Figure 5A ICC staining of CTC - ITB - 01 for ß - catenin ( green ) , E - cadherin ( orange ) , and DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 . B ICC staining of CTC - ITB - 01 for α - catenin ( green ) , visualization of actin filaments by phalloidin ( orange ) , and nuclei by DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 . C Generic EMT score of the adherent and non - adherently growing CTC - ITB - 01 fractions in relation to tumor samples from different cancer entities . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . The adherent fraction is shown with a black dot , the non - adherent fraction with a black triangle , and the mean score per cancer entity by a black line . D Generic EMT score of the adherent and non - adherently growing CTC - ITB - 01 fractions in relation to established cancer cell lines . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . The adherent fraction is shown with a black dot , the non - adherent fraction with a black triangle , and the mean score per cancer entity by a black line .

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Figure 5A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.C Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to tumor samples from different cancer entities. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line. D Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to established cancer cell lines. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.


Figure 6A In vivo bioluminescence imaging of CTC - ITB - 01 in four NSG mice . Blue indicates low radiance , red indicates high radiance . B Logarithmic plotting of in vivo radiance increase over time . Curves represent means ± SEM of measurements performed on multiple glands ( n = 8 ) . C H & E staining of xenografted mouse mammary glands show formation of primary tumors . D Stereoscope image of a mammary gland of immunodeficient NSG mouse . Fluorescently tagged CTC - ITB - 01 cells in green . E IHC staining for ER , confirms retained in vivo ER - positivity of formed tumors . Representative images of ER staining in higher ( left panel ) and lower ( right panel ) magnification of 2 different areas of a xenografted mammary gland . F Representative ex vivo luminescence images of indicated organs from engrafted mice . Organs include brain ( 1 ) , lung ( 2 ) , liver ( 3 ) and bones ( 4 ) . G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice ( M1 , M2 , M3 ) , indicating highest intensity in liver and bone . Data represent means ± SD of measurements performed on metastatic organs derived from three different mice .

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Figure 6A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice. Blue indicates low radiance, red indicates high radiance. B Logarithmic plotting of in vivo radiance increase over time. Curves represent means ± SEM of measurements performed on multiple glands (n=8). C H&E staining of xenografted mouse mammary glands show formation of primary tumors.D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green.E IHC staining for ER, confirms retained in vivo ER-positivity of formed tumors. Representative images of ER staining in higher (left panel) and lower (right panel) magnification of 2 different areas of a xenografted mammary gland.F Representative ex vivo luminescence images of indicated organs from engrafted mice. Organs include brain (1), lung (2), liver (3) and bones (4). G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice (M1, M2, M3), indicating highest intensity in liver and bone. Data represent means ± SD of measurements performed on metastatic organs derived from three different mice.


Figure 7A Equal numbers of CTC - ITB - 01 cells were seeded on 6 - well culture plates and grown in the presence or absence of different 17 - β - estradiol concentrations as indicated . After 10 days cells were fixed and stained with Coomassie blue staining ( right panel ) . Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations ( right panel ) . Scale bar 200 µm . B Stable knockdown of ER - alpha within CTC - ITB - 01 cells was performed by using lentiviral transfer of non - targeted ( scramble ) or two different targeted shRNAs against ER - alpha . CTC - ITB - 01 cells were seeded on 6 - well plates . After 10 days cells were fixed and stained ( left upper panel ) . Macroscopic photos are shown of the fixed cells ( left lower panel ) , scale bar 200 µm . Western blots of lysates from knockdown of ER - alpha expression in CTC - ITB - 01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies . The extracts were obtained 72h after lentiviral transduction ( right panel ) . Where indicated , western blots were visualized at long exposure ( l . e . ) and / or short exposure ( s . e . ) . C CTC - ITB - 01 and MCF - 7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated . 24h after E2 deprivation or E2 - treatment cells were harvested . Western blots of the protein lysates were probed with antibodies targeting ER - alpha , FOXM1 , Bcl - 2 , PR - A , PR - B and ID1 , actin was used as loading control , visualized at long exposure ( l . e . ) and / or short exposure ( s . e . ) . ( D ) Growth curves of CTC - ITB - 01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . E ) Growth curves of MCF - 7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . ( F ) Influence of Palbociclib on CTC - ITB - 01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide ( DMSO ) - . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . G ) Influence of Palbociclib on MCF - 7 after 80 hours . Data was chosen from one representative time point during the exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide ( DMSO ) - . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . ( H ) Effect of Palbociclib on the growth of CTC - ITB - 01 cells Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine the IC50 . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than the symbols are not displayed . I ) Effect of Palbociclib on the growth of MCF - 7 cells . Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine the IC50 . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than the symbols are not displayed .

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Figure 7A Equal numbers of CTC-ITB-01 cells were seeded on 6-well culture plates and grown in the presence or absence of different 17-β-estradiol concentrations as indicated. After 10 days cells were fixed and stained with Coomassie blue staining (right panel). Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations (right panel). Scale bar 200 µm.B Stable knockdown of ER-alpha within CTC-ITB-01 cells was performed by using lentiviral transfer of non-targeted (scramble) or two different targeted shRNAs against ER-alpha. CTC-ITB-01 cells were seeded on 6-well plates. After 10 days cells were fixed and stained (left upper panel). Macroscopic photos are shown of the fixed cells (left lower panel), scale bar 200 µm. Western blots of lysates from knockdown of ER-alpha expression in CTC-ITB-01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies. The extracts were obtained 72h after lentiviral transduction (right panel). Where indicated, western blots were visualized at long exposure (l.e.) and/or short exposure (s.e.).C CTC-ITB-01 and MCF-7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated. 24h after E2 deprivation or E2-treatment cells were harvested. Western blots of the protein lysates were probed with antibodies targeting ER-alpha, FOXM1, Bcl-2, PR-A, PR-B and ID1, actin was used as loading control, visualized at long exposure (l.e.) and/or short exposure (s.e.).(D) Growth curves of CTC-ITB-01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.E) Growth curves of MCF-7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.(F) Influence of Palbociclib on CTC-ITB-01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.G) Influence of Palbociclib on MCF-7 after 80 hours. Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.(H) Effect of Palbociclib on the growth of CTC-ITB-01 cells Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.I) Effect of Palbociclib on the growth of MCF-7 cells. Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.


