B ABR thresholds at 4w of WT , Syne4 - / - , Syne4 - / - mice injected with AAV . Syne4 , and Syne4 - / - mice injected with AAV . GFP , n = 7 for WT , n = 8 for Syne4 - / - , n = 20 for Syne4 - / - + AAV . Syne4 , n = 6 for Syne4 - / - + AAV . GFP . C Quantification of P1 - N1 amplitude delta from ( B ) , n = 4 for WT , n = 4 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 . D Quantification of P1 latency from ( B ) , n = 4 for WT , n = 2 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 .

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B ABR thresholds at 4w of WT, Syne4-/-, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=7 for WT, n=8 for Syne4-/-, n=20 for Syne4-/- + AAV.Syne4, n=6 for Syne4-/- + AAV.GFP. C Quantification of P1-N1 amplitude delta from (B), n=4 for WT, n=4 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4. D Quantification of P1 latency from (B), n=4 for WT, n=2 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4.


E DPOAE thresholds at 4w , n = 6 for WT , n = 5 for Syne4 - / - , n = 6 for Syne4 - / - + AAV . Syne4 . F ABR threshold at 12w , n = 6 for WT , n = 8 for Syne4 - / - , n = 16 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP .

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E DPOAE thresholds at 4w, n=6 for WT, n=5 for Syne4-/-, n=6 for Syne4-/- + AAV.Syne4. F ABR threshold at 12w, n=6 for WT, n=8 for Syne4-/-, n=16 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP.


A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4 - / - mouse injected with AAV . Syne4 mouse injected with AAV . Syne4 . B Whole mount immunofluorescence at 12w of the 12 kHz region of WT , Syne4 - / - mouse , and Syne4 - / - mouse injected with AAV . Syne4 .

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A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4-/- mouse injected with AAV.Syne4 mouse injected with AAV.Syne4. B Whole mount immunofluorescence at 12w of the 12 kHz region of WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4.


C IHC and OHC counts , n = 3 for WT , n = 4 for Mut , n = 6 for Mut + AAV . Syne4 . D Correlation between HC count and ABR threshold at 12kHz .

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C IHC and OHC counts, n=3 for WT, n=4 for Mut, n=6 for Mut + AAV.Syne4. D Correlation between HC count and ABR threshold at 12kHz.


E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , white arrows denote cuticular plate . F Image analysis of nucleus position of OHC . G Quantification of nuclear distance from the cuticular plate . A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4 - / - mice injected with AAV . Syne4 were measured .

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E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate. F Image analysis of nucleus position of OHC. G Quantification of nuclear distance from the cuticular plate. A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4-/- mice injected with AAV.Syne4 were measured.


B , C Quantification of activity level and cumulative freezing duration at 12w during seconds 90 - 210 of day 2 in WT , Syne4 - / - mice , Syne4 - / - mice injected with AAV . Syne4 , and Syne4 - / - mice injected with AAV . GFP , n = 14 for WT , n = 16 for Syne4 - / - , n = 9 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP . ( C ) Activity level . ( B ) Cumulative freezing duration .

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B, C Quantification of activity level and cumulative freezing duration at 12w during seconds 90-210 of day 2 in WT, Syne4-/- mice, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=14 for WT, n=16 for Syne4-/-, n=9 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP. (C) Activity level. (B) Cumulative freezing duration.


Distribution of 25 - 35 mer reads mapping to the mouse genome . Proportion of 30 - 35 nt genomic reads matching mouse tRNA sequences . The sequencing was done in duplicates . Error bars represent SEM , significance was calculated using unpaired t - test .

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Distribution of 25-35 mer reads mapping to the mouse genome. Proportion of 30-35 nt genomic reads matching mouse tRNA sequences. The sequencing was done in duplicates. Error bars represent SEM, significance was calculated using unpaired t-test.


Perbase sequence coverage of 30 - 35 nt reads across parental tRNA sequence . Anticodon positions are demarcated by discontinuous lines ( Blue = LIF - treated , Grey = Wnt3 + LIF - treated , Red = RA - treated mESCs ) . Perbase coverage of 30 - 35 nt reads across the 468 individual parental tRNAs plotted as heatmaps .

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Perbase sequence coverage of 30-35 nt reads across parental tRNA sequence. Anticodon positions are demarcated by discontinuous lines (Blue = LIF-treated, Grey = Wnt3+LIF- treated, Red = RA- treated mESCs). Perbase coverage of 30-35 nt reads across the 468 individual parental tRNAs plotted as heatmaps.


Northern blot of candidate tRNAs and tsRNAs tsGlnCTG , tsGlyGCC , tsGluTTC , tsValCAC and tsLysTTT . The black arrows represent the tRNAs and the red arrows mark the tsRNAs .

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Northern blot of candidate tRNAs and tsRNAs tsGlnCTG, tsGlyGCC, tsGluTTC, tsValCAC and tsLysTTT. The black arrows represent the tRNAs and the red arrows mark the tsRNAs.


Northern Blot of 5 ' - tsRNAs ; tsGlyGCC , tsGlnCTG , tsGluTTC , tsLysTTT , tsValCAC at various timepoints of RA - induced mESCs differentiation showing dynamic expression of these 5 ' - tsRNAs . Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates . The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs .

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Northern Blot of 5'-tsRNAs; tsGlyGCC, tsGlnCTG, tsGluTTC, tsLysTTT, tsValCAC at various timepoints of RA-induced mESCs differentiation showing dynamic expression of these 5'-tsRNAs. Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates. The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs.


Effect of ASO - mediated inhibition of 5 ' - tsRNAs on alkaline phosphatase activity in RA - treated mESCs . ( n = 3 ) , Error bars represent SEM , significance was calculated using unpaired t - test .

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Effect of ASO-mediated inhibition of 5'-tsRNAs on alkaline phosphatase activity in RA-treated mESCs. (n = 3), Error bars represent SEM, significance was calculated using unpaired t-test.


Relative expression of stemness markers in RA - induced differentiation mESCs blocked for 5 ' - tsRNA function with ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

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Relative expression of stemness markers in RA-induced differentiation mESCs blocked for 5'-tsRNA function with ASOs. (n = 2) Error bars represent SEM, significance was calculated using unpaired t-test.


Volcano plot of the transcriptome depicting the overexpressed transcripts ( in green ) and downregulated transcripts ( in pink ) in ASO treated - differentiating cells .

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Volcano plot of the transcriptome depicting the overexpressed transcripts (in green) and downregulated transcripts (in pink) in ASO treated-differentiating cells.


QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5 ' - tsRNA knockdowns using ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

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QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5'-tsRNA knockdowns using ASOs. (n =2) Error bars represent SEM, significance was calculated using unpaired t-test.


Scatter plot showing Log2 fold - change enrichment of protein interactome between LIF - and RA - treated mESCs . All peptides identified by LC - MS / MS had < 1 % FDR ( Fig EV4E ) . LC - MS / MS was conducted twice ( R2 = 0 . 85 between LIF duplicates and R2 = 0 . 82 between RA - treated mESCs duplicates ) .

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Scatter plot showing Log2 fold-change enrichment of protein interactome between LIF- and RA-treated mESCs. All peptides identified by LC-MS/MS had < 1% FDR (Fig EV4E). LC-MS/MS was conducted twice (R2 = 0.85 between LIF duplicates and R2 = 0.82 between RA-treated mESCs duplicates).


Scatter plot showing Log2 fold - change enrichment of mRNAs associated with tsGlnCTG over Scramble ( Scr1 ) in LIF - versus RA - treated mESCs .

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Scatter plot showing Log2 fold-change enrichment of mRNAs associated with tsGlnCTG over Scramble (Scr1) in LIF- versus RA-treated mESCs.


Bar plots and representative gene lists depicting association of tsGlnCTG with ' pluripotency - associated ' ( D ) and ' differentiation - responsive ' ( D ' ) genes in LIF - or RA - treated mESCs .

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Bar plots and representative gene lists depicting association of tsGlnCTG with 'pluripotency-associated' (D) and 'differentiation-responsive' (D') genes in LIF- or RA-treated mESCs.


In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo ( ASO ) against tsGlnCTG . ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1 . ( n = 2 ) , Error bars represent S . E . M .

