). The elution profile of each protein was quantified by densitometry analysis and normalized. Relative densitometry units were plotted against fraction number. Black arrows indicate the positions of the molecular weight size markers. Red arrows indicate the peak shift of the UVRAG eluates.


( a - c ) UVRAG interaction with beclin 1 and PtdIns ( 3 ) P , but not with RINT - 1 , is required for autophagy - induced Atg9 dispersal . ( a ) HeLa cells were stably transfected with control shRNA ( first row ) or UVRAG shRNA ( second - sixth rows ) . The UVRAG - depleted cells were then transfected with empty vector ( second row ) , Flag - UVRAG ( third row ) , UVRAGΔ270 − 442 mutant ( fourth row ) , UVRAGK78A / R82A mutant ( fifth row ) or UVRAGΔCCD mutant ( sixth row ) , and incubated in complete medium ( untreated , UT , first panel ) , starvation medium ( HBSS 2 h , third panel ) , or treated with rapamycin ( 50 nM 2 h , second panel ) or SMER28 ( 50 μM , 2 h , fourth panel ) . The distribution pattern of endogenous Atg9 ( green ) in the UVRAG ( wild - type or mutant ) - transduced cells ( red ) was analysed by confocal microscopy ( a ) . Asterisks denote condensed ( non - dispersed ) Atg9 in UVRAG - depleted HeLa cells and arrows denote the cells complemented with wild - type or mutant UVRAG .

O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O

(a-c) UVRAG interaction with beclin 1 and PtdIns(3)P, but not with RINT-1, is required for autophagy-induced Atg9 dispersal. (a) HeLa cells were stably transfected with control shRNA (first row) or UVRAG shRNA (second-sixth rows). The UVRAG-depleted cells were then transfected with empty vector (second row), Flag-UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row) or UVRAGΔCCD mutant (sixth row), and incubated in complete medium (untreated, UT, first panel), starvation medium (HBSS 2 h, third panel), or treated with rapamycin (50 nM 2 h, second panel) or SMER28 (50 μM, 2 h, fourth panel). The distribution pattern of endogenous Atg9 (green) in the UVRAG (wild-type or mutant)-transduced cells (red) was analysed by confocal microscopy (a). Asterisks denote condensed (non-dispersed) Atg9 in UVRAG-depleted HeLa cells and arrows denote the cells complemented with wild-type or mutant UVRAG.


( b , c ) The percentage of cells with Atg9 translocation was quantied ( c ) and the expression of UVRAG and actin was determined by immunoblotting ( b ) . Data represent the means : d : ; n D300 cells from three independent experiments ; NS , not signicant ; P < 0 : 01 ; P < 0 : 001 . Scale bars , 10 m .

O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b,c) The percentage of cells with Atg9 translocation was quantied (c) and the expression of UVRAG and actin was determined by immunoblotting (b). Data represent the means:d:; n D300 cells from three independent experiments; NS, not signicant; P <0:01; P <0:001. Scale bars, 10 m.


O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


( d ) Effect of knockdown of UVRAG , beclin 1 andRINT - 1 on the translocation of Atg9 to the LC3 - labelled autophagsomes during autophagy . Control - , UVRAG - , BECN1 - or RINT - 1 - knockdown HeLa cells were incubated in complete medium ( normal condition ) , starvation medium ( HBSS , 2 h ) , or treated with rapamycin ( 100 nM , 2 h ) or SMER28 ( 50 μM , 2 h ) . The cells were then stained for endogenous Atg9 ( green ) and LC3 ( red ) followed by confocal microscopy analysis ( top and bottom left panels ) . The percentage of Atg9 vesicles co - localized with LC3 + - autophagic puncta was quantified ( bottom middle panel ) . Endogenous UVRAG , beclin 1 and RINT - 1 protein expression are shown ( bottom right ) . Data represent mean ± s . d . ; n = 200 cells obtained from three independent experiments . NS , not significant ; * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 . Scale bars , 10 μm .

O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE 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-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Effect of knockdown of UVRAG, beclin 1 andRINT-1 on the translocation of Atg9 to the LC3-labelled autophagsomes during autophagy. Control-, UVRAG-, BECN1- or RINT-1-knockdown HeLa cells were incubated in complete medium (normal condition), starvation medium (HBSS, 2 h), or treated with rapamycin (100 nM, 2 h) or SMER28 (50 μM, 2 h). The cells were then stained for endogenous Atg9 (green) and LC3 (red) followed by confocal microscopy analysis (top and bottom left panels). The percentage of Atg9 vesicles co-localized with LC3+-autophagic puncta was quantified (bottom middle panel). Endogenous UVRAG, beclin 1 and RINT-1 protein expression are shown (bottom right). Data represent mean±s.d.; n = 200 cells obtained from three independent experiments. NS, not significant; *P0.05; **P0.01; ***P0.001. Scale bars, 10 μm.


( B ) H & amp ; amp ; E staining of representative atherosclerotic lesions in aortic root ( upper panel - 4x magnification ) and brachiocephalic arteries ( lower panel - 10x magnification ) at 14 week harvest . Lesion width of roots highlighted with black bars . ( C ) Quantification of root lesion size ( left panel , * p = 0 . 002 ) , and of brachiocephalic lesion size ( right panel , * p = 0 . 001 ) , both quantified as percent of total lumen area . Data in ( C ) are averaged lesion percentages from 4 - 6μm serial sections from each animal , n = 9 , 13 , 15 . All of the data represent the mean + SEM , * p < 0 . 05 versus LNA - control tissue . Level of significance was determined using one - way ANOVA with Bonferroni ' s post test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H&amp;E staining of representative atherosclerotic lesions in aortic root (upper panel-4x magnification) and brachiocephalic arteries (lower panel-10x magnification) at 14 week harvest. Lesion width of roots highlighted with black bars. (C) Quantification of root lesion size (left panel, *p=0.002), and of brachiocephalic lesion size (right panel, *p=0.001), both quantified as percent of total lumen area. Data in (C) are averaged lesion percentages from 4-6μm serial sections from each animal, n=9, 13, 15. All of the data represent the mean + SEM, *p<0.05 versus LNA-control tissue. Level of significance was determined using one-way ANOVA with Bonferroni's post test.


( A ) Movat pentachrome staining of aortic root ( upper panel - 4x magnification ) and brachiocephalic artery fibrous caps ( lower panel - 10x magnification ) at 14 week harvest , caps denoted with arrowheads and black bars ( roots only ) . ( B ) Quantification of aortic root ( left panel , * p = 0 . 005 , # p < 0 . 001 ) and brachiocephalic artery ( right panel * p = 0 . 011 , # p < 0 . 024 ) fibrous caps , graphed as percent of total plaque area .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O

(A) Movat pentachrome staining of aortic root (upper panel-4x magnification) and brachiocephalic artery fibrous caps (lower panel-10x magnification) at 14 week harvest, caps denoted with arrowheads and black bars (roots only). (B) Quantification of aortic root (left panel, *p=0.005, #p<0.001) and brachiocephalic artery (right panel*p=0.011, #p<0.024) fibrous caps, graphed as percent of total plaque area.


( C ) Representative immunofluorescence microscopy ( IF ) staining of smooth muscle actin ( red ) , denoted by white arrowheads , in aortic rootlesions shown at 10x magnification . IF images in ( C ) are representative of data quantified in ( D ) calculated from 3 - 6μm serial sections from animals in independent cohorts . Level of significance was determined using Kruskal - Wallace one - way ANOVA on ranks , * p = 0 . 002 ( D ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative immunofluorescence microscopy (IF) staining of smooth muscle actin (red), denoted by white arrowheads, in aortic rootlesions shown at 10x magnification. IF images in (C) are representative of data quantified in (D) calculated from 3-6μm serial sections from animals in independent cohorts. Level of significance was determined using Kruskal-Wallace one-way ANOVA on ranks, *p=0.002 (D).