Figure 1Western blotting of endogenous Egg immunopurified from OSCs . Anti - Egg antibodies were used for immunoprecipitation ( IP ) and western blotting . Input : OSC total lysates used for IP . n . i . : nonimmune IgG used as an IP negative control . M . W . indicates protein molecular weight marker . Western blotting of Myc - Egg expressed in OSCs . Anti - Myc ( Myc ) and anti - β - Tubulin ( Tub ) antibodies were used . Tub was detected as a loading control . Control : empty vector was used as a negative control . Western blotting of endogenous Egg immunopurified from OSCs . Anti - Egg antibodies were used for immunoprecipitation ( IP ) . Anti - Egg and anti - Ub antibodies were used for western blotting ( WB ) . Input : OSC total lysates used for IP . n . i . : nonimmune IgG used as an IP negative control . Western blotting of Egg immunopurified from total OSC lysates before ( - ) and after ( + ) phosphatase treatment . Anti - Egg antibodies were used for both immunoprecipitation ( IP ) and western blotting . Western blotting of Myc - Egg WT and its K1085R and S215 / T217 ( STAA ) mutants expressed in OSCs . Egg modifications identified in this study are summarized on the right . P : phosphorylation . Ub : monoubiquitination . In vitro histone methyltransferase assays . Top and second from the top : silver staining of purified Myc - Egg and Myc - EGFP . Second from the bottom : CBB staining of histone H3 . 1 . Bottom : 14C autoradiogram . EGFP : Myc - EGFP was used as a negative control . l . c . : light chain of antibody . Time : reaction time ( min ) of individual samples . Rescue assays . Myc - Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting ( WB ) was performed using anti - Egg and anti - Myc antibodies . RT - qPCR was used to quantify Stalker2 transposon expression level . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) . n . s . : not significant .

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Figure 1Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP) and western blotting. Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control. M.W. indicates protein molecular weight marker.Western blotting of Myc-Egg expressed in OSCs. Anti-Myc (Myc) and anti-β-Tubulin (Tub) antibodies were used. Tub was detected as a loading control. Control: empty vector was used as a negative control.Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP). Anti-Egg and anti-Ub antibodies were used for western blotting (WB). Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control.Western blotting of Egg immunopurified from total OSC lysates before (-) and after (+) phosphatase treatment. Anti-Egg antibodies were used for both immunoprecipitation (IP) and western blotting.Western blotting of Myc-Egg WT and its K1085R and S215/T217 (STAA) mutants expressed in OSCs. Egg modifications identified in this study are summarized on the right. P: phosphorylation. Ub: monoubiquitination.In vitro histone methyltransferase assays. Top and second from the top: silver staining of purified Myc-Egg and Myc-EGFP. Second from the bottom: CBB staining of histone H3.1. Bottom: 14C autoradiogram. EGFP: Myc-EGFP was used as a negative control. l.c.: light chain of antibody. Time: reaction time (min) of individual samples.Rescue assays. Myc-Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting (WB) was performed using anti-Egg and anti-Myc antibodies. RT-qPCR was used to quantify Stalker2 transposon expression level. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test). n.s.: not significant.


Figure 2Immunofluorescence analysis of OSCs using anti - Egg antibodies . Endogenous Egg is shown in red . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting of fractionated OSC materials . Anti - Egg , anti - β - Tubulin ( Tub ) , and anti - histone H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : the whole OSC lysates . Western blotting of Myc - Egg expressed in OSCs before ( siEGFP ) and after ( siUbc2 ) Ubc2 depletion . Anti - Myc - antibody was used for western blotting . The ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 43 ( siEGFP ) and 0 . 78 ( siUbc2 ) . Immunofluorescence analysis using anti - Flag antibody to identify Flag - Ubc2 ( green ) expressed in OSCs . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Immunofluorescence analysis of Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg expressed in OSCs using anti - Myc antibody . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting of Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg expressed in OSCs . Anti - Myc antibody was used . The ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 50 ( WT ) , 2 . 29 ( SV40 - NLS ) , and 0 . 95 ( PKI - NES ) . Rescue assays . Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . Stalker2 transposon expression was quantified using RT - qPCR . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) . In vitro HMT assays . Top : silver staining of purified Myc - Egg WT , Myc - Egg K1085R , Myc - SV40 - NLS - Egg WT , and Myc - SV40 - NLS - Egg K1085R . Middle : CBB staining of histone H3 . 1 . Bottom : 14C autoradiogram . Myc - EGFP was used as a negative control ( EGFP : not shown ) . Rescue assays . Myc - Egg WT , Myc - Egg K1085R , and Myc - SV40 - NLS - K1085R were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . RT - qPCR quantified the expression level of the Stalker2 transposon . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) .

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Figure 2Immunofluorescence analysis of OSCs using anti-Egg antibodies. Endogenous Egg is shown in red. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of fractionated OSC materials. Anti-Egg, anti-β-Tubulin (Tub), and anti-histone H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.Western blotting of Myc-Egg expressed in OSCs before (siEGFP) and after (siUbc2) Ubc2 depletion. Anti-Myc-antibody was used for western blotting. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.43 (siEGFP) and 0.78 (siUbc2).Immunofluorescence analysis using anti-Flag antibody to identify Flag-Ubc2 (green) expressed in OSCs. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Immunofluorescence analysis of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs using anti-Myc antibody. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs. Anti-Myc antibody was used. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.50 (WT), 2.29 (SV40-NLS), and 0.95 (PKI-NES).Rescue assays. Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. Stalker2 transposon expression was quantified using RT-qPCR. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).In vitro HMT assays. Top: silver staining of purified Myc-Egg WT, Myc-Egg K1085R, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R. Middle: CBB staining of histone H3.1. Bottom: 14C autoradiogram. Myc-EGFP was used as a negative control (EGFP: not shown).Rescue assays. Myc-Egg WT, Myc-Egg K1085R, and Myc-SV40-NLS-K1085R were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of the Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).


Figure 3Immunofluorescence analysis of endogenous Egg ( red ) in OSCs using anti - Egg antibody before ( siEGFP ) and after ( siWde ) Wde depletion . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting on fractionated materials of OSCs before ( siEGFP ) and after ( siWde ) Wde depletion . Anti - Egg , anti - Wde , anti - Tub , and anti - H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : whole OSC lysates . Immunofluorescence analysis of endogenous Egg ( red ) in OSCs upon Flag - Wde ( green ) co - expression . Anti - Egg and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Immunofluorescence analysis of Myc - Egg WT ( red ) in OSCs upon Wde co - expression ( green ) . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting on fractionated materials of Myc - Egg - expressed OSCs before ( - ) and after Wde WT and Wde ∆ CC co - expression . Anti - Myc , anti - Flag , anti - Tub , and anti - H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : the whole OSC lysates . Protein - protein interaction assays . Myc - Egg and Flag - Wde WT and its ∆ CC mutant were expressed in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) and anti - Flag ( lower ) antibodies were used for western blotting . Immunofluorescence analysis of Myc - Egg WT ( red ) in OSCs upon Flag - Wde WT and ∆ CC mutant ( green ) co - expression . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Figure 3Immunofluorescence analysis of endogenous Egg (red) in OSCs using anti-Egg antibody before (siEGFP) and after (siWde) Wde depletion. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting on fractionated materials of OSCs before (siEGFP) and after (siWde) Wde depletion. Anti-Egg, anti-Wde, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: whole OSC lysates.Immunofluorescence analysis of endogenous Egg (red) in OSCs upon Flag-Wde (green) co-expression. Anti-Egg and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Wde co-expression (green). Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting on fractionated materials of Myc-Egg-expressed OSCs before (-) and after Wde WT and Wde ∆CC co-expression. Anti-Myc, anti-Flag, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.Protein-protein interaction assays. Myc-Egg and Flag-Wde WT and its ∆CC mutant were expressed in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Flag-Wde WT and ∆CC mutant (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Figure 4Immunofluorescence analysis of Myc - Egg WT , Myc - Egg K987A , and Myc - Egg R988A mutants ( red ) in OSCs before and after Flag - Wde ( green ) co - expression . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting of Myc - Egg WT and its K987A and R988A mutants . Flag - Wde : Flag - Wde was co - expressed . SV40 - NLS : SV40 - NLS was added to Myc - Egg . Anti - Myc and anti - Flag antibodies were used . Immunofluorescence analysis of Myc - Egg K987A and Myc - Egg R988A mutants ( red ) in OSCs . Both mutants were fused to SV40 - NLS . Anti - Myc antibody was used for detection . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Protein - protein interaction assays . Myc - Egg WT , Myc - SV40 - NLS - Egg WT , and Myc - SV40 - NLS - Egg K1085R mutant were expressed with Flag - Wde in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) and anti - Flag ( lower ) antibodies were used for western blotting .