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In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. (n = 2), Error bars represent S.E.M.


Quantification of the Igf2bp1 bound c - Myc mRNA between LIF - versus RA - treated mESCs ( n = 2 ; see Fig . EV5F for duplicate data ) .

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Quantification of the Igf2bp1 bound c-Myc mRNA between LIF- versus RA-treated mESCs (n = 2; see Fig. EV5F for duplicate data).


Association of c - Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition . ( n = 2 ) , Error bars represent SD , significance was calculated by one - tailed unpaired t - test .

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Association of c-Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition. (n = 2), Error bars represent SD, significance was calculated by one-tailed unpaired t-test.


Relative enrichment of c - Myc mRNA in translating ( 80S and Polysome ) and non - translating ( mRNPs ) pools ( fractionated from polysome profiling ) between ASO - treated and Mock - treated RA - induced differentiating mESCs . ( n = 2 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

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Relative enrichment of c-Myc mRNA in translating (80S and Polysome) and non-translating (mRNPs) pools (fractionated from polysome profiling) between ASO-treated and Mock-treated RA-induced differentiating mESCs. (n =2) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Relative levels of c - Myc mRNA in ASO - treated and Mock - treated RA - induced differentiating mESCs compared to LIF treated mESCs . ( n = 3 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

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Relative levels of c-Myc mRNA in ASO-treated and Mock-treated RA-induced differentiating mESCs compared to LIF treated mESCs. (n = 3) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Epistatic analysis of 5 ' - tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity . ( n = 3 ) , Error bars represent SD , significance was calculated using unpaired t - test .

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Epistatic analysis of 5'-tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity. (n = 3), Error bars represent SD, significance was calculated using unpaired t-test.


( B ) Downstream polarization of VE - PTP ( red ) in endothelial cells in the thoracic aorta . Blood flow : left to right . VE - cadherin , green . ( C ) Higher magnification views of a region similar to B , showing variability of VE - PTP polarization ranging from conspicuous ( upper ) to subtle ( lower ) . Nuclei , blue ( lamin A / C ) . (

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(B) Downstream polarization of VE-PTP (red) in endothelial cells in the thoracic aorta. Blood flow: left to right. VE-cadherin, green. (C) Higher magnification views of a region similar to B, showing variability of VE-PTP polarization ranging from conspicuous (upper) to subtle (lower). Nuclei, blue (lamin A/C). (


( D ) Dot plot of proportions of VE - PTP staining in upstream and downstream halves of endothelial cells in thoracic aorta of wild - type mouse . Each dot is one endothelial cell . Mean ± SEM , n = 15 cells .

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(D) Dot plot of proportions of VE-PTP staining in upstream and downstream halves of endothelial cells in thoracic aorta of wild-type mouse. Each dot is one endothelial cell. Mean ± SEM, n = 15 cells.


( C ) VE - PTP internalization in HUVEC into EEA1 endosomes ( arrowheads ) under static conditions and laminar flow of 15 dyn / cm2 of shear force for 5 , 10 , and 30 min . ( D ) Quantification of VE - PTP staining signals of images shown in C , which co - localize with EEA1 . Mean ± SEM , n = 10 pooled in 3 independent experiments (

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(C) VE-PTP internalization in HUVEC into EEA1 endosomes (arrowheads) under static conditions and laminar flow of 15 dyn/cm2 of shear force for 5, 10, and 30 min. (D) Quantification of VE-PTP staining signals of images shown in C, which co-localize with EEA1. Mean ± SEM, n= 10 pooled in 3 independent experiments (


( E ) HUVEC exposed to same conditions as in C , followed by fixation with ( upper row ) or without ( lower row ) permeabilization with 0 . 3 % Triton X - 100 , and staining for VE - PTP ( red ) and nuclei ( blue , Hoechst 33342 ) . ( F ) VE - PTP on cell surface measured by cell - based ELISA assay of HUVEC after exposure as in C and D . Red and black lines show measurements with anti - VE - PTP antibody and isotype control antibody . Mean ± SEM , n = 6 independent experiments / group .

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(E) HUVEC exposed to same conditions as in C, followed by fixation with (upper row) or without (lower row) permeabilization with 0.3% Triton X-100, and staining for VE-PTP (red) and nuclei (blue, Hoechst 33342). (F) VE-PTP on cell surface measured by cell-based ELISA assay of HUVEC after exposure as in C and D. Red and black lines show measurements with anti-VE-PTP antibody and isotype control antibody. Mean ± SEM, n = 6 independent experiments/group. D


( C - D ) Strong Tie2 - pY992 ( green ) staining at cell borders and VE - PTP ( red ) downstream polarization in outer curvature in C in contrast to weak , dispersed staining for Tie2 - pY992 and VE - PTP in inner curvature in D of aortic arch of wild - type mouse .

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(C-D) Strong Tie2-pY992 (green) staining at cell borders and VE-PTP (red) downstream polarization in outer curvature in C in contrast to weak, dispersed staining for Tie2-pY992 and VE-PTP in inner curvature in D of aortic arch of wild-type mouse.


( E - F ) Tie2 - pY992 ( green ) at endothelial junctions marked by VE - cadherin ( red ) . ( E ) Contrasting patterns and intensity of Tie2 - pY992 in outer and inner curvatures . ( F ) Tie2 - pY992 mean fluorescence intensity at junctions ( normalized to outer curvature = 1 . 0 ) . Mean ± SEM , n = 6 mice / group .

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(E-F) Tie2-pY992 (green) at endothelial junctions marked by VE-cadherin (red). (E) Contrasting patterns and intensity of Tie2-pY992 in outer and inner curvatures. (F) Tie2-pY992 mean fluorescence intensity at junctions (normalized to outer curvature = 1.0). Mean ± SEM, n = 6 mice/group.


( E ) Comparison of distribution of fluorescence intensities of extravasated antibody showing significantly stronger fluorescence due to greater leakage in inner curvature . ( F ) Mean fluorescence intensity value for each mouse in the two groups shown in E . Mean ± SEM , n = 7 mice / group . (

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(E) Comparison of distribution of fluorescence intensities of extravasated antibody showing significantly stronger fluorescence due to greater leakage in inner curvature. (F) Mean fluorescence intensity value for each mouse in the two groups shown in E. Mean ± SEM, n = 7 mice/group. (


( G ) Localized distribution of intense leakage ( red ) around ostium of an intercostal artery ( arrow ) . VE - cadherin ( green ) . ( H ) Opposite distributions of extravasation and VE - PTP polarization in endothelial cells in thoracic aorta . Left and right panels are the same image with colors switched to highlight leakage ( red ) and polarized VE - PTP ( green , arrowheads ) on the left , and polarized VE - PTP ( red , arrowheads ) , VE - cadherin ( green ) , and leakage ( blue ) on the right .

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(G) Localized distribution of intense leakage (red) around ostium of an intercostal artery (arrow). VE-cadherin (green). (H) Opposite distributions of extravasation and VE-PTP polarization in endothelial cells in thoracic aorta. Left and right panels are the same image with colors switched to highlight leakage (red) and polarized VE-PTP (green, arrowheads) on the left, and polarized VE-PTP (red, arrowheads), VE-cadherin (green), and leakage (blue) on the right. D


( B ) Measurements of Tie2 - pY992 staining intensity at intercellular junctions in aortic arch endothelial cells showing lower values in the inner curvature than in the upper curvature of VE - PTPfl / fl mice but equally high values in both regions of VE - PTPiECKO mice . Normalized to mean value for outer curvature of VE - PTPfl / fl mice scaled to 1 . 0 . Mean ± SEM , n = 6 mice / group .

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(B) Measurements of Tie2-pY992 staining intensity at intercellular junctions in aortic arch endothelial cells showing lower values in the inner curvature than in the upper curvature of VE-PTPfl/fl mice but equally high values in both regions of VE-PTPiECKO mice. Normalized to mean value for outer curvature of VE-PTPfl/fl mice scaled to 1.0. Mean ± SEM, n = 6 mice/group.


