B Relative luciferase activity in HeLa shTRF2 following ionizing radiation ( 5 Gy ) or TRF2 knockdown upon doxycycline - induced shTRF2 expression ( n = 3 independent experiments ) . Error bars represent the s . e . m . * P < 0 . 05 . Two - way paired ANOVA .

B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. *P<0.05. Two-way paired ANOVA.


A Immunofluorescence showing gammaH2AX foci in Trf2F / F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

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A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce telomere uncapping ) or vehicle and injected with the indicated ASOs or PBS as control . Scale bar , 10 µm . D RT - qPCR detection of Ace2 mRNA levels in livers of mice treated as in C ( n = 5 - 8 mice per group ) .

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C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm. D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group).


E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 200 µm .

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E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm.


F Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive cells in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Atf7ip KO cells show increased expression of SETDB1 - regulated ERVs and the provirus reporter , MSCV - GFP as evidenced by RT - qPCR analysis . RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells . Data are mean ± SD ; n = 3 , technical replicates .

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A Atf7ip KO cells show increased expression of SETDB1-regulated ERVs and the provirus reporter, MSCV-GFP as evidenced by RT-qPCR analysis. RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells. Data are mean ± SD; n=3, technical replicates.


B Atf7ip KO mESCs ( TT # 2 - 12 ) show decreased H3K9me3 at the LTR of the SETDB1 - regulated reporter and the ERVs , as evidenced by Native ChIP followed by qPCR analysis . Gapdh was used as a negative control . Data are mean ± SEM ; n = 5 , independent experiments . * P < 0 . 05 and * * P < 0 . 01 by paired t - test .

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B Atf7ip KO mESCs (TT#2-12) show decreased H3K9me3 at the LTR of the SETDB1-regulated reporter and the ERVs, as evidenced by Native ChIP followed by qPCR analysis. Gapdh was used as a negative control. Data are mean ± SEM; n=5, independent experiments. *P < 0.05 and **P < 0.01 by paired t-test.


C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs . Endogenous SETDB1 - ATF7IP interaction is validated by anti - ATF7IP antibody co - IP experiment with anti - ATF7IP antibody .

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C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs. Endogenous SETDB1-ATF7IP interaction is validated by anti-ATF7IP antibody co-IP experiment with anti-ATF7IP antibody.


A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown , and the quantitative analyses are shown in B - D . Scale bar : 10 µm . B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test . C , D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated . Violin plot for SETDB1 ' s foci numbers from all the cells analysed in three independent experiments is shown in c . The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D . Over 100 cells were analysed per sample per experiment . * P < 0 . 05 by unpaired Student ' s t - test .

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A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown, and the quantitative analyses are shown in B-D. Scale bar: 10 µm. B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test. C, D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated. Violin plot for SETDB1's foci numbers from all the cells analysed in three independent experiments is shown in c. The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D. Over 100 cells were analysed per sample per experiment. *P < 0.05 by unpaired Student's t-test.


E , IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown . Arrows indicate the region used for line - plot analysis . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis . Scale bar : 10 µm . F The line - plot analysis for E . Relative intensities of SETDB1 signal were measured by Image J . G The quantitative analysis for E is shown . SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . NS : P > 0 . 05 , * * P < 0 . 01 versus " Mock , WT " group by Dunnett ' s test .

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E, IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown. Arrows indicate the region used for line-plot analysis. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis. Scale bar: 10 µm. F The line-plot analysis for E. Relative intensities of SETDB1 signal were measured by Image J. G The quantitative analysis for E is shown. SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. NS: P > 0.05, **P < 0.01 versus "Mock, WT" group by Dunnett's test.


A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype . Endogenous SETDB1 in HEK293T cells shows cytoplasm + nuclear localization profile . Scale bar : 10 µm .

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A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype. Endogenous SETDB1 in HEK293T cells shows cytoplasm+nuclear localization profile. Scale bar: 10 µm.


B Over - production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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B Over-production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells. The representative data of two independent experiments is shown. Scale bar: 10 µm.