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Figure 4Immunofluorescence analysis of Myc-Egg WT, Myc-Egg K987A, and Myc-Egg R988A mutants (red) in OSCs before and after Flag-Wde (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of Myc-Egg WT and its K987A and R988A mutants. Flag-Wde: Flag-Wde was co-expressed. SV40-NLS: SV40-NLS was added to Myc-Egg. Anti-Myc and anti-Flag antibodies were used.Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Protein-protein interaction assays. Myc-Egg WT, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R mutant were expressed with Flag-Wde in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.


Figure 5Rescue assays . Myc - Egg WT and Myc - SV40 - NLS - Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion ( siEgg and siWde ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . RT - qPCR quantified the expression level of Stalker2 transposon . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) . Western blotting of Myc - SV40 - NLS - Egg WT in whole ( Whole ) , cytoplasm / nucleoplasmic ( Cyto / Np ) and chromatin ( Chromatin ) fractions before ( - ) and after Flag - Wde co - expression . Anti - Myc and anti - Flag antibodies were used . Histone H3 was detected as a marker of chromatin fraction . Immunofluorescence analysis of H3K9me3 ( green ) , Myc - Egg WT ( red ) , and Myc - SV40 - NLS - Egg WT ( red ) in OSCs before and after Flag - Wde ( yellow ) co - expression . Anti - H3K9me3 , anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B ( p values based on unpaired Welch ' s t - test ) . Box plots show median ( line ) and 25th - 75th percentile ( box ) ± 1 . 5 interquartile range ( n > 25 cells for each box ) . Circles represent outliers .

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Figure 5Rescue assays. Myc-Egg WT and Myc-SV40-NLS-Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion (siEgg and siWde). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).Western blotting of Myc-SV40-NLS-Egg WT in whole (Whole), cytoplasm/nucleoplasmic (Cyto/Np) and chromatin (Chromatin) fractions before (-) and after Flag-Wde co-expression. Anti-Myc and anti-Flag antibodies were used. Histone H3 was detected as a marker of chromatin fraction.Immunofluorescence analysis of H3K9me3 (green), Myc-Egg WT (red), and Myc-SV40-NLS-Egg WT (red) in OSCs before and after Flag-Wde (yellow) co-expression. Anti-H3K9me3, anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B (p values based on unpaired Welch's t-test). Box plots show median (line) and 25th-75th percentile (box) ± 1.5 interquartile range (n > 25 cells for each box). Circles represent outliers.


Figure 6Heat map of Egg ChIP - seq within a 10kb window centered on Egg peaks . Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input . Heatmaps of Egg ChIP - seq signal within 10 kb windows centered on Egg peaks in control ( siEGFP ) and Wde - depleted ( siWde ) OSCs . Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input . Metapolts showing Egg ChIP - seq signals for genomic regions around transposon ( TE ) insertions . ChIP - qPCR was performed before ( siEGFP ) and after ( siWde ) Wde depletion . qPCR was performed for Piwi target transposons ( Stalker2 , mdg1 , and gypsy ) and normalized to % input of a PIwi non - target transposon ( roo ) . Error bars represent SD from three independent experiments . Density plots for Egg ChIP - seq signal on Stalker2 , mdg1 , and gypsy in control ( siEGFP ) and Wde - depleted ( siWde ) OSCs . Input : input DNA was used as a control . RPM : reads per million mapped reads .

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Figure 6Heat map of Egg ChIP-seq within a 10kb window centered on Egg peaks. Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input.Heatmaps of Egg ChIP-seq signal within 10 kb windows centered on Egg peaks in control (siEGFP) and Wde-depleted (siWde) OSCs.Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input. Metapolts showing Egg ChIP-seq signals for genomic regions around transposon (TE) insertions.ChIP-qPCR was performed before (siEGFP) and after (siWde) Wde depletion. qPCR was performed for Piwi target transposons (Stalker2, mdg1, and gypsy) and normalized to %input of a PIwi non-target transposon (roo). Error bars represent SD from three independent experiments.Density plots for Egg ChIP-seq signal on Stalker2, mdg1, and gypsy in control (siEGFP) and Wde-depleted (siWde) OSCs. Input: input DNA was used as a control. RPM: reads per million mapped reads.


Figure 1His - tagged GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD and SARS - CoV - 2 NTD proteins were incubated with BHK 21 cells expressing eGFP - fused ACE2s . Anti - His / APC antibody was used to detect cells expressing His - tagged protein . Cells stained with the GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD and SARS - CoV - 2 NTD proteins are shown in green , orange , marine and red , respectively . The SARS - CoV - 2 NTD was used as negative control .

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Figure 1His-tagged GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins were incubated with BHK 21 cells expressing eGFP-fused ACE2s. Anti-His/APC antibody was used to detect cells expressing His-tagged protein. Cells stained with the GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins are shown in green, orange, marine and red, respectively. The SARS-CoV-2 NTD was used as negative control.


Figure 2The mFc - tagged ACE2s were captured by anti - mIgG Fc antibodies immobilized on the CM5 chip , and sequentially tested the binding with serially diluted SARS - CoV - 2 RBD , GX / P2V / 2017 RBD and GD / 1 / 2019 RBD . The raw and fitted curves are displayed in dotted and solid lines , respectively . The binding affinity between the RBDs ( SARS - CoV - 2 RBD , GX / P2V / 2017 RBD and GD / 1 / 2019 RBD ) and the 13 ACE2s are shown . Mean ± SD represents the mean and standard deviation of three independent experiments .

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Figure 2The mFc-tagged ACE2s were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively. The binding affinity between the RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD) and the 13 ACE2s are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.