( C ) HUVEC transfected with control siRNA or VE - PTP siRNA and exposed to static or laminar shear force at 15 dyn / cm2 for 30 min , followed by immunoprecipitation of Tie2 . Left . Blot showing immunoprecipitates ( top ) or cell lysates ( bottom ) immunoblotted for pan - phosphotyrosine ( antibody 4G10 ) , Tie2 , or VE - PTP . Ratio of staining for phosphorylated Tie2 and total Tie2 ( Tie2 - pY to Tie2 , n = 4 experiments ) . Right . Relative Tie2 - pY levels in HUVEC after control siRNA or VE - PTP siRNA . Normalized to static control = 1 .

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(C) HUVEC transfected with control siRNA or VE-PTP siRNA and exposed to static or laminar shear force at 15 dyn/cm2 for 30 min, followed by immunoprecipitation of Tie2. Left. Blot showing immunoprecipitates (top) or cell lysates (bottom) immunoblotted for pan-phosphotyrosine (antibody 4G10), Tie2, or VE-PTP. Ratio of staining for phosphorylated Tie2 and total Tie2 (Tie2-pY to Tie2, n = 4 experiments). Right. Relative Tie2-pY levels in HUVEC after control siRNA or VE-PTP siRNA. Normalized to static control = 1.


( D ) Effect of shear stress on the phosphorylation levels of Tie2 Y992 at cellular junctions . HUVECs treated with either control siRNA ( left ) or Tie2 siRNA ( right ) , were exposed to 15 dyn / cm2 shear stress for 5 min . The resulting cells were fixed and stained for Tie2 - pY992 ( red ) and VE - cadherin ( green ) .

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(D) Effect of shear stress on the phosphorylation levels of Tie2 Y992 at cellular junctions. HUVECs treated with either control siRNA (left) or Tie2 siRNA (right), were exposed to 15 dyn/cm2 shear stress for 5 min. The resulting cells were fixed and stained for Tie2-pY992 (red) and VE-cadherin (green).


( E ) HUVECs cultured on biotinylated gelatin were treated either with control siRNA , VE - PTP siRNA , Tie2 siRNA or VE - cadherin siRNA , then exposed to 15 dyn / cm2 shear stress for 30 minutes followed by 3 min incubation with Streptavidin - Alexa647 ( red ) , washing , fixation and staining for VE - cadherin ( green ) . ( F and G ) Quantification of Streptavidin leakage area per image . Mean ± SEM , n = 19 pooled in ten independent experiments ( F ) , and n = 6 pooled in three independent experiments ( G ) .

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(E) HUVECs cultured on biotinylated gelatin were treated either with control siRNA, VE-PTP siRNA, Tie2 siRNA or VE-cadherin siRNA, then exposed to 15 dyn/cm2 shear stress for 30 minutes followed by 3 min incubation with Streptavidin-Alexa647 (red), washing, fixation and staining for VE-cadherin (green). (F and G) Quantification of Streptavidin leakage area per image. Mean ± SEM, n = 19 pooled in ten independent experiments (F), and n = 6 pooled in three independent experiments (G). D


( B ) Fractional area occupied by Oil Red O - stained atheromas in aortic arch , brachiocephalic artery , at intercostal artery ostia , and away from ostia . Each dot is value for one mouse . Mean ± SEM , n = 13 - 18 mice / group .

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(B) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 13-18 mice/group.


( D - E ) Fluorescence intensity distributions in D and mean intensities in E of extravasated anti - fibrinogen antibody in inner curvature of ApoE - / - / VE - PTPfl / fl mice and ApoE - / - / VE - PTPiECKO mice on high - fat diet for 7 wk . Mean ± SEM , n = 7 mice / group .

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(D-E) Fluorescence intensity distributions in D and mean intensities in E of extravasated anti-fibrinogen antibody in inner curvature of ApoE-/-/VE-PTPfl/fl mice and ApoE-/-/VE-PTPiECKO mice on high-fat diet for 7 wk. Mean ± SEM, n = 7 mice/group.


( C ) Measurements of Tie2 - pY992 intensity at intercellular junctions in aortic arch of mice shown in B . Mean ± SEM , n = 8 mice / group .

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(C) Measurements of Tie2-pY992 intensity at intercellular junctions in aortic arch of mice shown in B. Mean ± SEM, n = 8 mice/group.


( E ) Fractional area occupied by Oil Red O - stained atheromas in aortic arch , brachiocephalic artery , at intercostal artery ostia , and away from ostia . Each dot is value for one mouse . Mean ± SEM , n = 17 mice / group .

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(E) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 17 mice/group.


( E ) ( left ) Western blot of NANOG , OCT4 , and SOX2 . ( right ) Relative NANOG levels as estimated by densitometry ( n = 8 ) . NANOG was nearly 7 - fold more in PD , which is twice that of 2iL / SL2i . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± SEM * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns = not significant ( paired two - tailed student ' s t - test ) .

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(E) (left) Western blot of NANOG, OCT4, and SOX2. (right) Relative NANOG levels as estimated by densitometry (n=8). NANOG was nearly 7-fold more in PD, which is twice that of 2iL/SL2i. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).


( F ) Western blot of pERK and ERK at 0 , 1 , 4 , 8 , 12 , 16 , and 24 hrs after media change in indicated treatments ( n = 3 ) .

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(F) Western blot of pERK and ERK at 0, 1, 4, 8, 12, 16, and 24 hrs after media change in indicated treatments (n=3).


( G ) Western blot of pERK and ERK in SLPD , SLCHIR , 2iL , and SL2i after 8 , 12 , 16 , and 24 hrs of culture relative to SL , where the cells in SL were harvested 24 hrs after the media change ( n > 3 ) .

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(G) Western blot of pERK and ERK in SLPD, SLCHIR, 2iL, and SL2i after 8, 12, 16, and 24 hrs of culture relative to SL, where the cells in SL were harvested 24 hrs after the media change (n>3).


( H ) ( left ) Western blot of pERK and ERK in 1μM PD and increasing concentrations of CHIR in serum - free N2B27 media . ( right ) Relative pERK levels ( n = 3 ) . ( I ) ( left ) Western blot of pERK and ERK in 3μM CHIR and increasing concentrations of PD in serum - free N2B27 media . ( right ) Relative pERK levels ( n = 6 ) . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± SEM in H , and I . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns = not significant ( paired two - tailed student ' s t - test ) .

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(H) (left) Western blot of pERK and ERK in 1μM PD and increasing concentrations of CHIR in serum-free N2B27 media. (right) Relative pERK levels (n=3). (I) (left) Western blot of pERK and ERK in 3μM CHIR and increasing concentrations of PD in serum-free N2B27 media. (right) Relative pERK levels (n=6). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in H, and I. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).


( G ) ( left ) Western blot of pERK in TNERT , TNERTFgfr1 - / - , TNERTFgfr2 - / - , TNERTFgf4 - / - , and TNERTFgfbp1 - / - with or no OHT . ( Right ) relative pERK levels estimated by densitometry ( n = 4 ) . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± SEM in G , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and ns = not significant ( paired two - tailed student ' s t - test ) .

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(G) (left) Western blot of pERK in TNERT, TNERTFgfr1-/-, TNERTFgfr2-/-, TNERTFgf4-/-, and TNERTFgfbp1-/- with or no OHT. (Right) relative pERK levels estimated by densitometry (n=4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in G, *P < 0.05, **P < 0.01, ***P < 0.001, and ns = not significant (paired two-tailed student's t-test).


( E ) Western blot of FGFR1 , FGFR2 , and pERK in TNERT at one - hour intervals at indicated time points after OHT treatment ( n > 3 ) .

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(E) Western blot of FGFR1, FGFR2, and pERK in TNERT at one-hour intervals at indicated time points after OHT treatment (n>3).


( C ) Western blot of pERK and ERK in TNERT and TNERTNono - / - cells treated with or no OHT ( n = 3 ) .

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(C) Western blot of pERK and ERK in TNERT and TNERTNono-/- cells treated with or no OHT (n=3).


( D ) Immunoprecipitation analysis showing interactions between ERK and NONO in the presence or absence of Nanog induction by Doxycycline in TDiN cells .

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(D) Immunoprecipitation analysis showing interactions between ERK and NONO in the presence or absence of Nanog induction by Doxycycline in TDiN cells.