C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells , but no significant impact for ATF7IP KO HEK293T cells . Cells were treated with 10ng / ml of LMB for 5 h and then analysed with immunofluorescent staining . The representative data of three independent experiments is shown . Scale bar : 10 µm . D SETDB1 signal in the nucleus shown in C was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . * * P < 0 . 01 by Student ' s t - test ( two - sided test ) .

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C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells, but no significant impact for ATF7IP KO HEK293T cells. Cells were treated with 10ng/ml of LMB for 5 h and then analysed with immunofluorescent staining. The representative data of three independent experiments is shown. Scale bar: 10 µm. D SETDB1 signal in the nucleus shown in C was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. **P < 0.01 by Student's t-test (two-sided test).


E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells . The representative data of three independent experiments is shown . Scale bar : 10 µm . F SETDB1 signal in the nucleus shown in E was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells . * P < 0 . 05 and * * P < 0 . 01 by Dunnett ' s method .

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E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells. The representative data of three independent experiments is shown. Scale bar: 10 µm. F SETDB1 signal in the nucleus shown in E was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells. *P < 0.05 and **P < 0.01 by Dunnett's method.


A HEK293T cells were transfected with V5 - SETDB1 and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells analysed per sample were analyzed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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A HEK293T cells were transfected with V5-SETDB1 and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells analysed per sample were analyzed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


B HEK293T cells were transfected with V5 - SETDB1 d1 mutant and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments was shown . Scale bar : 10 µm . The yellow arrowheads indicate the cells that show nuclear ( and cytoplasmic ) - localization of introduced exogenous mSETDB1 .

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B HEK293T cells were transfected with V5-SETDB1 d1 mutant and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments was shown. Scale bar: 10 µm. The yellow arrowheads indicate the cells that show nuclear (and cytoplasmic)-localization of introduced exogenous mSETDB1.


A Co - transfection with ATF7IP augments the K885 ubiquitination of SETDB1 . HEK293T cells were transfected After the transfection , V5 - SETDB1 was immune - purified with anti - V5 antibody and used for WB analysis .

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A Co-transfection with ATF7IP augments the K885 ubiquitination of SETDB1. HEK293T cells were transfected After the transfection, V5-SETDB1 was immune-purified with anti-V5 antibody and used for WB analysis.


B ATF7IP regulates the ubiquitination of SETDB1 in mESCs . Endogenous SETDB1 was immune - purified from WT or Atf7ip KO mESCs cells and used for WB analysis .

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B ATF7IP regulates the ubiquitination of SETDB1 in mESCs. Endogenous SETDB1 was immune-purified from WT or Atf7ip KO mESCs cells and used for WB analysis.


C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis .

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C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis.


D HEK293T cells were transfected with either 3xF - or 3xF - NLS - SETDB1 . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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D HEK293T cells were transfected with either 3xF- or 3xF-NLS-SETDB1. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


E Nuclear localization itself promotes the ubiquitination of SETDB1 . HEK293T cells were transfected with 3xFLAG - or 3xFLAG - NLS - SETDB1 . After FLAG - IP , the purified SETDB1s were analyzed by WB .

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E Nuclear localization itself promotes the ubiquitination of SETDB1. HEK293T cells were transfected with 3xFLAG- or 3xFLAG-NLS-SETDB1. After FLAG-IP, the purified SETDB1s were analyzed by WB.


F , G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF - or 3xF - NLS - SETDB1 . Representative images are shown in F , and the quantitative analysis is shown in G . Scale bar : 10 µm . Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 50 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test .

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F, G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF- or 3xF-NLS-SETDB1. Representative images are shown in F, and the quantitative analysis is shown in G. Scale bar: 10 µm. Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 50 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test.


H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs . Atf7ip KO mESCs expressing 3xF - or 3xF - NLS - SETDB were used for FLAG - IP , and the purified SETDB1s were analyzed by WB .

H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs. Atf7ip KO mESCs expressing 3xF- or 3xF-NLS-SETDB were used for FLAG-IP, and the purified SETDB1s were analyzed by WB.


A ) Double fluorescence in situ hybridization using Nrg3 ( green ) and Gad1 ( red ) specific probes ; DAPI was used as a counter stain . Nrg3 is present in Gad1 - positive ( filled arrowheads ) and - negative neurons . ( A ' ) and ( A ' ' ) show higher magnifications of boxed areas indicated in ( A ) . Nrg3 - negative neurons are indicated by open arrowheads ; note that many but not all neurons express Nrg3 .