( E ) Movat pentachrome staining of aortic root ( upper panel - 4x magnification , * p < 0 . 001 ) and brachiocephalic artery necrotic areas ( lower panel - 10x magnification ) at 14 week harvest , representative necrotic zones denoted by highlighted areas . ( F ) Quantification of aortic root ( left panel , * p < 0 . 001 ) and brachiocephalic artery ( right panel , # p = 0 . 044 ) necrotic areas , graphed as percent of total plaque area .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O

(E) Movat pentachrome staining of aortic root (upper panel-4x magnification, *p<0.001) and brachiocephalic artery necrotic areas (lower panel-10x magnification) at 14 week harvest, representative necrotic zones denoted by highlighted areas. (F) Quantification of aortic root (left panel, *p<0.001) and brachiocephalic artery (right panel, #p=0.044) necrotic areas, graphed as percent of total plaque area.


ApoE - / - common carotid arteries ( CCA ) were dissected into plaque and adjacent non - plaque areas , then RNA isolated . ( A ) Graphed qPCR quantification of collagen 1A1 ( left panel ) and collagen 3A1 ( right panel ) mRNA expression in plaque ( P ) and non - plaque ( NP ) areas of ApoE - / - carotid arteries . Three assays were carried out , and the data represent mRNA expression normalized to NP , and the mean + SEM , n = 4 , 3 , 4 of mice / group . * represents p < 0 . 001 compared with LNA - miR - 29 non - plaque region .

B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O

ApoE-/- common carotid arteries (CCA) were dissected into plaque and adjacent non-plaque areas, then RNA isolated. (A) Graphed qPCR quantification of collagen 1A1 (left panel) and collagen 3A1 (right panel) mRNA expression in plaque (P) and non-plaque (NP) areas of ApoE-/- carotid arteries. Three assays were carried out, and the data represent mRNA expression normalized to NP, and the mean + SEM, n=4, 3, 4 of mice/group. * represents p<0.001 compared with LNA-miR-29 non-plaque region.


Picrosirius red staining of collagen in BCs ( 10x magnification ) ( B ) ; brightfield ( upper panel ) and polarized light ( lower panel ) imaging shown . Collagen quantification in BCs ( C , * p = 0 . 016 ) from polarized light images expressed as a percentage of plaque area .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

Picrosirius red staining of collagen in BCs (10x magnification) (B); brightfield (upper panel) and polarized light (lower panel) imaging shown. Collagen quantification in BCs(C, *p=0.016) from polarized light images expressed as a percentage of plaque area.


Picrosirius red staining of collagen in aortic roots ( 4x magnification ) ( D ) ; brightfield ( upper panel ) and polarized light ( lower panel ) imaging shown . Collagen quantification in roots ( E , * p = 0 . 003 , # p = 0 . 005 ) from polarized light images expressed as a percentage of plaque area .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Picrosirius red staining of collagen in aortic roots (4x magnification) (D); brightfield (upper panel) and polarized light (lower panel) imaging shown. Collagen quantification in roots (E, *p=0.003, #p=0.005) from polarized light images expressed as a percentage of plaque area.


Proteome studies analyze the frequency distribution of protein changes after the transfection of primary murine aortic VSMCs with pre - miR - 29b , anti - miR - 29b , pre - miR - 30c , or anti - miR - 30c . A log2 fold change of − 1 or 1 was used as cutoff . Log2 ( fold change ) is calculated using the normalized spectral counts and averaged for the 3 biological replicates . ( A ) The log ( fold change ) for differentially secreted proteins following overexpression or inhibition of pre - miR - 29b was analyzed , and significantly changed miR - 29 confirmed target transcripts are shown . FDR indicates false discovery rate . The complete list of significant proteins is in ( Appendix Table S1A - B ) .

O O O O O O O O O O O O O O B-ORGANISM B-TISSUE B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proteome studies analyze the frequency distribution of protein changes after the transfection of primary murine aortic VSMCs with pre-miR-29b, anti-miR-29b, pre-miR-30c, or anti-miR-30c. A log2 fold change of −1 or 1 was used as cutoff. Log2 (fold change) is calculated using the normalized spectral counts and averaged for the 3 biological replicates. (A) The log (fold change) for differentially secreted proteins following overexpression or inhibition of pre-miR-29b was analyzed, and significantly changed miR-29 confirmed target transcripts are shown. FDR indicates false discovery rate. The complete list of significant proteins is in (Appendix Table S1A-B).


( B ) Thirty microliters of conditioned medium from VSMCs transfected with pre - miR - 29b and anti - miR - 29b were separated by SDS - PAGE before validation of COL3A1 by immunoblotting . ( C ) Densitometry analysis of COL3A1 signal from blot in ( B ) ( pre - miR , left * p < 0 . 001 , anti - miR , right * p = 0 . 002 ) . All data in ( C ) represent the mean + SEM , * p < 0 . 05 versus other groups in anti - and pre - groups . Level of significance was determined using one - way ANOVA with Bonferroni ' s post test .

O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Thirty microliters of conditioned medium from VSMCs transfected with pre-miR-29b and anti-miR-29b were separated by SDS-PAGE before validation of COL3A1 by immunoblotting. (C) Densitometry analysis of COL3A1 signal from blot in (B) (pre-miR, left *p<0.001, anti-miR, right *p=0.002) . All data in (C) represent the mean + SEM, *p<0.05 versus other groups in anti- and pre- groups. Level of significance was determined using one-way ANOVA with Bonferroni's post test.


A , miR - 203 expression , as determined by qPCR , in five temporal different stages of normal early development : oocyte , 2 - cell embryo , morula , compacted morula and blastocyst . RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR . RNA expression is normalized by a housekeeping miRNA ( miR - 16 ) that maintained invariable during early embryogenesis . Data represent the mean of 6 different qPCR measures ( red bars ) . P = 0 . 05 ( Student ' s t - test ) comparing 2C / morula versus compacted morula / blastocyst .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O O O O B-ORGANISM O B-TISSUE O O B-TISSUE O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O B-TISSUE O

A, miR-203 expression, as determined by qPCR, in five temporal different stages of normal early development: oocyte, 2-cell embryo, morula, compacted morula and blastocyst. RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR. RNA expression is normalized by a housekeeping miRNA (miR-16) that maintained invariable during early embryogenesis. Data represent the mean of 6 different qPCR measures (red bars). P=0.05 (Student's t-test) comparing 2C/morula versus compacted morula/blastocyst.


F , Representative images of embryoid bodies ( EBs ) derived from wild - type iPSCs or ESCs , or from miiPSC and miESCs at day 30 of differentiation . Scale bars , 500 μm . G , Quantification of the percentage of EBs from panel ( F ) presenting internal large cavities and EBs beating during the indicated time course . Data are represented as mean ± s . e . m . ( n = 3 independent experiments ) . * P < 0 . 05 ; * * * P < 0 . 001 ( Student ´ s t - test ) .

O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-CELL_TYPE O B-CELL_TYPE O 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-TISSUE O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Representative images of embryoid bodies (EBs) derived from wild-type iPSCs or ESCs, or from miiPSC and miESCs at day 30 of differentiation. Scale bars, 500 μm. G, Quantification of the percentage of EBs from panel (F) presenting internal large cavities and EBs beating during the indicated time course. Data are represented as mean ± s.e.m. (n=3 independent experiments). *P<0.05; ***P<0.001 (Student´s t-test).