( E ) ( left ) Western blot of pERK and ERK in E14Tg2a cells treated with PD or FGF4 . ( Middle and right ) Relative levels of pERK and NONO in E14Tg2a cells treated with PD or FGF4 ( n > 4 ) . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± SEM in E * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 and ns = not significant ( paired two - tailed student ' s t - test ) .

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(E) (left) Western blot of pERK and ERK in E14Tg2a cells treated with PD or FGF4. (Middle and right) Relative levels of pERK and NONO in E14Tg2a cells treated with PD or FGF4 (n>4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in E *P < 0.05, **P < 0.01, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).


( L ) Cycloheximide chase assay of NANOG in SL , SLPD , and SLFGF4 in E14Tg2a cells .

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(L) Cycloheximide chase assay of NANOG in SL, SLPD, and SLFGF4 in E14Tg2a cells.


( A ) Immunofluorescence of pERK ( red ) and NANOG ( green ) in the indicated ESCs . Scale bars 25 μM . ( B ) The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG .

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(A) Immunofluorescence of pERK (red) and NANOG (green) in the indicated ESCs. Scale bars 25 μM. (B)The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG.


( C ) ( left ) Immunofluorescence of pERK and NANOG in 10 % NANOG - high NiRFP2A cells at indicated time cultured in the same media collected from cells before FACS . ( Right ) The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG at different time points . Scale bars 25 μM .

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(C) (left) Immunofluorescence of pERK and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FACS. (Right) The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG at different time points. Scale bars 25 μM.


( D ) ( left ) Western blot analysis of pERK , and NANOG in 10 % NANOG - high NiRFP2A cells at indicated time cultured in the same media collected from cells before FAC sorting . ( Right ) Expression levels of NANOG and pERK in 10 % NANOG - high NiRFP2A cells relative to 0 hrs during the time course of culture . The NANOG and pERK expression oscillate between high and low levels in the cells during culture . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± SEM in D . * P < 0 . 05 , * * P < 0 . 01 and ns = not significant ( paired two - tailed student ' s t - test ) .

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(D) (left) Western blot analysis of pERK, and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FAC sorting. (Right) Expression levels of NANOG and pERK in 10% NANOG-high NiRFP2A cells relative to 0 hrs during the time course of culture. The NANOG and pERK expression oscillate between high and low levels in the cells during culture. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in D. *P < 0.05, **P < 0.01 and ns = not significant (paired two-tailed student's t-test).


ACE2 - Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV . HIVs pseudotyped with the S glycoprotein from CoVs were incubated with different fusion proteins for 1 h before infection . Luciferase activities in target cells were measured , and the percent neutralization was calculated .

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ACE2-Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV.HIVs pseudotyped with the S glycoprotein from CoVs were incubated with different fusion proteins for 1 h before infection. Luciferase activities in target cells were measured, and the percent neutralization was calculated.


Inhibition of cell fusion by ACE2 - Ig . Potent inhibition of cell fusion mediated by the SARS - CoV ( left ) or 2019 nCoV ( right ) spike ( S ) glycoprotein . Cells expressing diffferent S glycoprotein were incubated with indicated fusion proteins and mixed with ACE2 - expressing cells . The activity of the reporter gene , β - gal , was measured as a correlate of fusion . The curves represent the best fit to the experimental data and were used for calculation of the IC50 . Bars indicate the S . E .

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Inhibition of cell fusion by ACE2-Ig.Potent inhibition of cell fusion mediated by the SARS-CoV (left) or 2019 nCoV (right) spike (S) glycoprotein. Cells expressing diffferent S glycoprotein were incubated with indicated fusion proteins and mixed with ACE2-expressing cells. The activity of the reporter gene, β-gal, was measured as a correlate of fusion. The curves represent the best fit to the experimental data and were used for calculation of the IC50. Bars indicate the S.E.


( B ) Representative immunofluorescence images of HeLa cells in various mitotic stages . Cells were extracted , fixed , and co - stained for Cyclin B2 ( green ) , ACA ( red ) , and DNA ( blue ) . Scale bar , 10 µm .

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(B) Representative immunofluorescence images of HeLa cells in various mitotic stages. Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue). Scale bar, 10 µm.


( C ) Representative immunofluorescence images of HeLa cells that were fixed and co - stained for Cyclin B2 ( green ) , mAb414 ( red ) , and DNA ( blue ) . Scale bar , 10 µm .

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(C) Representative immunofluorescence images of HeLa cells that were fixed and co-stained for Cyclin B2 (green), mAb414 (red), and DNA (blue). Scale bar, 10 µm.


Representative immunofluorescence images of HeLa cells treated with an indicated siRNA Cells were extracted , fixed , and co - stained for Cyclin B2 ( green ) , ACA ( red ) , and DNA ( blue ) .

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Representative immunofluorescence images of HeLa cells treated with an indicated siRNA Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


Representative immunofluorescence images of HeLa cells treated with an indicated various drugs ( E ) . Cells were extracted , fixed , and co - stained for Cyclin B2 ( green ) , ACA ( red ) , and DNA ( blue ) .

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Representative immunofluorescence images of HeLa cells treated with an indicated various drugs (E). Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


( F ) HeLa cells were synchronized by double thymidine , then released and collected at selected time points . Cell lysate samples were resolved by SDS - PAGE and analyzed by Western blotting using anti - Cyclin B1 antibody , anti - Cyclin B2 antibody , or anti - β - Actin antibody .

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(F) HeLa cells were synchronized by double thymidine, then released and collected at selected time points. Cell lysate samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B1 antibody, anti-Cyclin B2 antibody, or anti-β-Actin antibody.


( B ) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies . Nocodazole - arrested mitotic cells were collected and lysed . Immunoprecipitation was accomplished using control IgG , anti - Cyclin B2 - 1 ( Abcam , ab185622 ) , and anti - Cyclin B2 - 2 ( Proteintech , 21644 - 1 - AP ) . Immunoprecipitation samples were resolved by Western blotting using a mouse anti - Cyclin B2 antibody ( Santa Cruz , sc - 28303 ) and a mouse anti - Mad2 antibody ( Santa Cruz , sc - 47747 )

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(B) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies. Nocodazole-arrested mitotic cells were collected and lysed. Immunoprecipitation was accomplished using control IgG, anti-Cyclin B2-1 (Abcam, ab185622), and anti-Cyclin B2-2 (Proteintech, 21644-1-AP). Immunoprecipitation samples were resolved by Western blotting using a mouse anti-Cyclin B2 antibody ( Santa Cruz, sc-28303) and a mouse anti-Mad2 antibody (Santa Cruz, sc-47747)


( C and D ) Flag immunoprecipitation assay . 293T cells were co - transfected with Flag - C - Mad2 and GFP - Cyclin B1 / B2 ( C ) , and with Flag - Mad1 - 1 - 215 and GFP - Cyclin B1 / B2 ( D ) . After 36 h , the cells were collected and lysed , and immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitates were resolved by SDS - PAGE and analyzed by Western blotting using anti - Flag antibody and anti - GFP

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(C and D) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-C-Mad2 and GFP-Cyclin B1/B2 (C), and with Flag-Mad1-1-215 and GFP-Cyclin B1/B2 (D). After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-GFP antibody


( E ) GST pulldown assay . Purified GST , GST - Cyclin B1 - 165 - 433 , and GST - Cyclin B2 - 130 - 398 - bound agarose beads were used as affinity matrices to absorb purified Trx - 6 × His - tagged C - Mad2 recombinant protein . Absorbed proteins were analyzed by SDS - PAGE and Coomassie Brilliant Blue

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(E) GST pulldown assay. Purified GST, GST-Cyclin B1-165-433, and GST-Cyclin B2-130-398-bound agarose beads were used as affinity matrices to absorb purified Trx-6×His-tagged C-Mad2 recombinant protein. Absorbed proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining


( F - I ) Representative immunofluorescence images of HeLa cells stably expressing GFP - Cyclin B2 ( F ) or GFP - Cyclin B1 ( H ) . The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h . Then cells were fixed and co - stained for Mad2 ( red ) and DNA ( blue ) . Scale bar , 10 µm . Scatter graphs illustrating kinetochore intensity of GFP - Cyclin B2 ( G ) or GFP - Cyclin B1 ( I ) in cells treated as in F and H , respectively . Bars represent the mean kinetochore intensity ( ± SD ) normalized to values of the siControl group . Each dot represents one cell ( ≥ 30 cells from three independent experiments )

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(F-I) Representative immunofluorescence images of HeLa cells stably expressing GFP-Cyclin B2 (F) or GFP-Cyclin B1 (H). The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h. Then cells were fixed and co-stained for Mad2 (red) and DNA (blue). Scale bar, 10 µm. Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2 (G) or GFP-Cyclin B1 (I) in cells treated as in F and H, respectively. Bars represent the mean kinetochore intensity (±SD) normalized to values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments)


( B ) Flag immunoprecipitation assay . Flag - tagged Cyclin B2 - WT , Cyclin B2 - 4A , or Cyclin B1 were expressed in 293T cells and purified with anti - Flag M2 beads . Subsequently , the beads were incubated with mitotic HeLa lysates for 2 h , and , following extensive washes , immunoprecipitatates were resolved by SDS - PAGE and analyzed by Western blotting using anti - Flag antibody and anti - Mad2 antibody .

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(B) Flag immunoprecipitation assay. Flag-tagged Cyclin B2-WT, Cyclin B2-4A, or Cyclin B1 were expressed in 293T cells and purified with anti-Flag M2 beads. Subsequently, the beads were incubated with mitotic HeLa lysates for 2 h, and, following extensive washes, immunoprecipitatates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-Mad2 antibody.


( C ) Flag immunoprecipitation assay . 293T cells were co - transfected with Flag - Cyclin B2 - WT or Flag - Cyclin B2 - 4A together with GFP ( negative control ) and GFP - CDK1 . After 36 h , the cells were collected and lysed , and immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitation samples were resolved by SDS - PAGE and analyzed by Western blotting using anti - GFP antibody and anti - Flag antibody .

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(C) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-Cyclin B2-WT or Flag-Cyclin B2-4A together with GFP (negative control) and GFP-CDK1. After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitation samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-GFP antibody and anti-Flag antibody.


( D ) Representative immunofluorescence images of HeLa cells transfected with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for ACA ( red ) and DNA ( blue ) . ( E ) Scatter graphs illustrating kinetochore intensity of GFP - Cyclin B2 - WT / 4A treated as in D . Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of GFP - Cyclin B2 - WT . Each dot represents one cell ( ≥ 30 cells from three independent experiments ) .

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(D) Representative immunofluorescence images of HeLa cells transfected with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for ACA (red) and DNA (blue). (E) Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2-WT/4A treated as in D. Bars represent the mean kinetochore intensity (±SD) normalized to the values of GFP-Cyclin B2-WT. Each dot represents one cell (≥30 cells from three independent experiments).


( A ) HeLa cells were transfected with siControl or siCyclin B2 . At 36 h after transfection , cells were collected and lysed . The samples were resolved by SDS - PAGE and analyzed by Western blotting using anti - Cyclin B2 antibody , anti - Cyclin B1 antibody , and anti - tubulin

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(A) HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were collected and lysed. The samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B2 antibody, anti-Cyclin B1 antibody, and anti-tubulin antibody


( B ) Representative stills illustrating mitotic progression in mCherry - H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA . Images were acquired at the indicated time points after the start of NEBD . The arrow indicates lagging

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(B) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA. Images were acquired at the indicated time points after the start of NEBD. The arrow indicates lagging chromosomes


( C ) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B . Bars are means ± SD ( ≥ 30 cells from three independent experiments )

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(C) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B. Bars are means ±SD (≥30 cells from three independent experiments)


( D ) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B ( n = 30 cells )

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(D) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B (n = 30 cells)


( E ) Representative stills illustrating mitotic progression in mCherry - H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . Images were acquired at the indicated time points after the start of

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(E) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. Images were acquired at the indicated time points after the start of NEBD


( F ) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E . Bars are means ± SD ( ≥ 30 cells from three independent experiments )

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(F) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E. Bars are means ±SD (≥30 cells from three independent experiments)


Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2 . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for Mad2 ( green ) in D , ACA ( red ) , and DNA ( blue ) Representative immunofluorescence images of HeLa cells co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for Mad2 ( red ) in DNA ( blue )

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

Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (green) in D, ACA (red), and DNA (blue) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (red) in DNA (blue)


Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of the siControl

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Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2 . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained fo Cdc20 ( green ) in D , ACA ( red ) , and DNA ( blue ) E ) Representative immunofluorescence images of HeLa cells co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained fo Cdc20 ( red ) in E , and DNA ( blue )

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

Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (green) in D, ACA (red), and DNA (blue) E) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (red) in E, and DNA (blue)


Scatter graphs illustrating kinetochore intensity o Cdc20 ( F , cells treated as in D and E ) Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of the siControl

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Scatter graphs illustrating kinetochore intensity o Cdc20 (F, cells treated as in D and E) Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


( G ) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12

(G) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12 h


( H ) Immunoprecipitation assay performed with Cdc20 antibody . HeLa cells were transfected with siControl or siCyclin B2 . At 36 h after transfection , cells were arrested in mitosis , collected , and lysed . Cell lysates were incubated with Protein A / G beads bound with Cdc20 antibody . Following extensive washes , immunoprecipitates were resolved by SAS - PAGE and analyzed by Western blotting using anti - Cdc20 , anti - BubR1 , anti - Bub3 , and anti - Mad2

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(H) Immunoprecipitation assay performed with Cdc20 antibody. HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were arrested in mitosis, collected, and lysed. Cell lysates were incubated with Protein A/G beads bound with Cdc20 antibody. Following extensive washes, immunoprecipitates were resolved by SAS-PAGE and analyzed by Western blotting using anti-Cdc20, anti-BubR1, anti-Bub3, and anti-Mad2 antibodies


Presence of TOC proteins in the central vacuole of protoplasts . Arabidopsis protoplasts expressing the YFP - tagged chloroplast proteins were analyzed by confocal microscopy following ConA treatment for four hours . Representative images are shown ( A ) . Chlorophyll autofluorescence was used to determine the localization of the YFP fluorescence signals relative to the chloroplasts . Signals for SP1 - YFP and FAX1 - YFP , which here are control proteins , were only present at the chloroplast envelope . Accumulation of YFP puncta in the central vacuole is indicated by the arrowheads . Scale bar , 10 μm .

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

Presence of TOC proteins in the central vacuole of protoplasts. Arabidopsis protoplasts expressing the YFP-tagged chloroplast proteins were analyzed by confocal microscopy following ConA treatment for four hours. Representative images are shown (A). Chlorophyll autofluorescence was used to determine the localization of the YFP fluorescence signals relative to the chloroplasts. Signals for SP1-YFP and FAX1-YFP, which here are control proteins, were only present at the chloroplast envelope. Accumulation of YFP puncta in the central vacuole is indicated by the arrowheads. Scale bar, 10 μm.


Presence of TOC proteins in the central vacuole of Arabidopsis leaf and hypocotyl cells . Eight - day - old transgenic Arabidopsis seedlings stably expressing pToc33 : YFP - Toc33 or pToc75 : YFP - Toc75 were treated with ConA for two days , and then analyzed by confocal microscopy . Accumulation of YFP puncta in the central vacuole is indicated by the arrowheads . Scale bar , 10 μm .

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Presence of TOC proteins in the central vacuole of Arabidopsis leaf and hypocotyl cells. Eight-day-old transgenic Arabidopsis seedlings stably expressing pToc33:YFP-Toc33 or pToc75:YFP-Toc75 were treated with ConA for two days, and then analyzed by confocal microscopy. Accumulation of YFP puncta in the central vacuole is indicated by the arrowheads. Scale bar, 10 μm.


Lack of TOC proteins in the central vacuole of atg7 - 2 mutant hypocotyl cells . Eight - day - old transgenic Arabidopsis seedlings stably expressing pToc33 : YFP - Toc33 and pToc75 : YFP - Toc75 in the atg7 - 2 mutant background were treated with ConA for two days , and then analyzed by confocal microscopy . Note that YFP - Toc33 and YFP - Toc75 bodies ( arrowheads ) were retained at plastid bridges and extensions , and not released into the vacuole , as occurs in the WT background Scale bar , 10 μm .