Figure 3Hela - hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS - CoV - 2 , GX / P2V / 2017 and GD / 1 / 2019 , respectively . Green fluorescence indicates Hela - hACE2 cells infected with pseudovirus . Untransfected HeLa cell is used as negative control . The scale bar indicates 400 μm . Statistic for the infectivity of the three pseudoviruses . Data represent the results of five replicates . All data are presented as mean ± SD . * * * p < 0 . 001 ( Student ' s t - test ) . ns , no significant differences .

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Figure 3Hela-hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS-CoV-2, GX/P2V/2017 and GD/1/2019, respectively. Green fluorescence indicates Hela-hACE2 cells infected with pseudovirus. Untransfected HeLa cell is used as negative control. The scale bar indicates 400 μm. Statistic for the infectivity of the three pseudoviruses. Data represent the results of five replicates. All data are presented as mean ± SD. ***p < 0.001 (Student's t-test). ns, no significant differences.


Figure 6Flow cytometric assay of the SARS - CoV - 2 RBD and its mutants ( K417R , Q498H and K417R - Q498H ) binding to human , mouse , rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells . The SARS - CoV - 2 NTD was used as negative control . The mFc - tagged ACE2s from human , mouse , rat and European hedgehog were captured by anti - mIgG Fc antibodies immobilized on the CM5 chip , and sequentially tested the binding with serially diluted SARS - CoV - 2 RBD and its mutants ( K417R , Q498H and K417R - Q498H ) . The binding affinities of the four ACE2s to the SARS - CoV - 2 RBD and its mutants are shown . Mean ± SD represents the mean and standard deviation of three independent experiments .

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Figure 6Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H) binding to human, mouse, rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control.The mFc-tagged ACE2s from human, mouse, rat and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H).The binding affinities of the four ACE2s to the SARS-CoV-2 RBD and its mutants are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.


Figure 1B Western blot analysis ( probing with an anti - FLAG mAb ) to detect cleavage of the artificial substrate by wild - type ( WT ) / inactive ( S201A ) GlpG encoded on pBAD33 with / without arabinose ( Ara . ) . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red , arrows , respectively . Controls , empty pBAD33 ( empty ) C Western blot analysis to detect cleavage of the artificial substrate by wild - type ( WT ) / modified ( S133A or H187A ) Rhom7 encoded on pBAD33 with / without arabinose ( Ara . ) . , rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , and blue arrows , respectively . Controls , empty pBAD33 ( empty ) and wild - type S . sonnei ( Ss ) . D Activity of Rhom7 with / without its 7th TMD and / or C - terminal domain . rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , , and blue arrows , respectively . Controls , empty pBAD33 ( empty ) and wild - type S . sonnei ( Ss ) .

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Figure 1B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of the artificial substrate by wild-type (WT)/inactive (S201A) GlpG encoded on pBAD33 with/without arabinose (Ara.). rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, empty pBAD33 (empty)C Western blot analysis to detect cleavage of the artificial substrate by wild-type (WT)/modified (S133A or H187A) Rhom7 encoded on pBAD33 with/without arabinose (Ara.). , rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).D Activity of Rhom7 with/without its 7th TMD and/or C-terminal domain. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, , and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).


Figure 2C Western blot analysis of candidate substrates reproducibly cleaved by GlpG from the screen . Data information : In ( C rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red , arrows , respectively . Controls , inactive enzymes and wild - type S . sonnei ( Ss ) . D Western blot analysis of candidate substrates reproducibly cleaved by Rhom7 from the screen . Data information : In D ) rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , red , and blue arrows , respectively . Controls , inactive enzymes and wild - type S . sonnei ( Ss ) .

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Figure 2C Western blot analysis of candidate substrates reproducibly cleaved by GlpG from the screen. Data information: In (C rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).D Western blot analysis of candidate substrates reproducibly cleaved by Rhom7 from the screen. Data information: In D) rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, red, and blue arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).


Figure 3Western blot analysis ( probing with an anti - FLAG mAb ) to detect cleavage of HybO with ( + ) / without ( - ) chromosomal ( native ) or pBAD33 - encoded rhomboids ( plasmid ) . homboid substrates that are uncleaved , are marked by black , rrows , ild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 . Western blot analysis to detect cleavage of HybA with ( + ) / without ( - ) chromosomal ( native ) or pBAD33 - encoded rhomboids ( plasmid ) . rhomboid substrates that are uncleaved , cleaved by GlpG , or by Rhom7 , are marked by black , red , and blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 . Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33 - encoded rhomboid . Wild - type S . sonnei , Ss . rhomboid substrates that are uncleaved , cleaved by GlpG , or by Rhom7 , re marked by black , red , and blue arrows , espectively . ild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 . Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33 - encoded rhomboid . rhomboid substrates that are uncleaved , cleaved by GlpG , or by Rhom7 , are marked by black , red , and blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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

Figure 3Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of HybO with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). homboid substrates that are uncleaved, are marked by black, rrows, ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybA with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33-encoded rhomboid. Wild-type S. sonnei, Ss. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, re marked by black, red, and blue arrows, espectively. ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33-encoded rhomboid. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.


Figure 4A Bacterial two - hybrid analysis with HybA and / or HybB fused chromosomally with the T25 and T18 domains of B . pertussis CyaA , respectively in the absence of endogenous CyaA . Bacteria were grown on LB agar containing 20 µg / ml X - gal at 37 ° C for 14 hours in the presence / absence of O2 . Scale bar , 1 cm . B Western blot analysis to detect HybA cleavage in S . sonneiΔrhom7 by wild - type ( + ) or inactive ( S201A ) GlpG expressed chromosomally ( native ) or from pUC19 ( plasmid ) with ( + ) / without ( - ) HybB . HybA was expressed from its native locus or a plasmid ( pHybA ) . HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows , respectively . C Quantification of the ratio of cleaved / uncleaved HybA in strains with ( + ) or without ( - ) HybB . mean ± S . D . of three experiments . * , p < 0 . 05 ; * * * * , p < 0 . 0001 ( One - way ANOVA ) . D Western blot analysis to detect GlpG - mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB ( - ) . , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows , respectively . E Western blot analysis to detect GlpG - mediated cleavage of C - terminally sfCherry - 3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB ( + ) . , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows , respectively . F Growth of bacteria lacking hydrogenases ( ΔhyaA - F ΔhycE + / - ΔhybO - G ) or GlpG ( ΔglpG ) , or expressing uncleavable HybB ( hybAG296F ) in 5 % H2G H2 consumption by S . sonnei . Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0 . 5 % fumarate , 12 . 5 μg / ml nicotinic acid and 0 . 2 % casamino acids in a sealed glass chromatography vial . The headspace was purged with 10 % H2 / 90 % argon and cultures were incubated at 37 ° C with shaking at 180 rpm for 9 hours . H2 in the headspace was sampled and measured by gas chromatography . H S . sonnei growth in 10 % H2 with plasmid expressed wild - type or non - functional ( pglpGSAHA ) GlpG .