H , Representative images of EBs derived from human iPSCs transiently transfected with either control ( left ) or miR - 203 mimics ( right ) , at different time points during the differentiation process . Scale bars , 500 μm . I , Left panel shows the quantification of EBs size derived from human iPSCs transiently transfected with either control or miR - 203 mimics as in panel ( H ) at different time points during the differentiation process . The percentage of EBs presenting internal large cavities during the indicated time course of differentiation is shown in the right panel . Data are mean ± s . e . m . ( n = 3 independent experiments ) . * * * P < 0 . 001 ( Student ´ s t - test ) .

O O O O O B-TISSUE O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-TISSUE O O O B-ORGANISM B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, Representative images of EBs derived from human iPSCs transiently transfected with either control (left) or miR-203 mimics (right), at different time points during the differentiation process. Scale bars, 500 μm. I, Left panel shows the quantification of EBs size derived from human iPSCs transiently transfected with either control or miR-203 mimics as in panel (H) at different time points during the differentiation process. The percentage of EBs presenting internal large cavities during the indicated time course of differentiation is shown in the right panel. Data are mean ± s.e.m. (n=3 independent experiments). ***P<0.001 (Student´s t-test).


A , Teratoma volume ( mm3 ) 20 - 25 days after subcutaneous injection of wild - type iPSCs or miiPSCs expressing GFP . Data are represented as mean ± s . e . m . ( n = 8 tumors per genotype ; 292 , 8 ± 66 , 67 versus 1216 ± 140 , 1 ; difference between means : 923 , 5 ± 155 , 2 ; 95 % confidence interval : 1256 to - 590 , 7 ) ; * * * P < 0 . 001 ( Student ´ s t - test ) .

O O B-DISEASE O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Teratoma volume (mm3) 20-25 days after subcutaneous injection of wild-type iPSCs or miiPSCs expressing GFP. Data are represented as mean ± s.e.m. (n=8 tumors per genotype; 292,8 ± 66,67 versus 1216 ±140,1; difference between means: 923,5 ± 155,2; 95% confidence interval: 1256 to -590,7); ***P<0.001 (Student´s t-test).


B , Incidence and representative images of specific highly differentiated tissues in teratomas . The number of tumors included in the analysis is indicated in the panel . * * * P < 0 . 001 ( Student ´ s t - test ) . Scale bars , 50 μm .

O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O 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, Incidence and representative images of specific highly differentiated tissues in teratomas. The number of tumors included in the analysis is indicated in the panel. ***P<0.001 (Student´s t-test). Scale bars, 50 μm.


D , Representative example of E - Ls generated after i . p . injection of GFP - expressing miiPSCs . H / E , hematoxylin and eosin . The following antigens were detected by immunohistochemistry : Sox2 ( ectoderm ) , Cd34 ( mesoderm ) , Gata4 ( endoderm ) , AFP and CK8 ( visceral endoderm of the yolk sac ) and Ter119 ( nucleated erythroid cells ) . Scale bars , 500 μm and 100 μm for higher magnifications .

O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O B-GENEPROD O B-TISSUE O O B-GENEPROD O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-GENEPROD O B-SUBCELLULAR B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

D, Representative example of E-Ls generated after i.p. injection of GFP-expressing miiPSCs. H/E, hematoxylin and eosin. The following antigens were detected by immunohistochemistry: Sox2 (ectoderm), Cd34 (mesoderm), Gata4 (endoderm), AFP and CK8 (visceral endoderm of the yolk sac) and Ter119 (nucleated erythroid cells). Scale bars, 500 μm and 100 μm for higher magnifications.


B , Representative images showing the contribution of human control iPSCs or miiPSCs cells to mouse blastocysts . These structures were co - immunostained with ( human ) anti - OCT4 and anti - HuNu antibodies . Td - Tomato , red ; Scale bars , 50 μm .

O O O O O O O O B-ORGANISM O B-CELL_TYPE O O O O B-ORGANISM B-TISSUE O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

B, Representative images showing the contribution of human control iPSCs or miiPSCs cells to mouse blastocysts. These structures were co-immunostained with (human) anti-OCT4 and anti-HuNu antibodies. Td-Tomato, red; Scale bars, 50 μm.


C , Interspecies chimera assays in which the 8C - stage mouse embryos indicated in ( A , B ) , injected with one single human iPSCs and cultured in vitro for 48 h to reach the blastocyst stage , were then transfected to 2 . 5 days post coitum pseudo - pregnant females . The post - implantation mouse conceptuses were dissected at the E9 . 5 developmental stage and analyzed by immunofluorescence . Representative images showing the integration of control human iPSCs ( top ) or miiPSCs ( bottom ) into mouse E9 . 5 embryos . Anti - human HuNu antibody was co - stained with Td - Tomato direct fluorescence to detect human cells in E9 . 5 mouse embryos . Scale bars , 20 μm . D , E , Quantification of the Td - Tomato - ( D ) or HuNu - ( E ) positive area in 40 - 50 different cryosections of these embryos randomly and blindly selected for IF analysis . Data are mean ± s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ´ s t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O B-ORGANISM O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Interspecies chimera assays in which the 8C-stage mouse embryos indicated in (A, B), injected with one single human iPSCs and cultured in vitro for 48 h to reach the blastocyst stage, were then transfected to 2.5 days post coitum pseudo-pregnant females. The post-implantation mouse conceptuses were dissected at the E9.5 developmental stage and analyzed by immunofluorescence. Representative images showing the integration of control human iPSCs (top) or miiPSCs (bottom) into mouse E9.5 embryos. Anti-human HuNu antibody was co-stained with Td-Tomato direct fluorescence to detect human cells in E9.5 mouse embryos. Scale bars, 20 μm. D, E, Quantification of the Td-Tomato-(D) or HuNu-(E) positive area in 40-50 different cryosections of these embryos randomly and blindly selected for IF analysis. Data are mean ± s.e.m. **P<0.01; ***P< 0.001 (Student´s t-test).


B , C , Relative Luciferase Units ( RLU ; normalized to Renilla luciferase and relative to DNA amount ) in 293T cells transfected with DNA constructs carrying the wild - type 3 ´ UTRs from the indicated transcripts ( B ) or the mutated versions of Dnmt3a and Dnmt3b 3 ´ UTRs , downstream of the luciferase reporter ( C ) . Cells were co - transfected with Renilla luciferase as a control of transfection , and a plasmid expressing GFP or miR - 203 - GFP . Data are represented as mean ± s . d . ( n = 3 independent experiments ) .

O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O 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-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

B, C, Relative Luciferase Units (RLU; normalized to Renilla luciferase and relative to DNA amount) in 293T cells transfected with DNA constructs carrying the wild-type 3´UTRs from the indicated transcripts (B) or the mutated versions of Dnmt3a and Dnmt3b 3´UTRs, downstream of the luciferase reporter (C). Cells were co-transfected with Renilla luciferase as a control of transfection, and a plasmid expressing GFP or miR-203-GFP. Data are represented as mean ± s.d. (n=3 independent experiments).


E , Representative images of embryoid bodies ( EBs ) derived from wild - type iPSCs in which the expression of Dnmt3a and Dnmt3b was transiently repressed by siRNAs . Scale bars , 500 μm . Plots show the quantification of the size of EBs and the percentage of EBs with large cavities or beating at different time points during the differentiation process . Data are represented as mean ± s . e . m . ( n = 3 independent experiments ) .