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A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.


B ) Comparison of mRNA levels of Nrg3 , Nrg1β type III ( Nrg1β − III ) , Nrg2β , Nrg1β type II ( Nrg1β − II ) , Nrg2α and Nrg1β type I ( Nrg1β − I ) in the cortex ( Cx ) , hippocampus ( HC ) and striatum ( Str ) of juvenile mice ( P30 - 60 ) by quantitative RT - PCR . Data represent means ± SD of 4 biological replicates . 2 - way - ANOVA with Bonferroni ' s multiple comparisons test was performed to assess statistical significance ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , ns = not significant ) .

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B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant).


D ) Detection of Nrg3 by western blot analysis in fractions from brain lysates : S1 , crude lysate ; S2 , cytosol and light membranes ; S2 ' , cytosol ; LM , light membranes ; P2 ' , crude synaptosomes ; S3 ' , synaptosomal cytoplasm ; SV , synaptic vesicles ; P3 , synaptosomal membranes ; SPM , synaptic plasma membranes .

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D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes.


E , F ) Immunohistochemical analysis in the hippocampus of wildtype ( E , E ' , E ' ' ) and ErbB4 mutant mice ( F ) at 2 month of age using Nrg3 - , ErbB4 - and parvalbumin ( Parv ) - specific antibodies . The white box in ( E ) is displayed magnified in ( E ' , E ' ' ) . In control mice , Nrg3 is enriched on dendrites of ErbB4 + PV interneurons , compared to the surrounding neuropil ( E ' , E ' ' ) . In ErbB4 mutants , the Nrg3 enrichment on PV interneurons is not apparent ( F ) .

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E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F).


G ) Nrg3 , ErbB4 and GluA4 were identified by immunohistology and co - localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1 . Note that ( G ) displays Nrg3 / ErbB4 , ( G ' ) Nrg3 / GluA4 , and ( G ' ' ) ErbB4 / GluA4 signals of the same triple - stained image ; false colors were assigned for better signal visualization .

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G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization.


H ) Quantification of GluA4 puncta present on ErbB4 + dendrites in the CA1 stratum radiatum of adult wildtype ( wt ) and Nrg3 - / - mice ( age P90 - 120 ) . Data are presented as box plots with Tukey ' s whiskers and outliers ; means are indicated by a Plus symbols . n = 65 ( wt ) and n = 62 ( Nrg3 - / - ) from 5 animals each . Unpaired t - test ( 2 - tailed ) with Welch ' s correction was performed to assess statistical significance ( * * * * P < 0 . 0001 ) .

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H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).


A ) His6 - tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations . Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho - tyrosine ( pY ) and ErbB4 , and of whole lysates using antibodies against pErk1 / 2 and pAkt

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A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt


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C , D ) Neurons from Nrg3 - / - ; ErbB4 - / - ; vGAT - Cre mice were transduced with a lentivirus expressing either ( C ) ErbB4wt or ( D ) ErbB4kd ( kinase dead ErbB4 ) in a Cre - dependent manner , and with a second virus at a low titer expressing Nrg3 / SypCFP . Immunocytochemical analysis of ErbB4 and SypCFP ; ( C , C ' ) and ( D , D ' ) each show the same image , ( C ' , D ' ) show the SypCFP signal

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C, D) Neurons from Nrg3-/-;ErbB4-/-;vGAT-Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase dead ErbB4) in a Cre-dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C,C') and (D,D') each show the same image, (C',D') show the SypCFP signal


E - G ) Quantification of Synapse preference towards ErbB4 + neurons ( E ) , ErbB4 enrichment ( F ) and SypCFP enrichment ( G ) in synapses on neurons expressing wildtype ErbB4 ( wt ) or ErbB4kd ( kd )

O O O O O O O O O O O O O O O O O O O O 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-G) Quantification of Synapse preference towards ErbB4+ neurons (E), ErbB4 enrichment (F) and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd)