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Lack of TOC proteins in the central vacuole of atg7-2 mutant hypocotyl cells. Eight-day-old transgenic Arabidopsis seedlings stably expressing pToc33:YFP-Toc33 and pToc75:YFP-Toc75 in the atg7-2 mutant background were treated with ConA for two days, and then analyzed by confocal microscopy. Note that YFP-Toc33 and YFP-Toc75 bodies (arrowheads) were retained at plastid bridges and extensions, and not released into the vacuole, as occurs in the WT background Scale bar, 10 μm.


Autophagy inhibition affected accumulation of TOC proteins . Eight - day - old wild - type ( WT ) and atg7 - 2 plants were treated respectively with proteasome inhibitor , bortezomib ( Btz ) , cysteine protease inhibitor , E64d , or DMSO ( Mock ) for one day . Total protein extracts from these plants were analyzed by immunoblotting using antibodies as indicated . Actin acted as a loading control . Typical immunoblotting results are shown ( B ) . In B , the arrowhead indicates full - length Toc159 , and the hollow arrow indicates a truncated form of Toc159 . Molecular weight markers ( sizes in kD ) are indicated to the right of the images .

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

Autophagy inhibition affected accumulation of TOC proteins. Eight-day-old wild-type (WT) and atg7-2 plants were treated respectively with proteasome inhibitor, bortezomib (Btz), cysteine protease inhibitor, E64d, or DMSO (Mock) for one day. Total protein extracts from these plants were analyzed by immunoblotting using antibodies as indicated. Actin acted as a loading control. Typical immunoblotting results are shown (B). In B, the arrowhead indicates full-length Toc159, and the hollow arrow indicates a truncated form of Toc159. Molecular weight markers (sizes in kD) are indicated to the right of the images.


Autophagy inhibition affected accumulation of TOC proteins . Eight - day - old wild - type ( WT ) and atg7 - 2 plants were treated respectively with proteasome inhibitor , bortezomib ( Btz ) , cysteine protease inhibitor , E64d , or DMSO ( Mock ) for one day . Band intensities were quantified and normalized to equivalent data for Actin ( C ) . Data are means ± SEM from three biological replicates . Asterisks in C indicate significance according to an unpaired two - tailed Student ' s t - test ( * , significant at P < 0 . 05 ; * * , significant at P < 0 . 01 ) .

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

Autophagy inhibition affected accumulation of TOC proteins. Eight-day-old wild-type (WT) and atg7-2 plants were treated respectively with proteasome inhibitor, bortezomib (Btz), cysteine protease inhibitor, E64d, or DMSO (Mock) for one day. Band intensities were quantified and normalized to equivalent data for Actin (C). Data are means ± SEM from three biological replicates. Asterisks in C indicate significance according to an unpaired two-tailed Student's t-test (*, significant at P < 0.05; **, significant at P < 0.01).


Presence of autophagosome in the chloroplast envelope . Arabidopsis protoplasts expressing free YFP and YFP - ATG8 ( autophagosome marker ) were analyzed by confocal microscopy . Corresponding chlorophyll autofluorescence images were employed to orientate the YFP signal in relation to the chloroplasts . For eliminating interference from cytosolic signals , intact protoplasts ( upper panels ) were ruptured using 10 - μm nylon mesh to isolate the chloroplasts . Retention of YFP signals in association with chloroplasts upon rupturing the cell ( lower panels ) clearly indicated that the YFP - ATG8 was associated with chloroplasts . Representative images are shown ( A ) .

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Presence of autophagosome in the chloroplast envelope. Arabidopsis protoplasts expressing free YFP and YFP-ATG8 (autophagosome marker) were analyzed by confocal microscopy. Corresponding chlorophyll autofluorescence images were employed to orientate the YFP signal in relation to the chloroplasts. For eliminating interference from cytosolic signals, intact protoplasts (upper panels) were ruptured using 10-μm nylon mesh to isolate the chloroplasts. Retention of YFP signals in association with chloroplasts upon rupturing the cell (lower panels) clearly indicated that the YFP-ATG8 was associated with chloroplasts. Representative images are shown (A).


whole protein samples of protoplasts and purified chloroplasts were analyzed by SDS - PAGE and immunoblotting , using antibodies against the YFP tag and Tic110 ( a chloroplast marker ) . Typical immunoblotting results are shown ( B ) .

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whole protein samples of protoplasts and purified chloroplasts were analyzed by SDS-PAGE and immunoblotting, using antibodies against the YFP tag and Tic110 (a chloroplast marker). Typical immunoblotting results are shown (B).


Presence of NBR1 - HA in chloroplast fraction . The protoplasts were isolated from four - week - old transgenic Arabidopsis plants stably expressing p35S : NBR1 - HA ( NBR1 - HA ) and p35S : YFP - HA ( YFP - HA ) , followed by chloroplast purification , respectively . Protein samples of whole protoplasts and isolated chloroplasts of NBR1 - HA and YFP - HA were then analyzed by SDS - PAGE and immunoblotting , using antibodies against the HA tag and Tic40 . Typical immunoblotting results are shown ( G ) .

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

Presence of NBR1-HA in chloroplast fraction. The protoplasts were isolated from four-week-old transgenic Arabidopsis plants stably expressing p35S:NBR1-HA (NBR1-HA) and p35S:YFP-HA (YFP-HA), followed by chloroplast purification, respectively. Protein samples of whole protoplasts and isolated chloroplasts of NBR1-HA and YFP-HA were then analyzed by SDS-PAGE and immunoblotting, using antibodies against the HA tag and Tic40. Typical immunoblotting results are shown (G).


BiFC results showed interaction between NBR1 and TOC proteins . cYFP and nYFP refer to C - terminal YFP fragment and N - terminal YFP fragment , respectively . Reconstitution of YFP fluorescence was assessed after transient coexpression of the indicated pairs of fusion proteins . CDKA1 and OEP7 were used as negative controls for OEM and cytosolic proteins , respectively . Representative images are shown . Scale bars , 10 μm . The frequency of protoplasts showing a BiFC signal in C was quantified . All data are means ± SEM from three experiments .

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

BiFC results showed interaction between NBR1 and TOC proteins. cYFP and nYFP refer to C-terminal YFP fragment and N-terminal YFP fragment, respectively. Reconstitution of YFP fluorescence was assessed after transient coexpression of the indicated pairs of fusion proteins. CDKA1 and OEP7 were used as negative controls for OEM and cytosolic proteins, respectively. Representative images are shown. Scale bars, 10 μm. The frequency of protoplasts showing a BiFC signal in C was quantified. All data are means ± SEM from three experiments.


NBR1 coimmunoprecipitated with TOC proteins . Proteins were extracted from eight - day - old transgenic Arabidopsis seedlings stably expressing p35S : NBR1 - HA or p35S : NBR1 - ΔUBA - HA , or control seedlings only expressing p35S : YFP - HA , following UVB treatment for four hours . Obtained protein extract was analyzed by IP using anti - HA resin , and analyzed by immunoblotting of four endogenous proteins or HA - tagged proteins . Tic40 acted as negative controls . The asterisks indicate nonspecific cross - reacting bands . Arrow indicates the NBR1 - HA band . The arrowhead indicates full - length Toc159 protein . Molecular weight markers ( sizes in kD ) are indicated to the right of the images .

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

NBR1 coimmunoprecipitated with TOC proteins. Proteins were extracted from eight-day-old transgenic Arabidopsis seedlings stably expressing p35S:NBR1-HA or p35S: NBR1-ΔUBA-HA, or control seedlings only expressing p35S:YFP-HA, following UVB treatment for four hours. Obtained protein extract was analyzed by IP using anti-HA resin, and analyzed by immunoblotting of four endogenous proteins or HA-tagged proteins. Tic40 acted as negative controls. The asterisks indicate nonspecific cross-reacting bands. Arrow indicates the NBR1-HA band. The arrowhead indicates full-length Toc159 protein. Molecular weight markers (sizes in kD) are indicated to the right of the images.


Phenotypes of seedlings under UVB stress . Briefly , eight - day - old WT , atg7 - 2 , nbr1 - 2 and NBR1 - OX ( overexpressor ) seedlings grown under normal condition were subjected to UVB stress , with UVB treatment for 3 hours and recovery for 7 days . Representative plant images ( A ) and total chlorophyll content values ( B ) are shown ; the latter provide a convenient metric for biomass . The chlorophyll values for the mutants and overexpressor plants were statistically significantly different from that for the WT ( Student ' s t test ; * , significant at P < 0 . 05 ; * * * , significant at P < 0 . 001 ) .

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

Phenotypes of seedlings under UVB stress. Briefly, eight-day-old WT, atg7-2, nbr1-2 and NBR1-OX (overexpressor) seedlings grown under normal condition were subjected to UVB stress, with UVB treatment for 3 hours and recovery for 7 days. Representative plant images (A) and total chlorophyll content values (B) are shown; the latter provide a convenient metric for biomass. The chlorophyll values for the mutants and overexpressor plants were statistically significantly different from that for the WT (Student's t test; *, significant at P < 0.05; ***, significant at P < 0.001).


TOC protein levels were regulated by autophagy in response to short - term UVB stress . Eight - day - old plants of indicated genotypes were subjected to UVB stress for the indicated times , and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane ( C , upper ) . Coomassie staining indicated that the treatments did not change the overall protein profile ( C , lower ) . Band intensities were quantified and normalized to equivalent Actin data ( D ) Time 0 was taken as 100 % . Unpaired two - tailed Student ' s t - tests revealed that the Toc159 protein level in nbr1 - 2 ( P < 0 . 05 ) and NBR1 - OX ( P < 0 . 05 ) , the Toc75 protein level in atg7 - 2 ( P < 0 . 05 ) , nbr1 - 2 ( P < 0 . 01 ) , and NBR1 - OX ( P < 0 . 05 ) , and the Toc33 protein level in atg7 - 2 ( P < 0 . 05 ) , nbr1 - 2 ( P < 0 . 01 ) , and NBR1 - OX ( P < 0 . 05 ) , were significantly different from levels in the WT at the time of 1 d recovery from UVB stress .

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

TOC protein levels were regulated by autophagy in response to short-term UVB stress. Eight-day-old plants of indicated genotypes were subjected to UVB stress for the indicated times, and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane (C, upper). Coomassie staining indicated that the treatments did not change the overall protein profile (C, lower). Band intensities were quantified and normalized to equivalent Actin data (D) Time 0 was taken as 100%. Unpaired two-tailed Student's t-tests revealed that the Toc159 protein level in nbr1-2 (P < 0.05) and NBR1-OX (P < 0.05), the Toc75 protein level in atg7-2 (P < 0.05), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.05), and the Toc33 protein level in atg7-2 (P < 0.05), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.05), were significantly different from levels in the WT at the time of 1 d recovery from UVB stress.


Toc159 was modified by both K48 and K63 ubiquitin linkage types . Eight - day - old WT plants were grown under normal growth condition ( - ) , or subjected to UVB stress ( + ) , with UVB treatment for four hours . The protein extract from the plants was analyzed by IP using native Toc159 antibody , or its preimmune serum as a negative control , and analyzed by immunoblotting using antibodies as indicated to the left of the images ( E ) . Tic40 acted as a negative control . The asterisk indicates a truncated Toc159 form without A domain Molecular weight markers ( sizes in kD ) are indicated to the right of the images . Please note that ubiquitin antibodies tend to produce stronger signals for hyper - ubiquitinated species than for hypo - ubiquitinated or mono - ubiquitinated species ( based on the number of epitopes ) , which contrasts with the behaviour of the Toc159 antibody . Thus , the signals of the two antibodies are not expected to fully overlap . Poly - ubiquitinated Toc159 may not be detected by the Toc159 antibody due to the much lower abundance of these species compared with the mono - ubiquitinated or unmodified forms . This is why the poly - Ub K48 and K63 signals appear at higher molecular weight positions than the Toc159 signals . Relative Toc159 amounts modified by K48 or K63 ubiquitin linkage in plants with or without UVB stress were assessed by quantifying the smeary band intensity of the IP samples in α - K48 or α - K63 blots ( F ) . All data are means ± SEM from three experiments .

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

Toc159 was modified by both K48 and K63 ubiquitin linkage types. Eight-day-old WT plants were grown under normal growth condition (-), or subjected to UVB stress (+), with UVB treatment for four hours. The protein extract from the plants was analyzed by IP using native Toc159 antibody, or its preimmune serum as a negative control, and analyzed by immunoblotting using antibodies as indicated to the left of the images (E). Tic40 acted as a negative control. The asterisk indicates a truncated Toc159 form without A domain Molecular weight markers (sizes in kD) are indicated to the right of the images. Please note that ubiquitin antibodies tend to produce stronger signals for hyper-ubiquitinated species than for hypo-ubiquitinated or mono-ubiquitinated species (based on the number of epitopes), which contrasts with the behaviour of the Toc159 antibody. Thus, the signals of the two antibodies are not expected to fully overlap. Poly-ubiquitinated Toc159 may not be detected by the Toc159 antibody due to the much lower abundance of these species compared with the mono-ubiquitinated or unmodified forms. This is why the poly-Ub K48 and K63 signals appear at higher molecular weight positions than the Toc159 signals. Relative Toc159 amounts modified by K48 or K63 ubiquitin linkage in plants with or without UVB stress were assessed by quantifying the smeary band intensity of the IP samples in α-K48 or α-K63 blots (F). All data are means ± SEM from three experiments.


TOC protein levels were regulated by autophagy in response to short - term heat stress . Eight - day - old plants of indicated genotypes grown in vitro were subjected to short term heat stress , recovered for the indicated times , and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane ( B , upper ) . Coomassie staining indicated that the treatments did not change the overall protein profile ( B , lower ) . Band intensities were quantified and normalized to equivalent Actin data ( C ) . Time 0 was taken as 100 % . Data are means ± SEM from three biological replicates . Unpaired two - tailed Student ' s t - tests revealed that the Toc159 protein level in nbr1 - 2 ( P < 0 . 05 ) and NBR1 - OX ( P < 0 . 01 ) , and the Toc75 protein level in NBR1 - OX ( P < 0 . 05 ) , were significantly different from levels in the WT at Time 12 h ; and that the Toc159 protein level in atg7 - 2 ( P < 0 . 001 ) , nbr1 - 2 ( P < 0 . 01 ) , and NBR1 - OX ( P < 0 . 001 ) , and the Toc75 protein level in atg7 - 2 ( P < 0 . 01 ) , nbr1 - 2 ( P < 0 . 01 ) , and NBR1 - OX ( P < 0 . 01 ) , were significantly different from levels in the WT at Time 24 h .

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

TOC protein levels were regulated by autophagy in response to short-term heat stress. Eight-day-old plants of indicated genotypes grown in vitro were subjected to short term heat stress, recovered for the indicated times, and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane (B, upper). Coomassie staining indicated that the treatments did not change the overall protein profile (B, lower). Band intensities were quantified and normalized to equivalent Actin data (C). Time 0 was taken as 100%. Data are means ± SEM from three biological replicates. Unpaired two-tailed Student's t-tests revealed that the Toc159 protein level in nbr1-2 (P < 0.05) and NBR1-OX (P < 0.01), and the Toc75 protein level in NBR1-OX (P < 0.05), were significantly different from levels in the WT at Time 12 h; and that the Toc159 protein level in atg7-2 (P < 0.001), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.001), and the Toc75 protein level in atg7-2 (P < 0.01), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.01), were significantly different from levels in the WT at Time 24 h.


Analysis of protein import efficiency in vitro . Chloroplasts were isolated from 12 - day - old WT , nbr1 - 2 and NBR1 - OX seedlings grown in vitro under normal condition , and were subjected to in vitro protein import assay by using 35S - labeled Rubisco small subunit ( SSU ) precursor protein as the import substrate , followed by corresponding quantification of the maturation of the radiolabeled precursor protein . A representative phosphor screen image is shown ( upper left ) , and Coomassie staining of Rubisco large subunit ( LSU ) indicated equal loading ( lower left ) ; Times indicate minutes after the start of each import reaction . IVT , in vitro translation product used as a control . Together with similar images from two additional experiments , this was used to conduct the quantitative analysis shown ( right ) ; the amount of imported , mature protein in each sample was expressed as a percentage of that present at the final time point for the WT . Data are means ± SEM from three biological replicates .

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

Analysis of protein import efficiency in vitro. Chloroplasts were isolated from 12-day-old WT, nbr1-2 and NBR1-OX seedlings grown in vitro under normal condition, and were subjected to in vitro protein import assay by using 35S-labeled Rubisco small subunit (SSU) precursor protein as the import substrate, followed by corresponding quantification of the maturation of the radiolabeled precursor protein. A representative phosphor screen image is shown (upper left), and Coomassie staining of Rubisco large subunit (LSU) indicated equal loading (lower left); Times indicate minutes after the start of each import reaction. IVT, in vitro translation product used as a control. Together with similar images from two additional experiments, this was used to conduct the quantitative analysis shown (right); the amount of imported, mature protein in each sample was expressed as a percentage of that present at the final time point for the WT. Data are means ± SEM from three biological replicates.


Chloroplast development in WT , nbr1 - 2 and NBR1 - OX seedlings under UVB stress was studied . Cotyledons of eight - day - old plants grown in vitro under normal condition ( D ) or UVB stress ( E ) were analyzed by transmission electron microscopy , and representative images are shown . UVB stress was conducted in the same procedures as in C . In D and E , the upper images are at the same magnification ( scale bar , 2 μm ) ; and below these are high magnification ( 5 × ) images corresponding to the boxed regions above . These and similar micrographs of chloroplasts were used to determine chloroplast shape ( F ) , the number of thylakoid lamellae per granum ( G ) , and the number of starch granules per chloroplast ( H ) in each genotype , with or without UVB stress . Asterisks indicate significance according to an unpaired two - tailed Student ' s t - test ( * , significant at P < 0 . 05 ; * * * , significant at P < 0 . 001 ; * * * * , significant at P < 0 . 0001 ) . Values are means ± SEM from 30 - 50 chloroplasts .

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

Chloroplast development in WT, nbr1-2 and NBR1-OX seedlings under UVB stress was studied. Cotyledons of eight-day-old plants grown in vitro under normal condition (D) or UVB stress (E) were analyzed by transmission electron microscopy, and representative images are shown. UVB stress was conducted in the same procedures as in C. In D and E, the upper images are at the same magnification (scale bar, 2 μm); and below these are high magnification (5×) images corresponding to the boxed regions above. These and similar micrographs of chloroplasts were used to determine chloroplast shape (F), the number of thylakoid lamellae per granum (G), and the number of starch granules per chloroplast (H) in each genotype, with or without UVB stress. Asterisks indicate significance according to an unpaired two-tailed Student's t-test (*, significant at P < 0.05; ***, significant at P < 0.001; ****, significant at P < 0.0001). Values are means ± SEM from 30-50 chloroplasts.


Phenotypes of two - week - old WT , nbr1 - 2 , ppi1 and ppi1 nbr1 - 2 seedlings grown on soil . Representative plant images ( A ) and chlorophyll contents ( B ) are shown . The chlorophyll values for the ppi1 and ppi1 nbr1 - 2 plants were statistically significantly different ( Student ' s t test ; * * * * , significant at P < 0 . 0001 ) . Values are means ± SEM from 20 plants .

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

Phenotypes of two-week-old WT, nbr1-2, ppi1 and ppi1 nbr1-2 seedlings grown on soil. Representative plant images (A) and chlorophyll contents (B) are shown. The chlorophyll values for the ppi1 and ppi1 nbr1-2 plants were statistically significantly different (Student's t test; ****, significant at P < 0.0001). Values are means ± SEM from 20 plants.


Chloroplast development in ppi1 and ppi1 nbr1 - 2 seedlings was studied . Cotyledons of two - week - old plants grown under normal condition as in A were analyzed by transmission electron microscopy , and representative images are shown ( E ) . In E , the upper images are at the same magnification ( scale bar , 1 μm ) ; and below these are high magnification ( 3 × ) images corresponding to the boxed regions above . These and similar micrographs of chloroplasts were used to determine chloroplast size ( F ) and the number of thylakoid lamellae per granum ( G ) in each genotype . Asterisks indicate significance according to an unpaired two - tailed Student ' s t - test ( * * * * , significant at P < 0 . 0001 ) . Values are means ± SEM from 30 - 40 chloroplasts .

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

Chloroplast development in ppi1 and ppi1 nbr1-2 seedlings was studied. Cotyledons of two-week-old plants grown under normal condition as in A were analyzed by transmission electron microscopy, and representative images are shown (E). In E, the upper images are at the same magnification (scale bar, 1 μm); and below these are high magnification (3×) images corresponding to the boxed regions above. These and similar micrographs of chloroplasts were used to determine chloroplast size (F) and the number of thylakoid lamellae per granum (G) in each genotype. Asterisks indicate significance according to an unpaired two-tailed Student's t-test (****, significant at P < 0.0001). Values are means ± SEM from 30-40 chloroplasts.


( A ) Comparative expression during murine pre - implantation embryo development of Dppa2 ( red ) , Dppa4 ( blue ) , Dux ( black ) , and Zscan4c ( ZGA marker , green ) . Each dot represents the average value of single - cell RNA -

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

(A) Comparative expression during murine pre-implantation embryo development of Dppa2 (red), Dppa4 (blue), Dux (black), and Zscan4c (ZGA marker, green). Each dot represents the average value of single-cell RNA-seq


( C ) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes , early 2C , mid 2C and late 2C Arcs depict splicing events , and numbers their relative frequency ( i . e . reads across junctions ) .

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

(C) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes, early 2C, mid 2C and late 2C Arcs depict splicing events, and numbers their relative frequency (i.e. reads across junctions).


( A ) Average expression of Dppa2 and Dppa4 in a single - cell RNA - seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters , respectively , and the double - negative population .

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

(A) Average expression of Dppa2 and Dppa4 in a single-cell RNA-seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.


( B ) Fraction of GFP + cells in mESCs carrying a MERVL - GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control . Bars represent the average , error bars the SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - test .

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(B) Fraction of GFP+ cells in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


( C ) Dppa2 , Dppa4 , Dux and Zscan4 gene expression in mESCs carrying a MERVL - GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control . Expression was normalized to Actb . Bars represent the average , error bars the SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - test .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dppa2, Dppa4, Dux and Zscan4 gene expression in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


RNA - seq analysis of WT and Dppa2 ( D ) KO mESC clones . The dot plot displays the average gene expression of three independent clones from each cell type ; n = 3 .

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

RNA-seq analysis of WT and Dppa2 (D) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


RNA - seq analysis of WT and Dppa4 ( E ) KO mESC clones . The dot plot displays the average gene expression of three independent clones from each cell type ; n = 3 .

RNA-seq analysis of WT and Dppa4 (E) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


( F ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 and Tdpoz4 ) , and two DUX - independent targets of DPPA2 and DPPA4 ( Mael and Tdrd1 ) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control . Expression was normalized to Actb . Bars represent the average , error bars the SD ; n =

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

(F) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and two DUX-independent targets of DPPA2 and DPPA4 (Mael and Tdrd1) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3


( H ) Comparative expression of DUX - dependent , DPPA2 and DPPA4 - dependent , and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters , respectively , and the double - negative population ( unpaired t - test ) .

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

(H) Comparative expression of DUX-dependent, DPPA2 and DPPA4-dependent, and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population (unpaired t-test).


( A ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 and Tdpoz4 ) , and three targets of DPPA2 and DPPA4 independent of DUX ( Mael , Tdrd1 , Prex2 ) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2 , DPPA4 or GFP as a control Expression was normalized to Actb . Bars represent the average , error bars the SD ; n = 3 . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - test .

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

(A) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and three targets of DPPA2 and DPPA4 independent of DUX (Mael, Tdrd1, Prex2) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2, DPPA4 or GFP as a control Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.