O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, Representative images of embryoid bodies (EBs) derived from wild-type iPSCs in which the expression of Dnmt3a and Dnmt3b was transiently repressed by siRNAs. Scale bars, 500 μm. Plots show the quantification of the size of EBs and the percentage of EBs with large cavities or beating at different time points during the differentiation process. Data are represented as mean ± s.e.m. (n=3 independent experiments).


F , Expression levels of Dnmt3a or Dnmt3b transcripts after transfection of wild - type iPSCs with specific siRNAs either against Dnmt3a , Dnmt3b or a combination of both ( Dnmt3a / b ) ( as indicated in E ) . RNA expression was measured 24 hours after the transfection protocols and was normalized by GAPDH mRNA levels . Data are represented as mean ± s . e . m . ( n = 3 independent experiments ) .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

F, Expression levels of Dnmt3a or Dnmt3b transcripts after transfection of wild-type iPSCs with specific siRNAs either against Dnmt3a, Dnmt3b or a combination of both (Dnmt3a/b) (as indicated in E). RNA expression was measured 24 hours after the transfection protocols and was normalized by GAPDH mRNA levels. Data are represented as mean ± s.e.m. (n=3 independent experiments).


G , Representative images of EBs derived from miiPSCs that were transiently and simultaneously transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors , and simultaneously treated with Dox to induce miR - 203 expression . Scale bars , 500 μm . Plots show the quantification of EB size , percentage of EBs with large cavities , and beating EBs at different time points during differentiation . Data are represented as mean ± s . e . m . ( n = 3 independent experiments ) .

O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, Representative images of EBs derived from miiPSCs that were transiently and simultaneously transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors, and simultaneously treated with Dox to induce miR-203 expression. Scale bars, 500 μm. Plots show the quantification of EB size, percentage of EBs with large cavities, and beating EBs at different time points during differentiation. Data are represented as mean ± s.e.m. (n=3 independent experiments).


H , Expression levels of miR - 203 , Dnmt3a or Dnmt3b transcripts in iPSCs after induction of miR - 203 ( miiPSCs ) and transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors ( as indicated in G ) . RNA expression was measured 24 hours after the transfection protocols and was normalized by a control miRNA ( miR - 142 ) or GAPDH mRNA , respectively . Data are represented as mean ± s . e . m . ( n = 3 independent experiments ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

H, Expression levels of miR-203, Dnmt3a or Dnmt3b transcripts in iPSCs after induction of miR-203 (miiPSCs) and transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors (as indicated in G). RNA expression was measured 24 hours after the transfection protocols and was normalized by a control miRNA (miR-142) or GAPDH mRNA, respectively. Data are represented as mean ± s.e.m. (n=3 independent experiments).


C , DNA methylation distribution of the indicated samples , smoothed over 100 kb - blocks .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

C, DNA methylation distribution of the indicated samples, smoothed over 100 kb-blocks.


H , The specific differentially methylated regions ( DMR ) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite - modified DNA . Ten independent clones were sequenced per condition . The quantification of methylated ( filled circles ) vs . unmethylated ( empty circles ) CpGs is shown in the histogram in the indicated conditions .

O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

H, The specific differentially methylated regions (DMR) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite-modified DNA. Ten independent clones were sequenced per condition. The quantification of methylated (filled circles) vs. unmethylated (empty circles) CpGs is shown in the histogram in the indicated conditions.


I , Heatmaps representing the methylation levels at the top Differentially Methylated Regions ( DMRs ; n = 100 ; upper panel ) or the Imprinting Control Regions ( ICRs ; n = 103 different imprinting loci ; lower panel ) in the indicated samples ( referred to panels A , B ) . Color code and scale of DNA methylation , from 0 . 0 ( red ) to 1 . 0 ( green ) is applicable to both heatmaps .

O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

I, Heatmaps representing the methylation levels at the top Differentially Methylated Regions (DMRs; n=100; upper panel) or the Imprinting Control Regions (ICRs; n=103 different imprinting loci; lower panel) in the indicated samples (referred to panels A,B). Color code and scale of DNA methylation, from 0.0 (red) to 1.0 (green) is applicable to both heatmaps.


B , Representative immunofluorescences showing cardiac Troponin T ( cTnT , green ) and nuclei ( Hoechst , blue ) staining of in vitro - generated cardiomyocytes derived from wild - type iPSCs transiently transfected with either control mimics , miR - 203 mimics or miR - 203 mimics + Dnmt3a / b cDNAs . Pictures were taken at day 15 of differentiation . Scale bars , 68 μm . The cTnT - positive area in these cardiomyocytes is shown in the right histogram . Data are represented as mean ± s . d . ( n = 2 independent experiments with 6 replicates each ) .

O O O B-EXP_ASSAY O B-TISSUE B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, Representative immunofluorescences showing cardiac Troponin T (cTnT, green) and nuclei (Hoechst, blue) staining of in vitro-generated cardiomyocytes derived from wild-type iPSCs transiently transfected with either control mimics, miR-203 mimics or miR-203 mimics + Dnmt3a/b cDNAs. Pictures were taken at day 15 of differentiation. Scale bars, 68 μm. The cTnT-positive area in these cardiomyocytes is shown in the right histogram. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).


C , Beating frequency ( measured as number of beats per 5 seconds ) of these cardiomyocytes at day 15 of differentiation . Data are represented as mean ± s . d . ( n = 8 independent replicates ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O

C, Beating frequency (measured as number of beats per 5 seconds) of these cardiomyocytes at day 15 of differentiation. Data are represented as mean ± s.d. (n=8 independent replicates).


D , miRNA or mRNA levels as determined by quantitative PCR of the indicated transcripts at different time points during cardiomyocyte differentiation in the indicated samples . Data are represented as mean ± s . d . ( n = 2 independent experiments with 6 replicates each ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, miRNA or mRNA levels as determined by quantitative PCR of the indicated transcripts at different time points during cardiomyocyte differentiation in the indicated samples. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).


( A , B ) ELISA screen of the lectin - Fc library against full - length trimeric SARS - CoV - 2 Spike ( A ) or monomeric RBD ( B ) . Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value . Lectin - Fc fusion proteins with a normalized OD > 0 . 5 in either ( A ) or ( B ) are indicated in both panels .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A,B) ELISA screen of the lectin-Fc library against full-length trimeric SARS-CoV-2 Spike (A) or monomeric RBD (B). Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value. Lectin-Fc fusion proteins with a normalized OD > 0.5 in either (A) or (B) are indicated in both panels.


( C ) Lectin - Fc and human ACE2 - mIgG1 Fc - fusion protein ( hACE2 ) binding to untreated or heat - denatured full - length SARS - CoV - 2 Spike by ELISA . hACE2 - mIgG1 was used as control for complete denaturation of Spike protein . Results are shown as mean OD values ± SD normalized to the BSA control ( technical replicates , N = 3 ) .

O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(C) Lectin-Fc and human ACE2-mIgG1 Fc-fusion protein (hACE2) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike by ELISA. hACE2-mIgG1 was used as control for complete denaturation of Spike protein. Results are shown as mean OD values ± SD normalized to the BSA control (technical replicates, N=3).


( D ) Lectin - Fc binding to full - length SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with the Spike protein . Results are shown as mean OD values ± SD normalized to BSA controls ( technical replicates , N = 3 ) .

O O O O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(D) Lectin-Fc binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized to BSA controls (technical replicates, N=3).