H ) Nrg3 - / - neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4 , GFP and GluA ; ( H ) shows the triple stained image , while ( H ' ) and ( H ' ' ) only show ErbB4 and GluA signals , respectively

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nrg3-/- neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP and GluA; (H) shows the triple stained image, while (H') and (H'') only show ErbB4 and GluA signals, respectively


I ) Co - cultures of Nrg3 - / - neurons with HEK293 cells expressing Nrg3 . Cultures were stained with antibodies against Nrg3 , ErbB4 and GluA . Note that Nrg3 , ErbB4 and GluA are enriched at the contact sites between Nrg3 - expressing HEK293 cells and ErbB4 + neurons

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

I) Co-cultures of Nrg3-/- neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4 and GluA. Note that Nrg3, ErbB4 and GluA are enriched at the contact sites between Nrg3-expressing HEK293 cells and ErbB4+ neurons


C ) Example traces of evoked excitatory postsynaptic currents ( eEPSCs ) averaged over 50 sweeps . D , E ) Quantification of the peak amplitude of the first eEPSC ( D ) and of the paired pulse ratio ( E )

O O O O O O O O O O O O O O O O O O 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) Example traces of evoked excitatory postsynaptic currents (eEPSCs) averaged over 50 sweeps. D, E) Quantification of the peak amplitude of the first eEPSC (D) and of the paired pulse ratio (E)


F - I ) sEPSC recordings from CA1 pyramidal ( F , H ) and PV neurons ( G , I ) in acute slices . sEPSC frequencies ( F , G ) and amplitudes ( H , I ) are

F-I) sEPSC recordings from CA1 pyramidal (F,H) and PV neurons (G, I) in acute slices. sEPSC frequencies (F,G) and amplitudes (H,I) are shown


( B - C ) Fluorescent dye release from the mitochondria - mimic liposomes by the indicated wild - type ( wt ) or mutant Bax in the presence ( B ) or absence ( C ) of tBid or by tBid alone ( C ) was measured by quenching the fluorescence of the released dyes by the dye - specific antibodies outside the liposomes during a time course . The fraction of dye release was normalized to that by detergent . The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes .

(B-C) Fluorescent dye release from the mitochondria-mimic liposomes by the indicated wild-type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye-specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes.


( D ) Digitonin permeabilized BMK Bax - / - / Bak - / - cells expressing SMAC - mCherry in the mitochondria intermembrane space ( 50 μL , 500 , 000 total cells ) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37 ° C in a 96 - well plate . The samples were centrifuged for 10 min and separated into supernatant and pellet fractions . SMAC - mCherry release was calculated as the fraction of total ( supernatant + pellet ) mCherry fluorescence coming from the supernatant fraction . The data was normalized to the percent SMAC - mCherry release of wt Bax and cBid at 60 min . Each symbol represents the normalized SMAC - mCherry release for one single replicate ( n = 3 or more independent replicates ) . Where applicable the data was fit with the [ Agonist ] vs . response - Variable slope equation in Graphpad Prism 8 . 0 . 1 . The dotted lines represent the 95 % confidence interval of the fit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.


Fig . 2 Identification of protein complexes and communities in the cellular extracts . ( A ) Elution of selected protein complexes as a function of their retention times ( see Appendix Fig . S4 for their corresponding subunit elutions ) .

Fig. 2 Identification of protein complexes and communities in the cellular extracts. (A) Elution of selected protein complexes as a function of their retention times (see Appendix Fig. S4 for their corresponding subunit elutions).


( B ) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak ; 50 % of protein complexes are observed to have higher molecular weights than structurally characterized , indicating that they are organized in higher - order assemblies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak; 50% of protein complexes are observed to have higher molecular weights than structurally characterized, indicating that they are organized in higher-order assemblies.


Fig . 4 Higher - order assemblies identified by proteome - wide cross - linking mass spectrometry , biomolecular modeling and negative - stain electron microscopy . ( A ) Distance distributions of identified cross - links on top of the modeled protein complexes and identification of novel interactions . Satisfied distances are shown in blue and over - length cross - links are shown in red .

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

Fig. 4 Higher-order assemblies identified by proteome-wide cross-linking mass spectrometry, biomolecular modeling and negative-stain electron microscopy. (A) Distance distributions of identified cross-links on top of the modeled protein complexes and identification of novel interactions. Satisfied distances are shown in blue and over-length cross-links are shown in red.


( B ) Negatively stained electron micrographs of fractions 3 - 7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions . Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Negatively stained electron micrographs of fractions 3-7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions. Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted.


( C ) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative - staining electron micrographs ; shown for fatty acid synthase , 20S proteasome , 60S and 40S ribosome ( the number of particles per image per fraction is indicated below the class averages ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative-staining electron micrographs; shown for fatty acid synthase, 20S proteasome, 60S and 40S ribosome (the number of particles per image per fraction is indicated below the class averages).


( D ) Simplification of lysate ( collecting only the flow - through from anion exchange chromatography ) prior to SEC separation allows class - averaging of structural signatures from complex fractions that were previously too low abundant . Proteins identified in this simplified fraction are found in Table S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Simplification of lysate (collecting only the flow-through from anion exchange chromatography) prior to SEC separation allows class-averaging of structural signatures from complex fractions that were previously too low abundant. Proteins identified in this simplified fraction are found in Table S4.


Fig . 5 Visualization of transient interactions in fatty acid synthesis . ( A ) Communities in fatty acid metabolism and the quantification of intra - community distances within cryo - electron micrographs . Fatty acid synthase ( FAS ) frequently interacts with other sizeable protein complexes in a linear ' pearl - string - like ' arrangement and usually localizes at the edges of the community . Scale bars correspond to 25 nm . FAS particles ( circles ) and their nearest neighbors ( arrow heads ) are indicated .

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

Fig. 5 Visualization of transient interactions in fatty acid synthesis. (A) Communities in fatty acid metabolism and the quantification of intra-community distances within cryo-electron micrographs. Fatty acid synthase (FAS) frequently interacts with other sizeable protein complexes in a linear 'pearl-string-like' arrangement and usually localizes at the edges of the community. Scale bars correspond to 25 nm. FAS particles (circles) and their nearest neighbors (arrow heads) are indicated.


( C ) Related to ( A ) . Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non - FAS neighbors in comparison to random distributions . Whereas FAS molecules are randomly distributed , their binders are not , confirming physical interactions . Supervised picking means that all single - particles were manually picked from the images . Randomized distance means that these manually picked particles were assigned random coordinates in each image ( randomization of x , y coordinates considering image borders ) and then their distance is calculated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Related to (A). Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non-FAS neighbors in comparison to random distributions. Whereas FAS molecules are randomly distributed, their binders are not, confirming physical interactions. Supervised picking means that all single-particles were manually picked from the images. Randomized distance means that these manually picked particles were assigned random coordinates in each image (randomization of x,y coordinates considering image borders) and then their distance is calculated.


( D ) Cross - linking mass spectrometry data show that the binder is a carboxylase that is bound to the malonyl transacylase domain and acyl carrier protein ( ACP ) is in the vicinity , considering cross - link length and the positions of the lysine on the ctFAS structure . Cross - links come from both affinity - purified and fractionated cell extracts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross-linking mass spectrometry data show that the binder is a carboxylase that is bound to the malonyl transacylase domain and acyl carrier protein (ACP) is in the vicinity, considering cross-link length and the positions of the lysine on the ctFAS structure. Cross-links come from both affinity-purified and fractionated cell extracts.


Fig . 6 CryoEM structure of fatty acid synthase resolved to 4 . 7 Å as obtained from cryo electron micrographs of fraction numbers 7 - 9 . ( A ) The cryoEM of C . thermophilum fatty acid synthase ( ctFAS ) map is shown isosurface rendered and superimposed with the fitted X - ray structure of yeast FAS ( Jenni et al , 2007 ) . The domes and the cap show the unambiguous fit of α - helices and β - strands .

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

Fig. 6 CryoEM structure of fatty acid synthase resolved to 4.7 Å as obtained from cryo electron micrographs of fraction numbers 7-9. (A) The cryoEM of C. thermophilum fatty acid synthase (ctFAS) map is shown isosurface rendered and superimposed with the fitted X-ray structure of yeast FAS (Jenni et al, 2007). The domes and the cap show the unambiguous fit of α-helices and β-strands.


( B ) A slice through the central wheel of fungal FAS . The pitch of α - helices is resolved .

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

(B) A slice through the central wheel of fungal FAS. The pitch of α-helices is resolved.


( C ) As for ( B ) but sliced through the dome structure .

(C) As for (B) but sliced through the dome structure.


( D ) Location of acyl carrier protein ( ACP ) within the cryoEM map of ctFAS and the position of additional density outside the dome .

O O O 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) Location of acyl carrier protein (ACP) within the cryoEM map of ctFAS and the position of additional density outside the dome.


A Whole mount immunofluorescence of a P8 Syne4 - / - organ of Corti showing intact hair cells , as labelled with myosin VIIa . B Whole mount immunofluorescence of WT and Syne4 - / - organ of Corti from the 8 , 16 , and 32 kHz regions at P8 and P14 .

A Whole mount immunofluorescence of a P8 Syne4-/- organ of Corti showing intact hair cells, as labelled with myosin VIIa. B Whole mount immunofluorescence of WT and Syne4-/- organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14.


C Inner and outer hair cell counts of Syne4 - / - organ of Corti at P8 , P10 , P12 , and P14 .

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C Inner and outer hair cell counts of Syne4-/- organ of Corti at P8, P10, P12, and P14.


D FM1 - 43 uptake performed on P8 + 1 DIV ( days - in - vitro ) WT and Syne4 - / - organ of Corti . Top shows OHC plane and bottom shows IHC plane .

O O O O O O O O O O O O O O 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 FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4-/- organ of Corti. Top shows OHC plane and bottom shows IHC plane.


B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV . GFP at P1 showing complete transduction of both inner and outer HC . Myosin VIIa was used to label the hair cells . C Examples of 8 , 16 , and 32 kHz regions of an organ of Corti of a mouse injected with AAV . GFP . Top shows OHC plane , bottom shows IHC plane , right shows YZ orthogonal projection . Black asterisks show bright Deiters cells . D Quantification of GFP intensity of inner and outer HC from 3 injected mice , normalized to the average intensity of HC in a control , un - injected mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. C Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. D Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse.


E Transduction rates of AAV9 - PHP . B at 8 , 16 , and 32kHz regions based on GFP fluorescence . A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate .

O O O O O O O O O O O O O O O O O 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 Transduction rates of AAV9-PHP.B at 8, 16, and 32kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate.


F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV . Syne4 .

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F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4.


G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope . Eight OHC were measured .

G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured.


H 3D surface projection of two adjacent OHC from a mouse injected with AAV . Syne4 . In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining .

O O O O O O O O O O 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 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.


A Whole - mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT , Syne4 - / - , and Syne4 - / - mice injected with AAV . Syne4 . B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT , Syne4 - / - mice and Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , white arrows denote cuticular plate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4-/-, and Syne4-/- mice injected with AAV.Syne4. B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4-/- mice and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate.


C Image analysis of nucleus position of IHC and OHC . Images show an arc fitted through the apical surface , nucleus , and basal end of the cell in 3D . X , Y , and Z axes show position in μm .

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C Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm.


D Quantification of nuclear distance from the cuticular plate . A total of 45 IHC and 135 OHC from 3 WT mice , 45 IHC and 81 OHC from 3 Syne4 - / - mice , 30 IHC and 90 OHC from 2 Syne4 - / - mice injected with AAV . Syne4 were measured .

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D Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4-/- mice, 30 IHC and 90 OHC from 2 Syne4-/- mice injected with AAV.Syne4 were measured.


A Representative example of ABR traces obtained at 4w from a WT , Syne4 - / - mouse , and Syne4 - / - mouse injected with AAV . Syne4 in response to an 18 kHz stimulus .

A Representative example of ABR traces obtained at 4w from a WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4 in response to an 18 kHz stimulus.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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 B-MEASURED_VAR O O O O

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 .

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

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 .

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

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 .

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 ) .

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 B-CONTROLLED_VAR O O O O O O

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 .

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

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 .

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 B-CONTROLLED_VAR O O O O

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 .

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

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 .

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

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 .

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

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 .

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

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 ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O 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) 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 (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

(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 .

(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 .

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)