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

Figure 4A Bacterial two-hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 µg/ml X-gal at 37°C for 14 hours in the presence/absence of O2. Scale bar, 1 cm.B Western blot analysis to detect HybA cleavage in S. sonneiΔrhom7 by wild-type (+) or inactive (S201A) GlpG expressed chromosomally (native) or from pUC19 (plasmid) with (+)/without (-) HybB. HybA was expressed from its native locus or a plasmid (pHybA). HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.C Quantification of the ratio of cleaved/uncleaved HybA in strains with (+) or without (-) HybB. mean ± S.D. of three experiments. *, p<0.05; ****, p<0.0001 (One-way ANOVA).D Western blot analysis to detect GlpG-mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB (-). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.E Western blot analysis to detect GlpG-mediated cleavage of C-terminally sfCherry-3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB (+). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.F Growth of bacteria lacking hydrogenases (ΔhyaA-F ΔhycE +/- ΔhybO-G) or GlpG (ΔglpG), or expressing uncleavable HybB (hybAG296F) in 5% H2G H2 consumption by S. sonnei. Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0.5% fumarate, 12.5 μg/ml nicotinic acid and 0.2% casamino acids in a sealed glass chromatography vial. The headspace was purged with 10% H2/90% argon and cultures were incubated at 37 °C with shaking at 180 rpm for 9 hours. H2 in the headspace was sampled and measured by gas chromatography.H S. sonnei growth in 10% H2 with plasmid expressed wild-type or non-functional (pglpGSAHA) GlpG.


Figure 5B Western blot analysis ( probing with an anti - FLAG mAb ) to detect cleavage of FdoH cleavage in S . sonneiΔrhom7 with ( + ) / without ( - ) FdoI . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red arrows , respectively . C Western blot analysis to detect cleavage of FdnH in S . sonneiΔglpG with ( + ) or without ( - ) chromosomal Rhom7 ( native ) , or wild - type ( + ) / inactive ( SAHA ) Rhom7 expressed from pBAD33 in S . sonneiΔglpGΔrhom7 with ( + ) or without ( - ) FdnI . rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , and blue arrows , respectively . E Western blot analysis to detect cleavage of wild - type ( WT ) or uncleavable ( P300A ) HybA by active ( + ) or inactive ( S201A ) chromosomal GlpG in the presence ( + ) or absence ( - ) of HybB . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red arrows , respectively . F Western blot analysis to detect cleavage of wild - type ( WT ) or modified ( P259A ) FdoH by active ( + ) or inactive ( S201A ) chromosomal GlpG in the presence ( + ) or absence ( - ) of FdoI . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red arrows , respectively . G Western blot analysis to detect cleavage of wild - type ( WT ) or modified ( P259A ) FdnH with active ( + ) or inactive ( SAHA ) Rhom7 expressed from pBAD33 with ( + ) or without ( - ) FdnI . rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , blue arrows , respectively .

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

Figure 5B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of FdoH cleavage in S. sonneiΔrhom7 with (+)/without (-) FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.C Western blot analysis to detect cleavage of FdnH in S. sonneiΔglpG with (+) or without (-) chromosomal Rhom7 (native), or wild-type (+)/inactive (SAHA) Rhom7 expressed from pBAD33 in S. sonneiΔglpGΔrhom7 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively.E Western blot analysis to detect cleavage of wild-type (WT) or uncleavable (P300A) HybA by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of HybB. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.F Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdoH by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.G Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdnH with active (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.


Figure 6A Western blot analysis ( probing with an anti - V5 mAb ) to detect degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P300A ) HybA in S . sonneiΔrhom7 chromosomally expressing wild - type ( + ) or inactive ( S201A ) GlpG with ( + ) or without ( - ) HybB . Rhomboid substrates that are uncleaved , cleaved by GlpG , , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows . B Degradation of V5 - tagged HybA at times after blocking protein translation at T0 in the presence ( + ) or absence ( - ) of HybB . Rhomboid substrates that are uncleaved , cleaved by GlpG , black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows . C Western blot analysis of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdoH in S . sonneiΔrhom7 chromosomally expressing wild - type ( + ) or inactive ( S201A ) GlpG with ( + ) / without ( - ) FdoI . Rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red , arrows , respectively . D Western blot analysis of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdnH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( SAHA ) Rhom7 expressed from pBAD33 with ( + ) / without ( - ) FdnI . Rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , blue arrows , respectively . Degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdoH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( S201A , SAHA ) GlpG expressed chromosomally ( native ) or from pUC19 ( plasmid ) without FdoI , + / - exposure to 400 µM CuCl2 for 30 min . : Rhomboid substrates that are uncleaved , cleaved by GlpG , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows . F Degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdnH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( S201A , SAHA ) GlpG expressed from pUC19 ( plasmid ) without FdnI ( - ) , , + / - exposure to 400 µM CuCl2 for 30 min . Rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , and blue arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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

Figure 6A Western blot analysis (probing with an anti-V5 mAb) to detect degradation of N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+) or without (-) HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, , are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.B Degradation of V5-tagged HybA at times after blocking protein translation at T0 in the presence (+) or absence (-) of HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.C Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+)/without (-) FdoI. Rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively.D Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (-) FdnI. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI , +/- exposure to 400 µM CuCl2 for 30 min. : Rhomboid substrates that are uncleaved, cleaved by GlpG, are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.F Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed from pUC19 (plasmid) without FdnI (-), , +/- exposure to 400 µM CuCl2 for 30 min. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.


Figure 7A Western blot analysis probing the localisation and status of plasmid - encoded N - terminally V5 - tagged wild - type ( WT ) or modified ( P300A ) HybA in S . sonneiΔrhom7ΔhybB with wild - type ( + ) or inactive ( SAHA ) GlpG expressed from pUC19 . Whole cell lysate ( Whole cell . ) , Sol ( Sol . ) , Mem ( detergent - solubilised , Mem . ) , and the Aggregate ( Agg . ) fractions are shown . HybA that is uncleaved , cleaved only by GlpG , and further degraded post GlpG cleavage is marked by black , red , and green arrows , respectively .

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

Figure 7A Western blot analysis probing the localisation and status of plasmid-encoded N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7ΔhybB with wild-type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole cell lysate (Whole cell.), Sol (Sol.), Mem (detergent-solubilised, Mem.), and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG, and further degraded post GlpG cleavage is marked by black, red, and green arrows, respectively.


Figure 2 ( A ) Representative flow cytometry plots ( left ) and pooled data ( right ) of competition assays with sgControl and sgRnf31 B16 - F10 ova tumor cells after culture with the indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * * * * p < 0 . 0001 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM . ( B ) CRISPR / Cas9 screen showing depleted B16 - F10 genes after one week culture with TNF + IFN - γ ( both 10 ng / mL ) ; blue genes indicate overall genes with p < 0 . 001 , black genes indicate genes of interest with p < 0 . 05 . Significance assessed using MAGeCK test . ( H ) Representative flow cytometry plots ( left ) and pooled data ( right ) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM .

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

Figure 2(A) Representative flow cytometry plots (left) and pooled data (right) of competition assays with sgControl and sgRnf31 B16-F10 ova tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.(B) CRISPR/Cas9 screen showing depleted B16-F10 genes after one week culture with TNF + IFN-γ (both 10 ng/mL); blue genes indicate overall genes with p < 0.001, black genes indicate genes of interest with p < 0.05. Significance assessed using MAGeCK test.(H) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


Figure 3 ( A ) PI staining of sgControl or sgRnf31 B16 - F10 tumor cells treated with combined TNF / IFN - γ ( both 10 ng / mL ) for 24 hours in the absence and presence of QVD - OPh ( 10 μM ) ; n . s . : no significance , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM . ( B ) Western blot analysis of the indicated proteins on whole cell lysates for sgControl or sgRnf31 B16 - F10 tumor cells cultured with the indicated cytokine ( s ) ( 10 ng / mL for all ) for 0 , 3 , 6 , or 9 hours ; β - actin was used as a loading control . ( D ) CRISPR / Cas9 screen showing enriched genes from sgRnf31 B16 - F10 tumor cells after successive treatment with combined TNF / IFN - γ , as described in ( C ) ; red genes indicate overall genes with p < 0 . 0001 . Significance assessed using MAGeCK test . ( G ) PI staining of sgControl , sgRnf31 , sgRnf31 / Stat1 , sgRnf31 / Casp8 , sgRnf31 / Bid , or sgRnf31 / Rela B16 - F10 tumor cells treated with combined TNF / IFN - γ for 24 hours ; * p < 0 . 05 , * * * * p < 0 . 0001 , asterisk ( s ) indicated group vs TNF / IFN - γ - treated sgRnf31 group , Unpaired t - test for n > 3 biological replicates . Pooled data are represented as mean ± SEM .

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

Figure 3(A) PI staining of sgControl or sgRnf31 B16-F10 tumor cells treated with combined TNF/IFN-γ (both 10 ng/mL) for 24 hours in the absence and presence of QVD-OPh (10 μM); n.s.: no significance, ***p < 0.001, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.(B) Western blot analysis of the indicated proteins on whole cell lysates for sgControl or sgRnf31 B16-F10 tumor cells cultured with the indicated cytokine(s) (10 ng/mL for all) for 0, 3, 6, or 9 hours; β-actin was used as a loading control.(D) CRISPR/Cas9 screen showing enriched genes from sgRnf31 B16-F10 tumor cells after successive treatment with combined TNF/IFN-γ, as described in (C); red genes indicate overall genes with p < 0.0001. Significance assessed using MAGeCK test.(G) PI staining of sgControl, sgRnf31, sgRnf31/Stat1, sgRnf31/Casp8, sgRnf31/Bid, or sgRnf31/Rela B16-F10 tumor cells treated with combined TNF/IFN-γ for 24 hours; *p < 0.05, ****p < 0.0001, asterisk(s) indicated group vs TNF/IFN-γ-treated sgRnf31 group, Unpaired t-test for n > 3 biological replicates. Pooled data are represented as mean ± SEM.


Figure 4 ( B ) Representative flow cytometry plots ( left ) and pooled data ( right ) showing PI staining of parental B16 - F10 tumor cells left untreated or treated with supernatant ( Sup ) ( derived from a previous overnight OT - I / B16 - F10 ova co - culture at the indicated E : T ratio for 48 hours , following a 4 hour pre - treatment with vehicle or HOIPIN - 1 ( 50 μM ) ( the concentration of vehicle / HOIPIN - 1 was halved upon the addition of Sup ) ; * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM . ( C ) Representative flow cytometry plots ( left ) and pooled data ( right ) showing PI staining of parental A375 tumor cells left untreated or treated with TNF / IFN - γ ( both 10 ng / mL ) for 48 hours , following a 4 hour pre - treatment with vehicle or HOIPIN - 1 ( 50 μM ) ( the concentration of vehicle / HOIPIN - 1 was halved upon the addition of cytokines ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM .

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

Figure 4(B) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental B16-F10 tumor cells left untreated or treated with supernatant (Sup) (derived from a previous overnight OT-I/B16-F10 ova co-culture at the indicated E:T ratio for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of Sup); *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.(C) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental A375 tumor cells left untreated or treated with TNF/IFN-γ (both 10 ng/mL) for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of cytokines); *p < 0.05, **p < 0.01, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


Figure 5 ( B ) Individual ( left ) and average ( right ) in vivo growth of subcutaneous wild - type or sgRnf31 B16 - F10 ova tumors left untreated or treated with adoptively transferred OT - I CD8 + T cells ( on days indicated by black arrows ) ; * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - test for OT - I - treated wild - type vs OT - I - treated sgRnf31 with n = 5 / 6 mice per group . Pooled data are represented as mean ± SEM . ( C ) Overall survival of mice from ( B ) ; * p < 0 . 05 , * * p < 0 . 01 , Log - rank test .

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

Figure 5(B) Individual (left) and average (right) in vivo growth of subcutaneous wild-type or sgRnf31 B16-F10 ova tumors left untreated or treated with adoptively transferred OT-I CD8+ T cells (on days indicated by black arrows); *p < 0.05, **p < 0.01, Unpaired t-test for OT-I-treated wild-type vs OT-I-treated sgRnf31 with n = 5/6 mice per group. Pooled data are represented as mean ± SEM.(C) Overall survival of mice from (B); *p < 0.05, **p < 0.01, Log-rank test.


Figure 1 ( B ) Representative eTLCs ( left ) and corresponding elongation kinetics ( right ) for the CGA - CGA inhibitory pair ( red ) and the CGC optimal pair ( green ) . Product formation is normalized to the fraction of Met ICs that form Met - Puro when reacted with puromycin ( Fig EV1A ) . ( C ) Representative eTLCs ( left ) and corresponding elongation kinetics ( right ) for the CGA - CCG inhibitory pair ( red ) and the CGC - CCA optimal pair ( green ) . Product formation is normalized to the fraction of Met ICs that form Met - Puro when reacted with puromycin ( Fig EV1A ) .

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

Figure 1(B) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CGA inhibitory pair (red) and the CGC optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).(C) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CCG inhibitory pair (red) and the CGC-CCA optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).


Figure 2 ( A ) Comparison of observed rates of elongation for inhibitory pairs ( red ) and their optimal controls ( green ) at limiting tRNA concentrations ( hatched bars , 15 to 25 nM aa - tRNA ) and saturating tRNA concentrations ( solid bars , 150 to 250 nM aa - tRNA ) . ( B ) Comparison of total peptide formation for inhibitory pairs ( red ) and their optimal controls ( green ) at limiting tRNA concentrations ( hatched bars , 15 to 25 nM aa - tRNA ) and saturating tRNA concentrations ( solid bars , 150 to 250 nM aa - tRNA ) . ( C ) Elongation kinetics for the CGA - CGA inhibitory codon pair with the native arginine ICGtRNA ( red ) or the non - native arginine UCGtRNA ( pink ) and for the CGC - CGC optimal control pair with the native arginine ICGtRNA ( green ) .

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

Figure 2(A) Comparison of observed rates of elongation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).(B) Comparison of total peptide formation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).(C) Elongation kinetics for the CGA-CGA inhibitory codon pair with the native arginine ICGtRNA (red) or the non-native arginine UCGtRNA (pink) and for the CGC-CGC optimal control pair with the native arginine ICGtRNA (green).


Figure 3 ( A ) Meta - analysis of footprint size of all 17 inhibitory pairs identified aligning the first codon of the pair in the ribosomal P site . ( B ) Metacodon analysis of 21 nt RPFs centered at the first codon of each inhibitory pair ( red ) compared to their corresponding optimal pair ( green ) . P - values were determined by Student ' s t - test between each inhibitory and the corresponding optimal pair ( * * p < 0 . 01 , n = 2 ) . ( C ) Comparison of 21 nt RPFs aligned at all 17 inhibitory codons from libraries made with CHX / ANS ( blue ) and CHX / TIG ( black ) ( left ) to their corresponding optimal pairs with the same antibiotic combination ( right ) . No significant difference found ( n . s . : p = 0 . 9961 , n = 2 ) between CHX - TIG and CHX - ANS .

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

Figure 3(A) Meta-analysis of footprint size of all 17 inhibitory pairs identified aligning the first codon of the pair in the ribosomal P site.(B) Metacodon analysis of 21 nt RPFs centered at the first codon of each inhibitory pair (red) compared to their corresponding optimal pair (green). P-values were determined by Student's t-test between each inhibitory and the corresponding optimal pair (** p < 0.01, n=2).(C) Comparison of 21 nt RPFs aligned at all 17 inhibitory codons from libraries made with CHX/ANS (blue) and CHX/TIG (black) (left) to their corresponding optimal pairs with the same antibiotic combination (right). No significant difference found (n.s.: p=0.9961, n=2) between CHX-TIG and CHX-ANS.


Figure 1E - F , Candidate gene regions with neurogenesis - associated de novo methylation ( E ) and demethylation ( F ) . Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions . Stars indicate significantly differentially methylated CpGs with q < 0 . 05 ( logistic regression with multiple testing correction using SLIM method ) and methylation difference greater than 20 % .

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Figure 1E-F, Candidate gene regions with neurogenesis-associated de novo methylation (E) and demethylation (F). Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions. Stars indicate significantly differentially methylated CpGs with q < 0.05 (logistic regression with multiple testing correction using SLIM method) and methylation difference greater than 20 %.


Figure 2D , Dnmt3a / b - KO did not affect NPC proliferation . Depicted are data points for every sample with genotype means ± standard errors of the mean ( SEM ) . E , Reduced percentage of dead cells ( Annexin V / propidium iodide ( PI ) double - positive ) in KO cultures at 46 h after start of differentiation . Depicted are data points for every sample with genotype means ± SEM . F - G , NPCs from WT and KO mice differentiated into astrocytes ( GFAP - positive ) and neurons ( Tubb3 - positive ) . Increased numbers of both neurons and astrocytes were generated from KO NPCs . Depicted are data points for every sample with genotype means ± SEM . Scale bar : 100 µm . H , High magnification fluorescent image of differentiated neurons . Scale bar : 20 µm . I , Sholl analysis of Tubb3 - labeled WT and KO neurons ( n = 29 neurons , WT ; n = 30 neurons , KO ) . Reported p - value corresponds to genotype effect from two - way ANOVA . Asterisks highlight data points with p < 0 . 05 after multiple testing adjustment ( Holm method ) of repeated t - tests . Depicted are means ± SEM . J , High magnification fluorescent image of differentiated astrocytes . Scale bar : 20 µm . K , Astrocytes from KO cultures covered a larger area than WT astrocytes . Data points represent means of individual cultures ( n > 30 astrocytes per culture ) . Depicted are data points per sample with genotype means ± SEM .

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

Figure 2D, Dnmt3a/b-KO did not affect NPC proliferation. Depicted are data points for every sample with genotype means ± standard errors of the mean (SEM). E, Reduced percentage of dead cells (Annexin V/propidium iodide (PI) double-positive) in KO cultures at 46 h after start of differentiation. Depicted are data points for every sample with genotype means ± SEM. F-G, NPCs from WT and KO mice differentiated into astrocytes (GFAP-positive) and neurons (Tubb3-positive). Increased numbers of both neurons and astrocytes were generated from KO NPCs. Depicted are data points for every sample with genotype means ± SEM. Scale bar: 100 µm.H, High magnification fluorescent image of differentiated neurons. Scale bar: 20 µm. I, Sholl analysis of Tubb3-labeled WT and KO neurons (n = 29 neurons, WT; n = 30 neurons, KO). Reported p-value corresponds to genotype effect from two-way ANOVA. Asterisks highlight data points with p < 0.05 after multiple testing adjustment (Holm method) of repeated t-tests. Depicted are means ± SEM. J, High magnification fluorescent image of differentiated astrocytes. Scale bar: 20 µm. K, Astrocytes from KO cultures covered a larger area than WT astrocytes. Data points represent means of individual cultures (n > 30 astrocytes per culture). Depicted are data points per sample with genotype means ± SEM.


Figure 3H , Dnmt3a / b - dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons . Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS ( left ) and expression fold changes in KO versus WT neurons as determined by qRT - PCR ( right ; n = 4 cultures per genotype ; p - values from Mann - Whitney test ; depicted are data points for every sample with genotype means ± SEM ) .

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

Figure 3H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).


Figure 4A , No difference in numbers of Ki67 - positive , proliferating cells was observed between the subgranular zone ( SGZ ) of Dnmt3a / b - WT and Dnmt3a / b - KO mice one week after tamoxifen administration . Depicted are data points for every animal with genotype means ± SEM . B , Bright field image of Ki67 - positive cells stained using diaminobenzidine method . Scale bar : 100 µm . C , KO of Dnmt3a / b did not affect numbers of new - born neurons in the dentate gyrus . Mice were administered with tamoxifen and analyzed 5 weeks after the first injection . BrdU was injected 3 . 5 weeks before analysis . Depicted are data points for every animal with genotype means ± SEM . D , Fluorescence image of BrdU / NeuN - positive new - born neurons . Scale bar : 25 µm .

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

Figure 4A, No difference in numbers of Ki67-positive, proliferating cells was observed between the subgranular zone (SGZ) of Dnmt3a/b-WT and Dnmt3a/b-KO mice one week after tamoxifen administration. Depicted are data points for every animal with genotype means ± SEM. B, Bright field image of Ki67-positive cells stained using diaminobenzidine method. Scale bar: 100 µm. C, KO of Dnmt3a/b did not affect numbers of new-born neurons in the dentate gyrus. Mice were administered with tamoxifen and analyzed 5 weeks after the first injection. BrdU was injected 3.5 weeks before analysis. Depicted are data points for every animal with genotype means ± SEM. D, Fluorescence image of BrdU/NeuN-positive new-born neurons. Scale bar: 25 µm.


Figure 5B , Representative fluorescence image of mature and immature neurons . Scale bar : 50 µm . C , KO mice exhibit reduced total numbers of Calb2 - positive cells relative to Dcx - positive cells in the dentate gyrus . F , Representative fluorescence image of the immunostaining used for detection of mature neurons . Scale bar : 100 µm . G , Sholl analysis of Gfp - positive cells revealed reduced dendritic outgrowth of adult - born neurons in KO mice ( n = 20 neurons , WT ; n = 23 neurons , KO ) . Depicted p - value reports genotype effect from two - way ANOVA ( distance to soma : p < 0 . 001 ) . Asterisks highlight data points with p < 0 . 05 after multiple testing correction ( Holm method ) of repeated t - tests . Data points are means ± SEM . Experimental scheme as depicted in Fig . 4E . I , Adult - born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer ( n = 32 neurons from 3 mice , WT ; n = 46 neurons from 4 mice , KO ) . Depicted are individual data points for every neuron with genotype means ± SEM ( p - value from unpaired t - test ) . J , Mean spine size per animal was reduced in KO mice ( n = 50 spines per mouse ) . K , Mean percentage of mushroom spines ( diameter of spine head ≥ 0 . 45 µm ) per animal was lower in KO mice compared to WT mice . L , Representative fluorescence image of dendritic spines in WT and KO mice . Scale bar : 5 µm .

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

Figure 5B, Representative fluorescence image of mature and immature neurons. Scale bar: 50 µm. C, KO mice exhibit reduced total numbers of Calb2-positive cells relative to Dcx-positive cells in the dentate gyrus. F, Representative fluorescence image of the immunostaining used for detection of mature neurons. Scale bar: 100 µm. G, Sholl analysis of Gfp-positive cells revealed reduced dendritic outgrowth of adult-born neurons in KO mice (n = 20 neurons, WT; n = 23 neurons, KO). Depicted p-value reports genotype effect from two-way ANOVA (distance to soma: p < 0.001). Asterisks highlight data points with p < 0.05 after multiple testing correction (Holm method) of repeated t-tests. Data points are means ± SEM. Experimental scheme as depicted in Fig. 4E. I, Adult-born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer (n = 32 neurons from 3 mice, WT; n = 46 neurons from 4 mice, KO). Depicted are individual data points for every neuron with genotype means ± SEM (p-value from unpaired t-test). J, Mean spine size per animal was reduced in KO mice (n = 50 spines per mouse). K, Mean percentage of mushroom spines (diameter of spine head ≥ 0.45 µm) per animal was lower in KO mice compared to WT mice. L, Representative fluorescence image of dendritic spines in WT and KO mice. Scale bar: 5 µm.


Figure 6A - B , Quantification of adult - born ( BrdU - positive ) cells in the dentage gyrus ( DG ) of Dnmt3a / b - KO and WT mice after five weeks or three months of environmental enrichment ( ENR ) or standard housing ( STD ) . BrdU was injected 4 . 5 weeks before analysis . Depicted p - values correspond to genotype effects from two - way ANOVA . Influences of ENR were significant with p < 0 . 01 . Depicted are data points for every animal with group means ± SEM . D , Fluorescence images of c - Fos - positive cells in the DG and CA3 of WT and KO mice . Scale bar : 200 µm . E , Numbers of c - Fos - positive cells in the DG without ( STD ) and with acute stimulation in ENR for 1 h ( n = 2 , WT STD ; n = 2 , KO STD ; n = 5 , WT ENR ; n = 5 , KO ENR ) . F , Numbers of c - Fos - positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR ( n = 5 mice , WT ; n = 5 mice , KO ) . G , Percentage of new - born neurons ( BrdU / NeuN - positive ) that expressed c - Fos after ENR ( n = 5 mice , WT ; n = 5 mice , KO ) . H , Representative fluorescent image of c - Fos expression in BrdU / NeuN - positive cells in the DG . Scale bar : 25 µm . I , Percentage of c - Fos - positive cells that expressed Dcx in WT and KO mice ( n = 5 mice , WT ; n = 4 mice , KO ) . J , Representative fluorescent image of c - Fos - positive cells and Dcx - positive cells in the DG . Scale bar : 100 µm . L , Fluorescent image of c - Fos expression in Parvalbumin ( Pv ) - positive interneurons in CA3 . Scale bar : 50 µm . M , A higher percentage of Pv - positive interneurons in CA3 expressed c - Fos in KO mice ( n = 4 mice , WT ; n = 4 mice , KO ) .

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Figure 6A-B, Quantification of adult-born (BrdU-positive) cells in the dentage gyrus (DG) of Dnmt3a/b-KO and WT mice after five weeks or three months of environmental enrichment (ENR) or standard housing (STD). BrdU was injected 4.5 weeks before analysis. Depicted p-values correspond to genotype effects from two-way ANOVA. Influences of ENR were significant with p < 0.01. Depicted are data points for every animal with group means ± SEM.D, Fluorescence images of c-Fos-positive cells in the DG and CA3 of WT and KO mice. Scale bar: 200 µm. E, Numbers of c-Fos-positive cells in the DG without (STD) and with acute stimulation in ENR for 1 h (n = 2, WT STD; n = 2, KO STD; n = 5, WT ENR; n = 5, KO ENR). F, Numbers of c-Fos-positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR (n = 5 mice, WT; n = 5 mice, KO). G, Percentage of new-born neurons (BrdU/NeuN-positive) that expressed c-Fos after ENR (n = 5 mice, WT; n = 5 mice, KO). H, Representative fluorescent image of c-Fos expression in BrdU/NeuN-positive cells in the DG. Scale bar: 25 µm. I, Percentage of c-Fos-positive cells that expressed Dcx in WT and KO mice (n = 5 mice, WT; n = 4 mice, KO). J, Representative fluorescent image of c-Fos-positive cells and Dcx-positive cells in the DG. Scale bar: 100 µm. L, Fluorescent image of c-Fos expression in Parvalbumin (Pv)-positive interneurons in CA3. Scale bar: 50 µm. M, A higher percentage of Pv-positive interneurons in CA3 expressed c-Fos in KO mice (n = 4 mice, WT; n = 4 mice, KO).


Figure 7D , Average time animals needed to reach the platform per day . Note that KO mice showed increased path length and latency on days 4 and 5 . E , Time mice spent on day 5 in the quadrant in which the goal was located during the acquisition phase .

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Figure 7D, Average time animals needed to reach the platform per day. Note that KO mice showed increased path length and latency on days 4 and 5. E, Time mice spent on day 5 in the quadrant in which the goal was located during the acquisition phase.