( E ) High speed AFM of single trimeric Spike visualizing the real - time interaction dynamics with lectins . Top panel shows 5 frames of trimeric Spike alone imaged on mica . Middle and bottom panels show 5 sequential frames of trimeric Spike / Clec4g and trimeric Spike / CD209c complexes , acquired at a rate of 153 . 6 and 303 ms / frame , respectively . Association and dissociation events between lectin and Spike are indicated by white and red arrows , respectively . The blue dotted ellipses display the core of the complexes showing low conformational mobility . Color schemes indicate height of the molecules in nanometers ( nm ) . Volumes of single trimeric Spike , trimeric Spike / Clec4g and trimeric Spike / CD209c complexes are indicated , as well as numbers of lectins bound to trimeric Spike , averaged over the experimental recording period .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O 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-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

(E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top panel shows 5 frames of trimeric Spike alone imaged on mica. Middle and bottom panels show 5 sequential frames of trimeric Spike/Clec4g and trimeric Spike/CD209c complexes, acquired at a rate of 153.6 and 303 ms/frame, respectively. Association and dissociation events between lectin and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike, trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.


( A ) ELISA analyses of human lectin - hIgG1 Fc - fusion protein ( hCLEC4g , hCD209 , hCD299 ) binding to untreated or heat - denatured full - length SARS - CoV - 2 Spike . A human ACE2 - hIgG1 - Fc fusion protein ( hACE2 ) was used to control for the complete denaturation of Spike . Results are shown as mean OD values ± SD normalized to a BSA control ( technical replicates , N = 3 ) .

O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(A) ELISA analyses of human lectin-hIgG1 Fc-fusion protein (hCLEC4g, hCD209, hCD299) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike. A human ACE2-hIgG1-Fc fusion protein (hACE2) was used to control for the complete denaturation of Spike. Results are shown as mean OD values ± SD normalized to a BSA control (technical replicates, N=3).


( B ) hCLEC4g , hCD209 , and hCD299 binding to full - length SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with the Spike protein . Results are shown as mean OD values ± SD normalized against the BSA control ( technical replicates , N = 3 ) .

O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(B) hCLEC4g, hCD209, and hCD299 binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized against the BSA control (technical replicates, N=3).


( C , D ) SPR analysis with immobilized full - length trimeric Spike , probed with various concentrations of hCLEC4G ( C ) and hCD209 ( D ) .

O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(C, D) SPR analysis with immobilized full-length trimeric Spike, probed with various concentrations of hCLEC4G (C) and hCD209 (D).


( C ) Infectivity of mouse Clec4g , CD209c , Asgr1 and hCLEC4G pre - treated SARS - CoV - 2 virus in Vero E6 cells . Data represent two to three independent experiments for Clec4g , CD209c and hCLEC4G lectins ( N = 5 - 9 ) and , one experiment for Asgr1 ( N = 2 - 4 ) . ( D ) Infectivity of mouse Clec4g , CD209c , Asgr1 and hCLEC4G pre - treated SARS - CoV - 2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non - treated SARS - CoV - 2 infected Calu3 cells measured in quadruplicates ( technical replicates ) . : ( C ) and ( D ) Viral RNA was measured with qRT - PCR 15 hours after infection with 0 . 02 MOI ( 103 PFUs ) of SARS - CoV - 2 . Data are presented as fold changes of viral loads over mock ( only SARS - CoV - 2 was added ) treated controls ( mean log10 values ± SD ) . All no virus controls were negative for SARS - CoV - 2 .

O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM B-TISSUE I-TISSUE B-CELL_LINE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O

(C) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in Vero E6 cells. Data represent two to three independent experiments for Clec4g, CD209c and hCLEC4G lectins (N=5-9) and, one experiment for Asgr1 (N=2-4). (D) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non-treated SARS-CoV-2 infected Calu3 cells measured in quadruplicates (technical replicates). : (C) and (D) Viral RNA was measured with qRT-PCR 15 hours after infection with 0.02 MOI (103 PFUs) of SARS-CoV-2. Data are presented as fold changes of viral loads over mock (only SARS-CoV-2 was added) treated controls (mean log10 values ± SD). All no virus controls were negative for SARS-CoV-2.


A . Immunoblot analysis of nuclear and cytoplasmic extracts prepared from parasites synchronized at ring ( R ) , trophozoite ( T ) and schizont ( S ) stages with anti - histone H3 C - terminus antibodies identifies three bands : full - length histone H3 , an intermediate form ( H3 - int ) and a fully processed form ( H3p ) . Anti - histone H4 and anti - PfAldolase antibodies served as loading controls for the nuclear and cytoplasmic fraction , respectively .

O O B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O

A. Immunoblot analysis of nuclear and cytoplasmic extracts prepared from parasites synchronized at ring (R), trophozoite (T) and schizont (S) stages with anti-histone H3 C-terminus antibodies identifies three bands: full-length histone H3, an intermediate form (H3-int) and a fully processed form (H3p). Anti-histone H4 and anti-PfAldolase antibodies served as loading controls for the nuclear and cytoplasmic fraction, respectively.


B . Immunoblot assays of mononucleosomes with anti - histone H3 C - terminus or anti - histone H4 antibodies . The right panel shows a mononoucleosome preparation separated on a denaturing polyacrylamide gel and stained with Coomassie brilliant blue ( C . B . ) .

O O B-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

B. Immunoblot assays of mononucleosomes with anti-histone H3 C-terminus or anti-histone H4 antibodies. The right panel shows a mononoucleosome preparation separated on a denaturing polyacrylamide gel and stained with Coomassie brilliant blue (C.B.).


C . Immunoblot analysis of a schizont - stage histone preparation with antibodies against the indicated post - translational modification ( PTM ) in the N - terminal tail of histone H3 ( upper panel ) .

O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD O O O O O

C. Immunoblot analysis of a schizont-stage histone preparation with antibodies against the indicated post-translational modification (PTM) in the N-terminal tail of histone H3 (upper panel).


B . GST - PfH3 or GST - PfH2A were incubated with increasing amounts of nuclear extracts , the reactions resolved using denaturing gel electrophoresis and analyzed by immunoblotting with anti - GST antibodies .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O

B. GST-PfH3 or GST-PfH2A were incubated with increasing amounts of nuclear extracts, the reactions resolved using denaturing gel electrophoresis and analyzed by immunoblotting with anti-GST antibodies.


C . GST - PfH3 was incubated with 25 μg of nuclear extracts and the indicated protease inhibitor . The resulting products were analyzed by denaturing gel electrophoresis and immunoblotting with anti - GST antibodies . ( - ) indicates a reaction without nuclear extract and protease inhibitor .

O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O

C. GST-PfH3 was incubated with 25 μg of nuclear extracts and the indicated protease inhibitor. The resulting products were analyzed by denaturing gel electrophoresis and immunoblotting with anti-GST antibodies. (-) indicates a reaction without nuclear extract and protease inhibitor.


A . Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p - HA in ring ( R ) , trophozoite ( T ) and schizont ( S ) stages of P . falciparum asexual growth . PfH3p - HA was detected using anti - HA antibodies ( green ) and endogenous histone H3 with anti - histone H3 N - terminal antibodies ( red ) . DAPI ( blue ) was used to stain the nucleus . Scale bar = 5 μm .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O

A. Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p-HA in ring (R), trophozoite (T) and schizont (S) stages of P. falciparum asexual growth. PfH3p-HA was detected using anti-HA antibodies (green) and endogenous histone H3 with anti-histone H3 N-terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm.


B . Nuclei isolated from wild - type ( WT ) or PfH3p - HA - expressing ( WT + PfH3p - HA ) schizont stage parasites were treated with 4 U / ml of micrococcal nuclease ( MNase ) for the indicated amounts of time , the DNA purified and migrated on a 2 % agarose gel , and stained with ethidium bromide . Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue ( C . B . ) or visualized by immunoblotting with anti - HA ( α - HA ) or anti - C - terminal histone H3 ( α - H3c ) antibodies .

O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O

B. Nuclei isolated from wild-type (WT) or PfH3p-HA-expressing (WT + PfH3p-HA) schizont stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue (C.B.) or visualized by immunoblotting with anti-HA (α-HA) or anti-C-terminal histone H3 (α-H3c) antibodies.


C . Co - immunoprecipitation ( IP ) experiments of purified mononucleosomes obtained from wild - type ( WT ) or transfected ( WT + PfH3p - HA ) schizont stage parasites were performed with either anti - HA antibodies or mouse IgG . Immunoprecipitated products ( right panel ) were analyzed by immunoblotting using anti - HA or anti - histone H4 antibodies .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O

C. Co-immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild-type (WT) or transfected (WT + PfH3p-HA) schizont stage parasites were performed with either anti-HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti-HA or anti-histone H4 antibodies.


A . Genome - wide distribution of ectopically expressed PfH3p - HA in P . falciparum schizont stages is represented as fold - enrichment of PfH3p - HA ChIP - seq signal over Input ( PfH3p - HA / Input in blue ; y - axis scale 1 - 10 ) or over PfH3n ( PfH3p - HA / Input in teal ; y - axis scale 1 - 10 ) calculated using MACS2 . The coverage of PfH3p - HA ( red ; y - axis scale 0 - 50 ) , Input ( black ; y - axis scale 0 - 30 ) and PfH3n ( green ; y - axis scale 0 - 30 ) is also shown and represents average reads per million over 1000 nt bins of the genome . The x - axis represents chromosome size in Mb . Major MACS2 - derived peaks identified in all replicates are indicated using an orange arrowhead . B . Principal Component Analysis of the different ChIP - seq replicates ( bigwig files derived from deduplicated bam files ) was performed using the plotPCA function of deepTools on a multibigwig summary file , over 150 nt bins . The Eigen values of the top two principal components PC1 and PC2 are shown and meaningful clustering of replicates highlighted . Rep = Biological Replicate . C . Summary of the PfH3p - enriched peaks - and corresponding genes - identified in the PfH3p - HA versus Input or PfH3p - HA versus PfH3n comparisons using MACS2 peak - calling analysis . The overlap between the two comparisons is indicated using a Venn diagram . FE = Fold Enrichment ; q - value represents the Benjamini and Hochberg false discovery rate . D . The genomic context of the six peaks identified in all three PfH3p - HA ChIP - seq replicates is shown . The fold enrichment of PfH3p - HA ChIP - seq signal over Input ( PfH3p - HA / Input ; blue ) or PfH3n ( PfH3p - HA / PfH3n ; teal ) is shown , as are the coverage plots of PfH3p - HA ( red ) , PfH3n ( green ) and Input ( black ) , which are represented as average reads per million over 1000 nt bins of the genome . DNA replication genes : Replication Protein A 1 ( RPA1 ) , Proliferating Cell Nuclear Antigen 1 ( PCNA1 ) , Single Stranded DNA binding protein ( SSB ) , Topoisomerase I ( TOPO - I ) , DNA polymerase alpha subunit ( DNA pol a ) , and Topoisomerase II ( TOPO - II ) are highlighted and their directionality indicated . Note that the data in part D correspond to Biological Replicate 1 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O 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-GENEPROD O O O O O O O O O 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-SUBCELLULAR O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

A. Genome-wide distribution of ectopically expressed PfH3p-HA in P. falciparum schizont stages is represented as fold-enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input in blue; y-axis scale 1-10) or over PfH3n (PfH3p-HA / Input in teal; y-axis scale 1-10) calculated using MACS2. The coverage of PfH3p-HA (red; y-axis scale 0-50), Input (black; y-axis scale 0-30) and PfH3n (green; y-axis scale 0-30) is also shown and represents average reads per million over 1000 nt bins of the genome. The x-axis represents chromosome size in Mb. Major MACS2-derived peaks identified in all replicates are indicated using an orange arrowhead. B. Principal Component Analysis of the different ChIP-seq replicates (bigwig files derived from deduplicated bam files) was performed using the plotPCA function of deepTools on a multibigwig summary file, over 150 nt bins. The Eigen values of the top two principal components PC1 and PC2 are shown and meaningful clustering of replicates highlighted. Rep = Biological Replicate. C. Summary of the PfH3p-enriched peaks - and corresponding genes - identified in the PfH3p-HA versus Input or PfH3p-HA versus PfH3n comparisons using MACS2 peak-calling analysis. The overlap between the two comparisons is indicated using a Venn diagram. FE = Fold Enrichment; q-value represents the Benjamini and Hochberg false discovery rate. D. The genomic context of the six peaks identified in all three PfH3p-HA ChIP-seq replicates is shown. The fold enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input; blue) or PfH3n (PfH3p-HA / PfH3n; teal) is shown, as are the coverage plots of PfH3p-HA (red), PfH3n (green) and Input (black), which are represented as average reads per million over 1000 nt bins of the genome. DNA replication genes: Replication Protein A 1 (RPA1), Proliferating Cell Nuclear Antigen 1 (PCNA1), Single Stranded DNA binding protein (SSB), Topoisomerase I (TOPO-I), DNA polymerase alpha subunit (DNA pol a), and Topoisomerase II (TOPO-II) are highlighted and their directionality indicated. Note that the data in part D correspond to Biological Replicate 1.


( D ) smFISH images showing LAT RNA in Ntrk1 - positive neurons . SCG - derived cultures were infected with HSV - 1 in the presence of ACV and allowed to establish latency over 7 days before being processed for smFISH using probes for HSV - 1 LAT ( green ) and neuronal marker Ntrk1 ( TrkA ) ( red ) or fibroblast marker Col3a1 ( red ) . Signal intensities for the LAT probe were quantified in neurons and fibroblasts and plotted by ImageJ . The percentage of LAT - positive cells were quantified and displayed as bar graphs with mean ± SEM . Left : 3 biological replicates , signal intensities of 50 cells were quantified for each replicate . Middle : 3 biological replicates , signal intensities of 30 cells were quantified for each replicate . Right : the blue and red dots represent the number of biological replicates done for quantification of each cell type . Scale bar , 10 μm . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) smFISH images showing LAT RNA in Ntrk1-positive neurons. SCG-derived cultures were infected with HSV-1 in the presence of ACV and allowed to establish latency over 7 days before being processed for smFISH using probes for HSV-1 LAT (green) and neuronal marker Ntrk1 (TrkA) (red) or fibroblast marker Col3a1 (red). Signal intensities for the LAT probe were quantified in neurons and fibroblasts and plotted by ImageJ. The percentage of LAT-positive cells were quantified and displayed as bar graphs with mean ± SEM. Left: 3 biological replicates, signal intensities of 50 cells were quantified for each replicate. Middle: 3 biological replicates, signal intensities of 30 cells were quantified for each replicate. Right: the blue and red dots represent the number of biological replicates done for quantification of each cell type. Scale bar, 10 μm. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( A ) smFISH images showing selected viral mRNAs in reactivating neurons probed for ICP27 ( immediate - early ) , UL30 ( early ) , UL36 ( true - late ) or cellular markers , Ntrk ( neurons ) , or Col3a1 ( fibroblasts ) . Nuclear DNA was visualized using DAPI ( blue ) . To induce reactivation , latently - infected SCG - derived cultures were simultaneously treated with LY294002 ( 20 μM ) and WAY - 150138 ( 20 μg / mL ) and cultured for 48 hrs prior to processing for smFISH . Scale bar , 10 μm .

O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-CELL_TYPE O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

(A) smFISH images showing selected viral mRNAs in reactivating neurons probed for ICP27 (immediate-early), UL30 (early), UL36 (true-late) or cellular markers, Ntrk (neurons), or Col3a1 (fibroblasts). Nuclear DNA was visualized using DAPI (blue). To induce reactivation, latently-infected SCG-derived cultures were simultaneously treated with LY294002 (20 μM) and WAY-150138 (20 μg/mL) and cultured for 48 hrs prior to processing for smFISH. Scale bar, 10 μm.


( B ) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM . Latently - infected cultures were untreated ( latent ) or treated with LY294002 and WAY - 150138 for 18 , 48 , or 72 hrs . Each dot corresponds to the value from each biological replicate . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

O O O O O O O O O O O O O O B-GENEPROD O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM. Latently-infected cultures were untreated (latent) or treated with LY294002 and WAY-150138 for 18, 48, or 72 hrs. Each dot corresponds to the value from each biological replicate. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( C - D ) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs . Signal overlap is evident as white or yellow nuclear foci in the merge . Bar , 10 μm . In ( D ) , the percentage of positive nuclei with overlapping UL30 and UL36 smFISH signals were quantified as the mean ± SEM ( n = 3 biological replicates ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O 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-SUBCELLULAR O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs. Signal overlap is evident as white or yellow nuclear foci in the merge. Bar, 10 μm. In (D), the percentage of positive nuclei with overlapping UL30 and UL36 smFISH signals were quantified as the mean ± SEM (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( B ) Differential gene expression analysis of cellular transcripts showed a significant upregulation of at least 19 viral transcript units ( TU ) and a subset of cellular genes in neurons undergoing reactivation Three host transcripts were also downregulated . Gadd45b is highlighted with a green asterisk . Vertical hashed blue lines indicate Log2 fold changes > 1 . 5 and < - 1 . 5 , while the horizontal hashed blue line indicates an adjusted p - value of 0 . 01 . Significantly regulated genes are named and shown as magenta circles .

O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Differential gene expression analysis of cellular transcripts showed a significant upregulation of at least 19 viral transcript units (TU) and a subset of cellular genes in neurons undergoing reactivation Three host transcripts were also downregulated. Gadd45b is highlighted with a green asterisk. Vertical hashed blue lines indicate Log2 fold changes >1.5 and < -1.5, while the horizontal hashed blue line indicates an adjusted p-value of 0.01. Significantly regulated genes are named and shown as magenta circles.


( A ) smFISH images showing nuclear Gadd45b mRNA in latent ( UL36 - negative ) neurons and nuclear / cytoplasmic Gadd45b mRNA in viral UL36 - positive neurons . Latently - infected SCG neurons treated with LY294002 and WAY - 150138 for 48 hrs were processed using smFISH to detect Gadd45b ( green ) and viral UL36 ( red ) mRNA levels and colocalization . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . ( B ) smFISH images processed as in ( A ) showing nuclear Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with LY294002 . Scale bar , 10 μm .

O O O B-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O B-TISSUE B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O

(A) smFISH images showing nuclear Gadd45b mRNA in latent (UL36-negative) neurons and nuclear/cytoplasmic Gadd45b mRNA in viral UL36-positive neurons. Latently-infected SCG neurons treated with LY294002 and WAY-150138 for 48 hrs were processed using smFISH to detect Gadd45b (green) and viral UL36 (red) mRNA levels and colocalization. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (B) smFISH images processed as in (A) showing nuclear Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with LY294002. Scale bar, 10 μm.


( E ) The percentage of HSV - 1 latently - infected neurons or reactivating neurons at 48 hrs post treatment with LY294002 and WAY - 150138 were scored for either predominantly nuclear or nuclear / cytoplasmic Gadd45b mRNA localization and displayed as mean ± SEM . Each purple dot represents a biological replicate experiment . ( F ) Quantification of nuclear or nuclear / cytoplasmic Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO . Each light blue dot represents a biological replicate experiment . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * * , p < 0 . 0001 ) .

O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) The percentage of HSV-1 latently-infected neurons or reactivating neurons at 48 hrs post treatment with LY294002 and WAY-150138 were scored for either predominantly nuclear or nuclear/cytoplasmic Gadd45b mRNA localization and displayed as mean ± SEM. Each purple dot represents a biological replicate experiment. (F) Quantification of nuclear or nuclear/cytoplasmic Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO. Each light blue dot represents a biological replicate experiment. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ****, p<0.0001).


( H ) Combined smFISH and immunofluorescence ( IF ) images showing high GFP - expressing neurons ( GFP - positive ) or low GFP - expressing ( GFP absent ) neurons simultaneously probed for Gadd45b mRNA . Reactivation was induced by treatment with LY294002 and WAY - 150138 for 60 hrs and processed for simultaneous smFISH and IF staining . Right panel shows quantification ( as in G ) of the smFISH signal intensity of Gadd45b mRNA and IF signal intensity of GFP protein at 48 hrs post treatment with LY294002 and WAY - 150138 ( n = 3 biological replicates ) . The signal intensity was quantified , plotted , and analysed by ImageJ . Bar , 10 μm . Data information : : R and R square values were calculated using Correlation analysis by Prism 8 .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Combined smFISH and immunofluorescence (IF) images showing high GFP-expressing neurons (GFP-positive) or low GFP-expressing (GFP absent) neurons simultaneously probed for Gadd45b mRNA. Reactivation was induced by treatment with LY294002 and WAY-150138 for 60 hrs and processed for simultaneous smFISH and IF staining. Right panel shows quantification (as in G) of the smFISH signal intensity of Gadd45b mRNA and IF signal intensity of GFP protein at 48 hrs post treatment with LY294002 and WAY-150138 (n=3 biological replicates). The signal intensity was quantified, plotted, and analysed by ImageJ. Bar, 10 μm. Data information: : R and R square values were calculated using Correlation analysis by Prism 8.


( A ) Viral DNA polymerase inhibitor phosphonoacetic acid ( PAA , 300 μg / ml ) treatment suppressed Gadd45b mRNA expression . Latently - infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time - points indicated . RNA was then collected and levels of Gadd45b mRNA quantified by RT - qPCR ( n = 3 biological replicates , the bars and error bars are mean ± SD ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

O O O B-ORGANISM B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 μg/ml) treatment suppressed Gadd45b mRNA expression. Latently-infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time-points indicated. RNA was then collected and levels of Gadd45b mRNA quantified by RT-qPCR (n=3 biological replicates, the bars and error bars are mean ± SD). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( C ) Immunofluorescence images showing nuclear - enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002 . Scale bar , 20 μm . ( D ) The signal intensity quantification showing the ratio of nuclear / cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY - 150138 neurons . The signal intensity was quantified by ImageJ . 3 biological replicates , 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate . The horizonal line and error bars are mean ± SD . ( E ) The percentage of Gadd45b nuclear puncta - localized neurons were quantified from latent neurons treated with LY294002 and WAY - 150138 for 72 hrs . Each dot represents each biological replicate ; 20 cells were quantified for each replicate . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O 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-GENEPROD B-SUBCELLULAR O O B-EXP_ASSAY B-CELL_TYPE O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Immunofluorescence images showing nuclear-enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002. Scale bar, 20 μm. (D) The signal intensity quantification showing the ratio of nuclear/cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY-150138 neurons. The signal intensity was quantified by ImageJ. 3 biological replicates, 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate. The horizonal line and error bars are mean ± SD. (E) The percentage of Gadd45b nuclear puncta-localized neurons were quantified from latent neurons treated with LY294002 and WAY-150138 for 72 hrs. Each dot represents each biological replicate; 20 cells were quantified for each replicate. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( G ) Latent SCG neurons treated with LY294002 and WAY - 150138 for 72 hrs , the Gadd45b nuclear puncta - presenting neurons were ICP4 negative ( none observed in over 60 Gadd45b nuclear puncta - positive neurons ) , but a small population ( ~ 5 % ) in Gadd45b nuclear - enriched neurons were ICP4 positive . Scale bar , 20 μm .

O O O O B-TISSUE B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD B-SUBCELLULAR O O O B-CELL_TYPE O B-GENEPROD O O O O O O O B-GENEPROD B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O B-CELL_TYPE O B-GENEPROD O O O O O O O O

(G) Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs, the Gadd45b nuclear puncta-presenting neurons were ICP4 negative (none observed in over 60 Gadd45b nuclear puncta-positive neurons), but a small population (~5%) in Gadd45b nuclear-enriched neurons were ICP4 positive. Scale bar, 20 μm.


( A ) Depletion of Gadd45b expression using lentiviral - transduced shRNAs in latently - infected SCG neurons case cause HSV - 1 reactivation . HSV1 - GFP - Us11 latently - infected cultures were infected with lentiviruses expressing either two different shRNAs against Gadd45b mRNA or a non - silencing shRNA ( NS ) . Reactivation was quantified by scoring the percentage of wells expressing GFP after 5 days . Knockdown efficiencies for individual shRNAs in latently - infected SCG neurons were confirmed by RT - qPCR using RNA collected in parallel . Reactivation rates were quantified from 30 wells for each condition , 3 biological replicates . RT - qPCR were quantified from 3 biological replicates . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-TISSUE B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Depletion of Gadd45b expression using lentiviral-transduced shRNAs in latently-infected SCG neurons case cause HSV-1 reactivation. HSV1-GFP-Us11 latently-infected cultures were infected with lentiviruses expressing either two different shRNAs against Gadd45b mRNA or a non-silencing shRNA (NS). Reactivation was quantified by scoring the percentage of wells expressing GFP after 5 days. Knockdown efficiencies for individual shRNAs in latently-infected SCG neurons were confirmed by RT-qPCR using RNA collected in parallel. Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates. RT-qPCR were quantified from 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( B ) smFISH shows that depletion of Gadd45b leads to increased UL36 expression . smFISH was performed 3 days post - lentiviral transduction . smFISH signals were quantified and plotted using ImageJ and Prism . Left : 3 biological replicates , 30 cells were quantified for each replicate . The horizonal lines are mean . Right : 3 biological replicates . The horizonal lines and error bars are mean ± SD .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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) smFISH shows that depletion of Gadd45b leads to increased UL36 expression. smFISH was performed 3 days post-lentiviral transduction. smFISH signals were quantified and plotted using ImageJ and Prism. Left: 3 biological replicates, 30 cells were quantified for each replicate. The horizonal lines are mean. Right: 3 biological replicates. The horizonal lines and error bars are mean ± SD.


( C ) Ectopic expression of Gadd45b - Myc - Flag inhibits viral mRNA expression . Lentivirus - delivered ectopic expression of Gadd45b - Myc - Flag in SCG neurons was confirmed by immunoblotting . Latently - infected SCG neurons were transduced with Gadd45b - Myc - Flag for 3 days and then treated with LY294002 and WAY - 150138 to induce reactivation . RNA was collected at the indicated time - points ( LY treatment for 18 or 72 hours ) and levels of viral UL36 mRNA quantified by RT - qPCR . 3 biological replicates . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE B-CELL_TYPE O O O B-EXP_ASSAY O O O O B-TISSUE B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Ectopic expression of Gadd45b-Myc-Flag inhibits viral mRNA expression. Lentivirus-delivered ectopic expression of Gadd45b-Myc-Flag in SCG neurons was confirmed by immunoblotting. Latently-infected SCG neurons were transduced with Gadd45b-Myc-Flag for 3 days and then treated with LY294002 and WAY-150138 to induce reactivation. RNA was collected at the indicated time-points (LY treatment for 18 or 72 hours) and levels of viral UL36 mRNA quantified by RT-qPCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( D ) Ectopic expression of Gadd45b - Myc - Flag antagonizes LY294002 - induced HSV - 1 reactivation . Latently - infected SCG neurons were either transduced with vector control ( Ctrl ) or Gadd45b - Myc - Flag from lentivirus for 3 days prior to addition of LY294002 . Reactivation was quantified by scoring the percentage of wells expressing GFP fluorescence at the indicated days . The percentage of wells expressing GFP - positive was scored as the mean ± SEM ( n = 3 biological replicates ) . Reactivation rates were quantified from 30 wells for each condition , 3 biological replicates .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Ectopic expression of Gadd45b-Myc-Flag antagonizes LY294002-induced HSV-1 reactivation. Latently-infected SCG neurons were either transduced with vector control (Ctrl) or Gadd45b-Myc-Flag from lentivirus for 3 days prior to addition of LY294002. Reactivation was quantified by scoring the percentage of wells expressing GFP fluorescence at the indicated days. The percentage of wells expressing GFP-positive was scored as the mean ± SEM (n=3 biological replicates). Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates.


( F ) Ectopic expression of Gadd45b - Myc - Flag suppressed viral ICP4 mRNA expression . Latently - infected SCG neurons were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY - 150138 for 72 hrs . Viral ICP4 mRNA was quantified by qRT - PCR . 3 biological replicates . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Ectopic expression of Gadd45b-Myc-Flag suppressed viral ICP4 mRNA expression. Latently-infected SCG neurons were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY-150138 for 72 hrs. Viral ICP4 mRNA was quantified by qRT-PCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( G ) Immunoblots for viral ICP4 protein in latent SCG neurons treated with LY294002 and WAY - 150138 in the presence or absence of Gadd45b - Myc - Flag was quantified by ImageJ ( n = 3 biological replicates ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O B-EXP_ASSAY O O B-GENEPROD O O O B-TISSUE B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunoblots for viral ICP4 protein in latent SCG neurons treated with LY294002 and WAY-150138 in the presence or absence of Gadd45b-Myc-Flag was quantified by ImageJ (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( A ) THP1 cells were infected with the shown strains of HSV - 1 ( MOI 3 ) . Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity .

O O O B-CELL_LINE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(A) THP1 cells were infected with the shown strains of HSV-1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.


( B ) THP1 cells were treated with 0 . 1 μg / ml of acyclovir ( ACV ) and infected with the KOS HSV - 1 and F HSV - 1 strains ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(B) THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS HSV-1 and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( C ) THP1 cells were treated with infectious or UV - inactivated HSV - 1 ( KOS ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

O O O B-CELL_LINE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(C) THP1 cells were treated with infectious or UV-inactivated HSV-1 (KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( D ) MDMs were infected with ICP0 - deficient or revertant HSV - 1 ( KOS ) , MOI 3 . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

O O O B-CELL_TYPE O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(D) MDMs were infected with ICP0-deficient or revertant HSV-1 (KOS), MOI 3. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( E ) THP1 - derived cells deficient for cGAS or STING were infected with the shown strains of HSV - 1 ( MOI 3 ) or stimulated with poly ( I : C ) ( 2 µg / ml ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(E) THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 µg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( A ) MDMs were infected for 18 h with HSV - 1 ( KOS ) , MOI 1 . Cytosolic viral proteins were detected by mass spectrometry using iTRAQ - labeling . Identified viral proteins are shown .

O O O B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O

(A) MDMs were infected for 18 h with HSV-1 (KOS), MOI 1. Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown.