β - catenin regulates the expression of MYBL2 in HPAF - II cells . HPAF - II cells were either treated with ETC - 159 ( 100 nM ) or transfected with siRNA against β - catenin alone or in the presence of ETC - 159 for 48 hours . The relative expression of MYBL2 as measured by qRT - PCR is shown . The horizontal lines represent mean of replicates .

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β-catenin regulates the expression of MYBL2 in HPAF-II cells. HPAF-II cells were either treated with ETC-159 (100 nM) or transfected with siRNA against β-catenin alone or in the presence of ETC-159 for 48 hours. The relative expression of MYBL2 as measured by qRT-PCR is shown. The horizontal lines represent mean of replicates.


Expression of DNA repair genes in Wnt - addicted cells is regulated by MYBL2 . HPAF - II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC - 159 ( 100 nM ) for 48 hours . Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT - PCR . Data are representative of three independent experiments . The horizontal lines represent mean of replicates .

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Expression of DNA repair genes in Wnt-addicted cells is regulated by MYBL2. HPAF-II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC-159 (100 nM) for 48 hours. Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT-PCR. Data are representative of three independent experiments. The horizontal lines represent mean of replicates.


H - K . Expression of MYBL2 is downregulated upon ETC - 159 or G007 - LK treatment in multiple Wnt - high tumors . Expression of MYBL2 in AsPC - 1 xenograft tumors and colorectal cancer PDX ( as measured by RNA - seq ) or in pancreatic cancer PDX with RNF43 mutation ( measured by qRT - PCR ) is reduced with ETC - 159 treatment . G007 - LK reduces MYBL2 expression in COL320HSR cells ( measured by qRT - PCR ) . p - values were calculated with Mann - Whitney U test . Each data point represents an individual tumor or replicate . n = 4 - 6 samples / group . The horizontal lines represent median of 4 - 6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1 . 5x interquartile range .

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H-K. Expression of MYBL2 is downregulated upon ETC-159 or G007-LK treatment in multiple Wnt-high tumors. Expression of MYBL2 in AsPC-1 xenograft tumors and colorectal cancer PDX (as measured by RNA-seq) or in pancreatic cancer PDX with RNF43 mutation (measured by qRT-PCR) is reduced with ETC-159 treatment. G007-LK reduces MYBL2 expression in COL320HSR cells (measured by qRT-PCR). p-values were calculated with Mann-Whitney U test. Each data point represents an individual tumor or replicate. n = 4-6 samples/group. The horizontal lines represent median of 4-6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1.5x interquartile range.


F , Wnt inhibition induces senescence , which is further enhanced by co - treatment with olaparib . ( F ) Representative images of tumor sections from the four treatment groups ( treated for 21 days ) stained for SA - β - galactosidase , a senescence marker , and counterstained with nuclear fast red . Blue color indicates positive staining for senescent cells . The horizontal lines represent mean of replicates . p - values were calculated by Mann - Whitney U test .

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F, Wnt inhibition induces senescence, which is further enhanced by co-treatment with olaparib. (F) Representative images of tumor sections from the four treatment groups (treated for 21 days) stained for SA-β-galactosidase, a senescence marker, and counterstained with nuclear fast red. Blue color indicates positive staining for senescent cells. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.


Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment . ( H ) HPAF - II cells were treated with DMSO , ETC - 159 ( 50 nM ) , olaparib ( 20 µM ) or both for 7 days . 53BP1 foci per cell were then assessed by immunofluorescence . The horizontal lines represent mean of replicates . p - values were calculated by Mann - Whitney U test .

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Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (H) HPAF-II cells were treated with DMSO, ETC-159 (50 nM), olaparib (20 µM) or both for 7 days. 53BP1 foci per cell were then assessed by immunofluorescence. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.


I . Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment . ( I ) Representative immunofluorescence images of 53BP1 foci ( red ) and nuclei counterstained with DAPI ( blue ) are shown for each group .

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I. Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (I) Representative immunofluorescence images of 53BP1 foci (red) and nuclei counterstained with DAPI (blue) are shown for each group.


Immunohistochemical staining of sections of small intestine from C57BL / 6J mice for DNA repair proteins shows that the expression of BRCA1 , FANCD2 and RAD51 was high in the crypts ( Wnt high compartment ) whereas the villi did not show any staining . Small intestinal adenomas from APCmin / + mice displayed positive nuclear immunostaining for HR and FA pathway proteins BRCA1 , FANCD2 and RAD51 . The bottom panels are higher magnification images of the areas marked in the top panels .

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Immunohistochemical staining of sections of small intestine from C57BL/6J mice for DNA repair proteins shows that the expression of BRCA1, FANCD2 and RAD51 was high in the crypts (Wnt high compartment) whereas the villi did not show any staining. Small intestinal adenomas from APCmin/+ mice displayed positive nuclear immunostaining for HR and FA pathway proteins BRCA1, FANCD2 and RAD51. The bottom panels are higher magnification images of the areas marked in the top panels.


PORCN inhibition reduces the expression of HR and FA pathway proteins in the normal mouse intestine . C57BL / 6J mice were treated with vehicle or ETC - 159 ( 60 mg / kg ) for 3 days . Small intestines were sectioned and analyzed by immunostaining for BRCA1 , FANCD2 , RAD51 and phosphorylated - BRCA1 .

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PORCN inhibition reduces the expression of HR and FA pathway proteins in the normal mouse intestine. C57BL/6J mice were treated with vehicle or ETC-159 (60 mg/kg) for 3 days. Small intestines were sectioned and analyzed by immunostaining for BRCA1, FANCD2, RAD51 and phosphorylated-BRCA1.


C . Representative images of calyx synapses from littermate control and Nrxn123 TKO mice . Brainstem sections were labeled with antibodies to vGluT1 ( red ) and Syt2 ( green ) . Scale bar , 10 μm . D . Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity ( normalized to control ) .

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C. Representative images of calyx synapses from littermate control and Nrxn123 TKO mice. Brainstem sections were labeled with antibodies to vGluT1 (red) and Syt2 (green). Scale bar, 10 μm. D. Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity (normalized to control).


E - H . Representative traces of spontaneous EPSCs ( sEPSCs ) ( E ) , and summary graphs of the sEPSC frequency ( F ) , amplitude ( G ) , and kinetics ( H ) recorded from littermate control and Nrxn123 TKO mice .

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E-H. Representative traces of spontaneous EPSCs (sEPSCs)(E), and summary graphs of the sEPSC frequency (F), amplitude (G), and kinetics (H) recorded from littermate control and Nrxn123 TKO mice.


A . Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s , recorded in a standard bath solution containing 2 mM Ca2 + . B . Pan - neurexin deletion suppresses synaptic transmission . Summary graphs show the amplitude ( left ) and charge transfer ( right ) of the first EPSC recorded in response to the paired stimuli . C . Pan - neurexin deletion more than doubles the paired - pulse ratio ( PPR ) , suggesting a large decrease in release probability . D . Pan - neurexin deletion decelerates the EPSC time course . Summary graphs show the rise time ( left ) and decay time constants ( right ) of the first EPSC in response to the paired stimuli .

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A. Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s, recorded in a standard bath solution containing 2 mM Ca2+. B. Pan-neurexin deletion suppresses synaptic transmission. Summary graphs show the amplitude (left) and charge transfer (right) of the first EPSC recorded in response to the paired stimuli. C. Pan-neurexin deletion more than doubles the paired-pulse ratio (PPR), suggesting a large decrease in release probability. D. Pan-neurexin deletion decelerates the EPSC time course. Summary graphs show the rise time (left) and decay time constants (right) of the first EPSC in response to the paired stimuli.


E - F . Pan - neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high - frequency stimulus train ( 100 Hz for 0 . 5 sec ) . Left , representative ESPC traces ; right , cumulative summary plot of the EPSC amplitudes during the train ( dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back - extrapolation to zero time , which is used to correct for vesicle replenishment during the train ) .

E-F. Pan-neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high-frequency stimulus train (100 Hz for 0.5 sec). Left, representative ESPC traces; right, cumulative summary plot of the EPSC amplitudes during the train (dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back-extrapolation to zero time, which is used to correct for vesicle replenishment during the train).


G . Pan - neurexin deletion does not decrease the readily - releasable pool of vesicles , but lowers the initial release probability during a high - frequency stimulus train . Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily - releasable pool size ( left ) , and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability ( right ) .

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G. Pan-neurexin deletion does not decrease the readily-releasable pool of vesicles, but lowers the initial release probability during a high-frequency stimulus train. Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily-releasable pool size (left), and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability (right).


A - D . Same as Fig 2A - D , except that ACSF contained only 1 mM Ca2 + , which is close to the estimated physiological concentration of extracellular Ca2 + ( Forsberg et al . , 2019 ) .

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A-D. Same as Fig 2A-D, except that ACSF contained only 1 mM Ca2+, which is close to the estimated physiological concentration of extracellular Ca2+ (Forsberg et al., 2019).


E - H . Same as Fig 2A - D , except that the slice were incubated with 0 . 2 mM EGTA - AM for 30 mins before recording , which introduces EGTA into the presynaptic terminals .

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E-H. Same as Fig 2A-D, except that the slice were incubated with 0.2 mM EGTA-AM for 30 mins before recording, which introduces EGTA into the presynaptic terminals.


A . Example traces of presynaptic Ca2 + - currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV . B . Summary graphs of the I - V relationship for the peak Ca2 + - current amplitude ( left ) and of the peak Ca2 + - current density ( right ) .

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A. Example traces of presynaptic Ca2+-currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV. B. Summary graphs of the I-V relationship for the peak Ca2+-current amplitude (left) and of the peak Ca2+-current density (right).


C . Example traces ( left ) and summary graphs ( right ) of the Ca2 + - current activation and deactivation kinetics . Currents were induced by a 50 ms step depolarization from - 80 mV to + 10 mV .

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C. Example traces (left) and summary graphs (right) of the Ca2+-current activation and deactivation kinetics. Currents were induced by a 50 ms step depolarization from -80 mV to +10 mV.


D . Example traces of Ca2 + - currents evoked by an action potential - equivalent depolarization ( APe , from - 80 mV to + 17 mV for 1 ms ; left ) , and summary graph of the APe - evoked Ca2 + - current peak amplitude and charge ( right ) .

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D. Example traces of Ca2+-currents evoked by an action potential-equivalent depolarization (APe, from -80 mV to +17 mV for 1 ms; left), and summary graph of the APe-evoked Ca2+-current peak amplitude and charge (right).


E . Example traces of presynaptic Ca2 + - currents induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV before and after perfusion of 200 nM agatoxin ( Agtx ) , a selective P / Q - type Ca2 + channel blocker .

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E. Example traces of presynaptic Ca2+-currents induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q-type Ca2+ channel blocker.


F . Summary graph of the relative contribution of P / Q - type Ca2 + channels to the total presynaptic Ca2 + currents , as quantified by the relative reduction in Ca2 + currents evoked by step depolarization to + 10 mV , in control and Nrxn123 TKO synapses .

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F. Summary graph of the relative contribution of P/Q-type Ca2+ channels to the total presynaptic Ca2+ currents, as quantified by the relative reduction in Ca2+ currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.


B . Example traces of the presynaptic capacitance and Ca2 + - currents at the calyx terminals induced by a 20 ms step depolarization from − 80 mV to + 10 mV with an extracellular solution containing 2 mM Ca2 + , and an internal pipette solution containing a standard 0 . 05 mM BAPTA . C . Summary graphs of the depolarization - induced capacitance jump that monitors synaptic vesicle exocytosis ( left ) , and the peak Ca2 + - current density ( right ) . D , E . Same as ( B , C ) , except that 0 . 5 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution . F , G . Same as ( B , C ) , except that 1 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution . H , I . Same as ( B , C ) , except that 10 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution .

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B. Example traces of the presynaptic capacitance and Ca2+-currents at the calyx terminals induced by a 20 ms step depolarization from −80 mV to +10 mV with an extracellular solution containing 2 mM Ca2+, and an internal pipette solution containing a standard 0.05 mM BAPTA. C. Summary graphs of the depolarization-induced capacitance jump that monitors synaptic vesicle exocytosis (left), and the peak Ca2+-current density (right). D, E. Same as (B, C), except that 0.5 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. F, G. Same as (B, C), except that 1.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. H, I. Same as (B, C), except that 10.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution.


J , K . Summary plots of the relationship between the total capacitance jump ( J ) or between the Ca2 + - current density ( K ) and the concentration of EGTA in the patch pipette .

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J, K. Summary plots of the relationship between the total capacitance jump (J) or between the Ca2+-current density (K) and the concentration of EGTA in the patch pipette.


A . Representative traces of Ca2 + - currents and 4 - AP insensitive K + - currents evoked by step depolarizations ( from − 50 mV to + 10 mV in 10 mV increments ) , recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12 - P14 . B . Same as ( A ) but after addition of 200 nM iberiotoxin ( IbTx ) by perfusion . C . Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in ( B ) from currents shown in ( A ) . D . Summary graphs of the capacitance , Ca2 + - current density , and BK - current density .

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A. Representative traces of Ca2+-currents and 4-AP insensitive K+-currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12-P14. B. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion. C. Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in (B) from currents shown in (A). D. Summary graphs of the capacitance, Ca2+-current density, and BK-current density.


A . Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 ( green ) , CaV2 . 1 - type Ca2 + - channels ( red ) , and the active zone protein Bassoon ( purple ) . Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12 - 14 . Scale bar , 10 μm .

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A. Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 (green), CaV2.1-type Ca2+-channels (red), and the active zone protein Bassoon (purple). Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12-14. Scale bar, 10 μm.


B . Summary graphs of the vGluT1 , CaV2 . 1 , and Bassoon immunostaining intensity ( normalized to control ; CaV2 . 1 and Bassoon signals were quantified over the vGluT1 - positive area to measure active zone - localized proteins ) .

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B. Summary graphs of the vGluT1, CaV2.1, and Bassoon immunostaining intensity (normalized to control; CaV2.1 and Bassoon signals were quantified over the vGluT1-positive area to measure active zone-localized proteins).


C . Summary graphs of the diameter and total size of the vGluT1 - positive presynaptic area of the calyx of Held .

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C. Summary graphs of the diameter and total size of the vGluT1-positive presynaptic area of the calyx of Held.


lines . Analysis of 2019 - nCoV - S expression ( A ) by Western blot . Representative blots from three experiments are shown . ß - Actin ( cell lysates )

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lines.Analysis of 2019-nCoV-S expression (A) by Western blot. Representative blots from three experiments are shown. ß-Actin (cell lysates)


ines . Analysis of 2019 - nCoV - S pseudotype incorporation ( B ) by Western blot . Representative blots from three experiments are shown . VSV - M ( particles ) served as loading controls .

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ines.Analysis of 2019-nCoV-S pseudotype incorporation (B) by Western blot. Representative blots from three experiments are shown. VSV-M (particles) served as loading controls.


( C ) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV - G , SARS - S or 2019 - nCoV - S . At 16 h postinoculation , pseudotype entry was analyzed . Shown are the combined data of three experiments . Error bars indicate SEM .

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(C) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV-G, SARS-S or 2019-nCoV-S. At 16 h postinoculation, pseudotype entry was analyzed. Shown are the combined data of three experiments. Error bars indicate SEM.


( C ) 293T cells transiently expressing ACE2 of human ( dark blue ) or bat ( light blue ) origin , human APN ( purple ) or hDPP4 ( green ) were inoculated with pseudotyped VSV harboring VSV - G , SARS - S , 2019 - nCoV - S , MERS - S or 229E - S . At 16 h postinoculation , pseudotype entry was analyzed . The average of three independent experiments is shown . Error bars indicate SEM .

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(C) 293T cells transiently expressing ACE2 of human (dark blue) or bat (light blue) origin, human APN (purple) or hDPP4 (green) were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, 2019-nCoV-S, MERS-S or 229E-S. At 16 h postinoculation, pseudotype entry was analyzed. The average of three independent experiments is shown. Error bars indicate SEM.


priming . Ammonium chloride ( A ) , were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins .

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priming.Ammonium chloride (A), were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.


E64d ( CatB / L inhibitor ) ( B ) and / or camostat ( TMPRSS2 inhibitor ) ( B ) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins .

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E64d (CatB/L inhibitor) (B) and/or camostat (TMPRSS2 inhibitor) (B) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.


( C ) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB / L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins . The average of three independent experiments is shown in panels A - C . Error bars indicate SEM . Statistical significance was tested by two - way ANOVA with Dunnett posttest .

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(C) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB/L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins. The average of three independent experiments is shown in panels A-C. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest.


entry . Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross - neutralization . The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment . Error bars indicate SD . Statistical significance was tested by two - way ANOVA with Dunnett posttest .

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entry.Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross-neutralization. The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate SD. Statistical significance was tested by two-way ANOVA with Dunnett posttest.


A , B . Comparisons of RNAseq and ChIPseq data . Wt strain ( A ) and ∆ rnjA ( B ) . The x - axis shows the relative RNAP occupancy at a given gene ( normalized ChIPseq coverage ) . The y - axis shows the relative abundance of the transcript ( normalized RNAseq coverage ; each dot is one gene ) . The genes are color - coded , ranging from yellow ( low RNA abundance ) to red ( high RNA abundance ) in wt . The ∆ rnjA strain has higher RNAP occupancy mainly among the genes with less abundant transcripts . The color coding in ( B ) reveals no dramatic overall changes in the vertical direction ( RNA abundance ) . If anything , some of the low abundance transcripts decreased further in level in the mutant strain . The main difference ( mostly among the low abundance transcripts ) is their shift in the horizontal direction to the right ( towards higher occupancy with RNAP ) . Data represent mean values of three independent experiments .

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A, B. Comparisons of RNAseq and ChIPseq data. Wt strain (A) and ∆rnjA (B). The x-axis shows the relative RNAP occupancy at a given gene (normalized ChIPseq coverage). The y-axis shows the relative abundance of the transcript (normalized RNAseq coverage; each dot is one gene). The genes are color-coded, ranging from yellow (low RNA abundance) to red (high RNA abundance) in wt. The ∆rnjA strain has higher RNAP occupancy mainly among the genes with less abundant transcripts. The color coding in (B) reveals no dramatic overall changes in the vertical direction (RNA abundance). If anything, some of the low abundance transcripts decreased further in level in the mutant strain. The main difference (mostly among the low abundance transcripts) is their shift in the horizontal direction to the right (towards higher occupancy with RNAP). Data represent mean values of three independent experiments.


C . Relative expression of all sigma factors in wt ( LK1371 ) versus ΔrnjA ( LK1381 ) . Wt levels of each sigma factor were set as 1 ( indicated with the horizontal line ) .

C. Relative expression of all sigma factors in wt (LK1371) versus ΔrnjA (LK1381). Wt levels of each sigma factor were set as 1 (indicated with the horizontal line).


D . Sigma - dependent genes and correlation with expression of sigma factors ( significantly changed ) in ΔrnjA ( LK1381 ) . The most down - regulated sigma factor was sigD and almost all sigD - dependent genes were downregulated . A similar trend is visible for the rest of sigma factors and their respective dependent genes . The x - axis shows expression of each sigma factor , the y - axis shows expression of genes for each sigma regulon . The violet ( dark blue ) line : regression line .

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D. Sigma-dependent genes and correlation with expression of sigma factors (significantly changed) in ΔrnjA (LK1381). The most down-regulated sigma factor was sigD and almost all sigD-dependent genes were downregulated. A similar trend is visible for the rest of sigma factors and their respective dependent genes. The x-axis shows expression of each sigma factor, the y-axis shows expression of genes for each sigma regulon. The violet (dark blue) line: regression line.


Average gene profiles of normalized RNA - seq and RNAP ChIPseq coverages from wt ( solid lines ) and ∆ rnjA ( dashed lines ) strains were plotted for gene classes I - IV ( n = 762 , 315 , 553 and 1654 genes , respectively ) . Open reading frames were rescaled to 1 kb , upstream and downstream regions of 0 . 2 kb were also included in the plots . Data represent mean values of 3 independent experiments . The pie - chart shows the overall distribution of class I - IV and other genes .

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Average gene profiles of normalized RNA-seq and RNAP ChIPseq coverages from wt (solid lines) and ∆rnjA (dashed lines) strains were plotted for gene classes I-IV (n = 762, 315, 553 and 1654 genes, respectively). Open reading frames were rescaled to 1 kb, upstream and downstream regions of 0.2 kb were also included in the plots. Data represent mean values of 3 independent experiments. The pie-chart shows the overall distribution of class I-IV and other genes.


A . SIM of exponential B . subtilis cells . RNase J1 was fused to GFP ( green ) , RNAP to mCherry ( red ) . The graph shows relative fluorescence intensities at the cell midsection ( along the long axis ) ; SIM of the cell is below . Yellow indicates colocalization of the two proteins .

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A. SIM of exponential B. subtilis cells. RNase J1 was fused to GFP (green), RNAP to mCherry (red). The graph shows relative fluorescence intensities at the cell midsection (along the long axis); SIM of the cell is below. Yellow indicates colocalization of the two proteins.


B . Pull - down with RNase J1 - 8xHis tag and RNAP - 10xHis tag and detection of the proteins with antibodies . Lanes 1 , 2 - RNase J1 - 8xHis was used to pull down RNAP ; lanes 3 , 4 - RNAP - 10xHis was used to pull down RNase J1 ; lanes 5 , 6 - purified proteins were used as markers ; lanes 7 , 8 - strains without His - tagged proteins were used as negative controls to demonstrate the specificity of the interaction . M , molecular size marker ; EX , exponential phase ; STA , stationary phase .

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B. Pull-down with RNase J1-8xHis tag and RNAP-10xHis tag and detection of the proteins with antibodies. Lanes 1, 2 - RNase J1-8xHis was used to pull down RNAP; lanes 3, 4 - RNAP-10xHis was used to pull down RNase J1; lanes 5, 6 - purified proteins were used as markers; lanes 7, 8 - strains without His-tagged proteins were used as negative controls to demonstrate the specificity of the interaction. M, molecular size marker; EX, exponential phase; STA, stationary phase.


C . Pull - down with RNase J1 - 8xHis tag from stationary phase cells The samples then either were ( lane 9 ) or were not ( lane 10 ) treated with RNase A to detect whether the interaction was via RNA . Lanes 11 , 12 - purified proteins were used as markers . The experiment was performed three times ( biological replicates ) with the same result .

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C. Pull-down with RNase J1-8xHis tag from stationary phase cells The samples then either were (lane 9) or were not (lane 10) treated with RNase A to detect whether the interaction was via RNA. Lanes 11, 12 - purified proteins were used as markers. The experiment was performed three times (biological replicates) with the same result.


B . Representative primary data - polyacrylamide gel ( the experiment was performed 3x with the same results ) . Lane 1 , the full length ( 33 nt ) labeled RNA ; lane 2 , the same as lane 1 but it included also incubation with RNase J1 ( J1 ) , 17 - 18 nt long fragments ( RNase J1 stopped by RNAP ; indicated with asterisks ) and < 5 nt fragments ( RNA released from TC into buffer - indicative of TC disassembly ) are shown ; lane 3 , the same as lane 1 but included also incubation with RNase R ( R ) ; lanes 4 and 5 , TCs were denatured by heat prior to RNase addition to demonstrate the activity and cleavage patterns of both enzymes ; lanes 6 and 7 , the same as lanes 2 and 3 but the buffer was washed off ( TCs were retained by streptavidin beads ) to demonstrate which RNA fragments were associated with TC ; lanes 8 , 9 ( M1 , M2 ) Mw marker generated by treating the 30 nt RNA with alkali and formamide ( M1 - 4 min treatment , M2 - 7 min treatment ) . As reported in ( Costanzo et al . , 2016 ) [ and references therein ] , the cleavage by alkali or formamide leaves the phosphate group of the attacked phosphodiester bond bound at 3 ' , initially in the 2 ' , 3 ' cyclic form ( upper band in the band couples ) . This successively opens ( lower band in the band couples ) yielding a double - banded pattern for short oligoribonucleotides .

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B. Representative primary data - polyacrylamide gel (the experiment was performed 3x with the same results). Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), 17-18 nt long fragments (RNase J1 stopped by RNAP; indicated with asterisks) and <5 nt fragments (RNA released from TC into buffer - indicative of TC disassembly) are shown; lane 3, the same as lane 1 but included also incubation with RNase R (R); lanes 4 and 5, TCs were denatured by heat prior to RNase addition to demonstrate the activity and cleavage patterns of both enzymes; lanes 6 and 7, the same as lanes 2 and 3 but the buffer was washed off (TCs were retained by streptavidin beads) to demonstrate which RNA fragments were associated with TC; lanes 8, 9 (M1, M2) Mw marker generated by treating the 30 nt RNA with alkali and formamide (M1-4 min treatment, M2-7 min treatment). As reported in (Costanzo et al., 2016) [and references therein], the cleavage by alkali or formamide leaves the phosphate group of the attacked phosphodiester bond bound at 3', initially in the 2',3' cyclic form (upper band in the band couples). This successively opens (lower band in the band couples) yielding a double-banded pattern for short oligoribonucleotides.


A . Primary data - polyacrylamide gel - a representative result . For description of bands / fragments see next panel legend . Asterisks indicate STUBS ( 16 - 19 nt ) . B . A schematic representation of quantitation of fragments . OUTSIDE ( 16 - 30 nt ) are RNA fragments that originated by digestion of the full length RNA by RNases that were stopped either before reaching or at the point of reaching RNAP ; STUBS ( 16 - 19 nt ) are RNA fragments that originated by digestion of the full length RNA that were stopped at the point of reaching RNAP ; INSIDE ( < 16 nt ) are RNA fragments ( oligonucleotides ) that could be only generated after the disassembly of the complex by the torpedo mechanism . The red color indicates parts of RNA that were digested . C . Quantitation of three independent experiments . " Full length " indicates the remaining undigested RNA . " OUTSIDE " , " INSIDE " are fragments as explained in ( B ) and indicated in ( A ) . The bars represent 100 % ( all fragments ) . The black - grey - white boxes indicate the percentage of each fragment group ( in % ) . The error bars indicate ± SEM for each group of fragments calculated from three biological replicates .

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A. Primary data - polyacrylamide gel - a representative result. For description of bands/fragments see next panel legend. Asterisks indicate STUBS (16-19 nt). B. A schematic representation of quantitation of fragments. OUTSIDE (16-30 nt) are RNA fragments that originated by digestion of the full length RNA by RNases that were stopped either before reaching or at the point of reaching RNAP; STUBS (16-19 nt) are RNA fragments that originated by digestion of the full length RNA that were stopped at the point of reaching RNAP; INSIDE (< 16 nt) are RNA fragments (oligonucleotides) that could be only generated after the disassembly of the complex by the torpedo mechanism. The red color indicates parts of RNA that were digested. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments as explained in (B) and indicated in (A). The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments calculated from three biological replicates.


A . TCs were assembled on DNA - RNA scaffolds ( DNA was biotinylated and attached to streptavidin coated magnetic beads ) ; TCs were then divided into three tubes and challenged with buffer ( " - " , mock treatment ) or RNase J1 of Xrn1 . Dissociation of RNAP from DNA was monitored by detecting RNAP with anti - β antibody in two fractions : B - beads ( RNAP still bound to DNA ) , R - released ( free in buffer ) . The gel strip shows representative primary data ( Western blot ) . The graph shows averages ( the bars ) of two experiments ( biological replicates ) and the error bars show the range . The combined signal for B + R for each treatment was set as 100 % .

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A. TCs were assembled on DNA-RNA scaffolds (DNA was biotinylated and attached to streptavidin coated magnetic beads); TCs were then divided into three tubes and challenged with buffer ("-", mock treatment) or RNase J1 of Xrn1. Dissociation of RNAP from DNA was monitored by detecting RNAP with anti-β antibody in two fractions: B - beads (RNAP still bound to DNA), R - released (free in buffer). The gel strip shows representative primary data (Western blot). The graph shows averages (the bars) of two experiments (biological replicates) and the error bars show the range. The combined signal for B+R for each treatment was set as 100 %.


B . Primary data - polyacrylamide gel . RNAP from E . coli . Lane 1 , the full length ( 33 nt ) labeled RNA ; lane 2 , the same as lane 1 but it included also incubation with RNase J1 ( J1 ) , lane 3 , the same as lane 1 but included also incubation with Xrn1 . C . Quantitation of three independent experiments . " Full length " indicates the remaining undigested RNA . " OUTSIDE " , " INSIDE " are fragments The bars represent 100 % ( all fragments ) . The black - grey - white boxes indicate the percentage of each fragment group ( in % ) . The error bars indicate ± SEM for each group of

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

B. Primary data - polyacrylamide gel. RNAP from E. coli. Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), lane 3, the same as lane 1 but included also incubation with Xrn1. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments


A . Exponential cells of indicated strains ( below the bars ) were plated onto LB agar and either were , or were not UV - irradiated . After overnight incubation , CFU were counted . UV sensitivity of the mutant strains ( KO ) was then calculated as the ratio between irradiated vs non - irradiated cells and normalized to this ratio from the wt strain As a consequence , the wt ratio is 1 . The experiment was conducted 4x ( biological replicates ) and the bars show the geometric mean . The p - values , shown above the bars , were computed using two - tailed unpaired t - test for logarithms of the ratios . The error bars show ± SEM ( computed on the log scale ) .

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A. Exponential cells of indicated strains (below the bars) were plated onto LB agar and either were, or were not UV-irradiated. After overnight incubation, CFU were counted. UV sensitivity of the mutant strains (KO) was then calculated as the ratio between irradiated vs non-irradiated cells and normalized to this ratio from the wt strain As a consequence, the wt ratio is 1. The experiment was conducted 4x (biological replicates) and the bars show the geometric mean. The p-values, shown above the bars, were computed using two-tailed unpaired t-test for logarithms of the ratios. The error bars show ±SEM (computed on the log scale).


B . Relative expression ( mRNA ) of helD , mfd , rho , greA , nusA , nusB , and nusG in the ∆ rnjA strain ( normalized to wt [ set as 1 ] ) .

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B. Relative expression (mRNA) of helD, mfd, rho, greA, nusA, nusB, and nusG in the ∆rnjA strain (normalized to wt [set as 1]).


D . Representative immunofluorescence images of MEFs 48h after transgene induction stained for the indicated reprogramming factors . Scale bar represents 50 μm ( n = 3 biological replicates ) .

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D. Representative immunofluorescence images of MEFs 48h after transgene induction stained for the indicated reprogramming factors. Scale bar represents 50 μm (n = 3 biological replicates).


E . Log fluorophore expression superimposed on Uniform Manifold Approximation and Projection ( UMAP ) embedding of the dataset . Shown are fluorophore expression levels ( gene exp . ) on a logarithmic ( ln ) scale . F . Log transgene expression superimposed on a UMAP embedding of the dataset . Shown are transgene expression levels ( gene exp . ) on an a logarithmic ( ln ) scale .

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E. Log fluorophore expression superimposed on Uniform Manifold Approximation and Projection (UMAP) embedding of the dataset. Shown are fluorophore expression levels (gene exp.) on a logarithmic (ln) scale. F. Log transgene expression superimposed on a UMAP embedding of the dataset. Shown are transgene expression levels (gene exp.) on an a logarithmic (ln) scale.


G . UMAP embedding of scRNA - seq dataset colored by technical replicate . Orange : Replicate 1 , Blue : Replicate

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G. UMAP embedding of scRNA-seq dataset colored by technical replicate. Orange: Replicate 1, Blue: Replicate 2


H . Cell cycle score superimposed on UMAP embedding of the dataset . See unsupervised analysis of single - cell RNA - seq section in Methods for further details .

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H. Cell cycle score superimposed on UMAP embedding of the dataset. See unsupervised analysis of single-cell RNA-seq section in Methods for further details.


D . Log - normalized expression z - score of genes up - ( top panels ) and downregulated ( bottom panels ) by Ascl1 ( left , blue violins ) and MyoD1 ( right , yellow violins ) versus their scaled expression levels ( x - axis ) binned into 10 % intervals . For each box , the centerline defines the median , the height of the box is given by the interquartile range ( IQR ) , the whiskers are given by 1 . 5 * IQR and outliers are given as points beyond the minimum or maximum whisker .

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D. Log-normalized expression z-score of genes up- (top panels) and downregulated (bottom panels) by Ascl1 (left, blue violins) and MyoD1 (right, yellow violins) versus their scaled expression levels (x-axis) binned into 10% intervals. For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker.


E . Fate titration plot of Ascl1 and MyoD1 double positive cells Shown are decision boundaries for indicated fates according to transcription factor levels . MyoD1 and Ascl1 expression shown on the x - and y - axis are log - normalized expression values scaled into the dynamic range of the single - positive condition Cells are colored according to their lineage endpoint as determined by CellRank .

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E. Fate titration plot of Ascl1 and MyoD1 double positive cells Shown are decision boundaries for indicated fates according to transcription factor levels. MyoD1 and Ascl1 expression shown on the x- and y-axis are log-normalized expression values scaled into the dynamic range of the single-positive condition Cells are colored according to their lineage endpoint as determined by CellRank.


F . Representative immunofluorescence images of cells 3 days after transgene induction . Cells were stained for the fluorescent reporter present on the PiggyBac construct of the reprogramming factor . Arrows indicate multinucleated cells . Scale bar represents 50 µm . mutAscl1 = mutant Ascl1 . G . Quantification of the percentage of cells with more than one nucleus among all transfected cells within a given condition . Data points represent biological replicates ( n = 4 ) . For each box , the centerline defines the median , the height of the box is given by the interquartile range ( IQR ) , the whiskers are given by 1 . 5 * IQR and outliers are given as points beyond the minimum or maximum whisker . Pairwise comparisons performed with Mann - Whitney U test , correction for multiple testing performed with Benjamini - Hochberg correction . * : p < 0 . 05 ( MyoD1 vs Ascl1 : p = 0 . 03 , MyoD1 vs mutAscl1 : p = 0 . 03 , MyoD1 vs Ascl1 & MyoD1 : p = 0 . 03 , MyoD1 vs mutAscl1 & MyoD1 : p = 0 . 03 , Ascl1 vs Ascl1 & MyoD1 : p = 0 . 03 , Ascl1 vs mutAscl1 & MyoD1 : p = 0 . 03 , mutAscl1 vs Ascl1 & MyoD1 : p = 0 . 03 , mutAscl1 vs mutAscl1 & MyoD1 : p = 0 .

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F. Representative immunofluorescence images of cells 3 days after transgene induction. Cells were stained for the fluorescent reporter present on the PiggyBac construct of the reprogramming factor. Arrows indicate multinucleated cells. Scale bar represents 50 µm. mutAscl1 = mutant Ascl1. G. Quantification of the percentage of cells with more than one nucleus among all transfected cells within a given condition. Data points represent biological replicates (n = 4). For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker. Pairwise comparisons performed with Mann-Whitney U test, correction for multiple testing performed with Benjamini-Hochberg correction. *: p < 0.05 (MyoD1 vs Ascl1: p = 0.03, MyoD1 vs mutAscl1: p = 0.03, MyoD1 vs Ascl1 & MyoD1: p = 0.03, MyoD1 vs mutAscl1 & MyoD1: p = 0.03, Ascl1 vs Ascl1 & MyoD1: p = 0.03, Ascl1 vs mutAscl1 & MyoD1: p = 0.03, mutAscl1 vs Ascl1 & MyoD1: p = 0.03, mutAscl1 vs mutAscl1 & MyoD1: p = 0.03


A . Triple RNAscope in situ hybridization showing Engrailed - 1 ( En1 ) , Choline acetyltransferase ( ChAT ) and Calbindin - 1 ( Calb1 ) expression in the lumbar spinal cord . En1 is expressed in V1 interneurons , dorsal ( V1P ) and ventral ( V1R ) to the main ChAT - expressing motoneuron pool . Ventral interneurons correspond to Renshaw cells as shown by Calb1 expression . Scale bar : 500 μm .

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A. Triple RNAscope in situ hybridization showing Engrailed-1 (En1), Choline acetyltransferase (ChAT) and Calbindin-1 (Calb1) expression in the lumbar spinal cord. En1 is expressed in V1 interneurons, dorsal (V1P) and ventral (V1R) to the main ChAT-expressing motoneuron pool. Ventral interneurons correspond to Renshaw cells as shown by Calb1 expression. Scale bar: 500 μm.


B . RT - qPCR of RNA from the lumbar enlargement at 4 . 5 - and 16 - months of age shows stable En1 expression in WT at both ages and a two - fold reduction of expression in heterozygous mice . Unpaired two - sided t - test . * * p < 0 . 005 ; * * * p < 0 . 0005 . n = 3 . Values are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-qPCR of RNA from the lumbar enlargement at 4.5- and 16-months of age shows stable En1 expression in WT at both ages and a two-fold reduction of expression in heterozygous mice. Unpaired two-sided t-test. **p<0.005; ***p<0.0005. n=3. Values are mean ± SD.


C . Triple staining EN1 IHC ( green ) , En1 RNAscope ISH ( red ) and ChAT RNAscope ( blue ) demonstrating the double - staining of En1 mRNA and protein ( EN1 ) in the V1 interneuron population ( left panel insets , arrowheads point towards examples of double - stained V1 interneurons ) , and the presence of EN1 protein in large cells not expressing En1 mRNA ( left panel ) but expressing ChAT ( right panel insets ) . Scale bar : 500 µm , 30 µm for high magnification insets . D . Left : EN1 ( red ) detected with the LSBio antibody is localized in ChAT - expressing neurons ( green ) in the ventral horns of the spinal cord . Right : EN1 signal is lost upon preincubation of the antibody with 1 . 5 M excess of recombinant hEN1 . Scale bar : 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Triple staining EN1 IHC (green), En1 RNAscope ISH (red) and ChAT RNAscope (blue) demonstrating the double-staining of En1 mRNA and protein (EN1) in the V1 interneuron population (left panel insets, arrowheads point towards examples of double-stained V1 interneurons), and the presence of EN1 protein in large cells not expressing En1 mRNA (left panel) but expressing ChAT (right panel insets). Scale bar: 500 µm, 30 µm for high magnification insets. D. Left: EN1 (red) detected with the LSBio antibody is localized in ChAT-expressing neurons (green) in the ventral horns of the spinal cord. Right: EN1 signal is lost upon preincubation of the antibody with 1.5 M excess of recombinant hEN1. Scale bar: 50 µm.


A . Analysis of the number of En1 + , Calb1 + and ChAT + neurons ( triple RNAscope ) . At 4 . 5 months , WT and En1 - Het mice show no difference in the number of cells expressing En1 , Calb1 or ChAT . Unpaired two - sided t - test . n = 5 - 6 . Data information : The analysis of neuron number in A were performed once . Values are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the number of En1+, Calb1+ and ChAT+ neurons (triple RNAscope). At 4.5 months, WT and En1-Het mice show no difference in the number of cells expressing En1, Calb1 or ChAT. Unpaired two-sided t-test. n=5-6. Data information: The analysis of neuron number in A were performed once. Values are mean ± SD.


C . Cresyl violet and ChAT staining at the lumbar level show no medium size ( 200 - 299 µm2 ) cell loss ( γMNs ) and a decrease of about 50 % in the number of large size ( > 300 µm2 ) cells ( αMNs ) . Unpaired two - sided t - test . * * p < 0 . 005 ; * * * * p < 0 . 0001 . n = 5 . Data information : The direct comparison of Cresyl violet and ChAT in C were performed once . Values are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cresyl violet and ChAT staining at the lumbar level show no medium size (200-299 µm2) cell loss (γMNs) and a decrease of about 50% in the number of large size (>300 µm2) cells (αMNs). Unpaired two-sided t-test. **p<0.005; ****p<0.0001. n=5. Data information: The direct comparison of Cresyl violet and ChAT in C were performed once. Values are mean ± SD.


D . Lumbar level Cresyl violet staining shows that , in contrast with small and medium size neurons ( interneurons and γMNs , left and center panels ) , large size neurons ( αMNs , right panel ) undergo progressive death first measured at 4 . 5 months . The values represent the average number of cells per ventral horn . For the small neurons ( 100 - 199 µm2 ) , there was no main effect , two - way ANOVA for repeated measures for treatment group : F ( 1 , 43 ) = 0 . 0017 , p = 0 . 968 , ns . For the medium sized neurons ( 200 - 299 µm2 ) two - way ANOVA for repeated measures for treatment group : showed no main effect F ( 1 , 43 ) = 2 . 085 , ns . For the large neurons ( > 300 µm2 ) , two - way ANOVA for repeated measures showed a significant main effect F ( 1 , 43 ) = 59 . 99 , p < 0 . 0001 . Post - hoc comparisons were performed by unpaired two - sided t - test with equal SD comparing WT with En1 - Het at each time point ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 ) . n = 5 to 6 . Data information : The longitudinal studies in D were performed once . Values are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Lumbar level Cresyl violet staining shows that, in contrast with small and medium size neurons (interneurons and γMNs, left and center panels), large size neurons (αMNs, right panel) undergo progressive death first measured at 4.5 months. The values represent the average number of cells per ventral horn. For the small neurons (100-199 µm2), there was no main effect, two-way ANOVA for repeated measures for treatment group: F(1,43)=0.0017, p=0.968, ns. For the medium sized neurons (200-299 µm2) two-way ANOVA for repeated measures for treatment group: showed no main effect F(1,43)=2.085, ns. For the large neurons (>300 µm2), two-way ANOVA for repeated measures showed a significant main effect F(1,43)=59.99, p<0.0001. Post-hoc comparisons were performed by unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=5 to 6. Data information: The longitudinal studies in D were performed once. Values are mean ± SD.


E . Compared to WT mice , En1 - Het mice experience gradual strength loss . This loss is observed with the forepaw grip strength ( left panel ) , the inverted grid test ( center panel and the hindlimb extensor reflex test ( right panel ) . Strength loss is first observed between 2 and 3 months , thus before measurable αMN cell body loss . Two - way ANOVA showed significant main effects for grip strength ( F ( 1 , 136 ) = 19 . 18 , p < 0 . 0001 ) , inverted grid test ( F ( 1 , 103 ) = 143 . 1 , p < 0 . 0001 ) and extensor score ( F ( 1 , 103 ) = 10 . 1 , p < 0 . 0001 ) . Comparisons were made by unpaired two - sided t - test with equal SD comparing WT with En1 - Het at each time point ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 ) . n = 4 to 20 . Data information : The longitudinal studies in E were performed once . Values are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Compared to WT mice, En1-Het mice experience gradual strength loss. This loss is observed with the forepaw grip strength (left panel), the inverted grid test (center panel and the hindlimb extensor reflex test (right panel). Strength loss is first observed between 2 and 3 months, thus before measurable αMN cell body loss. Two-way ANOVA showed significant main effects for grip strength (F(1,136)=19.18, p<0.0001), inverted grid test (F(1,103)=143.1, p<0.0001) and extensor score (F(1,103)=10.1, p<0.0001). Comparisons were made by unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=4 to 20. Data information: The longitudinal studies in E were performed once. Values are mean ± SD.


A . NMJs of En1 - Het and WT mice show similar numbers of AChR clusters ( left panel ) and late - occurring ( 15 . 5 months ) decrease in endplate area ( center panel ) and percentage of perforated endplates ( right panel ) . The number of AChR clusters seems to decrease between 2 and 3 months , but two - way ANOVA showed no significant genotype effect ( F ( 1 , 47 ) = 0 . 1291 , ns ) . There was a significant main effect for endplate area ( F ( 1 , 47 ) = 5 . 778 , p = 0 . 0202 ) and for the percentage of perforated endplates ( F ( 1 , 47 ) = 13 . 82 , p = 0 . 0005 ) . Post hoc analysis revealed significant genotype differences at 15 . 5 month of age . Unpaired two - sided t - test with equal SD comparing WT with En1 - Het at each time point ( * * p < 0 . 005 ) . n = 4 to 8 . Data information : The longitudinal study in A was performed once . Values are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. NMJs of En1-Het and WT mice show similar numbers of AChR clusters (left panel) and late-occurring (15.5 months) decrease in endplate area (center panel) and percentage of perforated endplates (right panel). The number of AChR clusters seems to decrease between 2 and 3 months, but two-way ANOVA showed no significant genotype effect (F(1,47)=0.1291, ns). There was a significant main effect for endplate area (F(1,47)=5.778, p=0.0202) and for the percentage of perforated endplates (F(1,47)=13.82, p=0.0005). Post hoc analysis revealed significant genotype differences at 15.5 month of age. Unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (**p<0.005). n=4 to 8. Data information: The longitudinal study in A was performed once. Values are mean ± SD.


B . Left panel illustrates the use of Alexa Fluor 488 - conjugated α - bungarotoxin ( α - BTX , in green ) and of neurofilament and synaptic vesicle glycoprotein antibodies ( 2H3 and SV2A , in red ) to evaluate the percentage of fully occupied endplates ( > 80 % occupancy ) . The right panel shows that the % of fully occupied endplates decreases progressively in the En1 - Het mouse , starting between 3 and 4 . 5 months of age . Scale bar : 50µm . Two - way ANOVA showed a significant main effect ( F ( 1 , 47 ) = 45 . 45 , p < 0 . 0001 ) . Unpaired two - sided t - test with equal SD comparing WT with En1 - Het at each time point ( * p < 0 . 05 ; * * p < 0 . 005 ) . n = 4 to 8 . See details of analysis in the Methods section . Data information : The longitudinal study in B was performed once . Values are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left panel illustrates the use of Alexa Fluor 488-conjugated α-bungarotoxin (α-BTX, in green) and of neurofilament and synaptic vesicle glycoprotein antibodies (2H3 and SV2A, in red) to evaluate the percentage of fully occupied endplates (>80% occupancy). The right panel shows that the % of fully occupied endplates decreases progressively in the En1-Het mouse, starting between 3 and 4.5 months of age. Scale bar: 50µm. Two-way ANOVA showed a significant main effect (F(1,47)=45.45, p<0.0001). Unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (*p<0.05; **p<0.005). n=4 to 8. See details of analysis in the Methods section. Data information: The longitudinal study in B was performed once. Values are mean ± SD.


C . Left panel illustrates that expressing the scFvEN1 , but not scFvMUT , abolishes EN1 staining by LSBio anti - EN1 antibody in ventral horn ChAT + cells and right panel quantifies this inhibition 1 - way ANOVA followed by Tukey corrected post - hoc comparisons . ( n = 5 mice per group , * * * p < 0 . 0005 , * * * * p < 0 . 0001 ) . Scale bar : 100 µm .

C. Left panel illustrates that expressing the scFvEN1, but not scFvMUT, abolishes EN1 staining by LSBio anti-EN1 antibody in ventral horn ChAT+ cells and right panel quantifies this inhibition 1-way ANOVA followed by Tukey corrected post-hoc comparisons. (n=5 mice per group, ***p<0.0005, ****p<0.0001). Scale bar: 100 µm.


E . Six months following infection ( 7 - month - old mice ) , extracellular EN1 neutralization does not modify the number of AChR clusters , nor the endplate surface area , nor the percentage of perforated endplates . In contrast , the percentage of fully occupied endplates is diminished ( right end panel ) . 1 - way ANOVA followed by Tukey corrected post - hoc comparisons . ( * * p < 0 . 005 ) . n = 5 per treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Six months following infection (7-month-old mice), extracellular EN1 neutralization does not modify the number of AChR clusters, nor the endplate surface area, nor the percentage of perforated endplates. In contrast, the percentage of fully occupied endplates is diminished (right end panel). 1-way ANOVA followed by Tukey corrected post-hoc comparisons. (**p<0.005). n=5 per treatment.


F . Six months following infection , extracellular EN1 neutralization does not globally modify the total neuron number of cells at the lumbar level ( left panel ) . A separate analysis of small ( 100 - 199 µm2 ) , medium ( 200 - 299 µm2 ) , and large ( > 300 µm2 ) neurons demonstrate a specific ( p < 0 . 0557 ) loss of the latter category ( αMNs ) . 1 - way ANOVA followed by Tukey corrected post - hoc comparisons . N = 5 per treatment .

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

F. Six months following infection, extracellular EN1 neutralization does not globally modify the total neuron number of cells at the lumbar level (left panel). A separate analysis of small (100-199 µm2), medium (200-299 µm2), and large (>300 µm2) neurons demonstrate a specific (p<0.0557) loss of the latter category (αMNs). 1-way ANOVA followed by Tukey corrected post-hoc comparisons. N=5 per treatment.


A . Mice were tested for strength at three months of age , before the onset of αMN loss , but when strength has already decreased in En1 - het mice as measured in the forepaw grip strength , inverted grid , and extensor reflex tests ( left side of each graph ) . The next day , the En1 - het mice were separated into two groups . One group received buffer and the other group recombinant hEN1 ( 1 µg in 5 µl ) , injected at the L5 level . One and a half months later ( 4 . 5 months of age ) En1 - het mice injected with hEN1 have recovered normal strength , in contrast with non - injected mice or mice injected with buffer . Unpaired two - sided t - test used at three months of age . * * * * p < 0 . 0001 . For 4 . 5 - month comparisons , 1 - way ANOVA followed by Tukey corrected post - hoc comparisons . * * * p < 0 . 0005 ; * * * * p < 0 . 0001 . n = 9 to

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mice were tested for strength at three months of age, before the onset of αMN loss, but when strength has already decreased in En1-het mice as measured in the forepaw grip strength, inverted grid, and extensor reflex tests (left side of each graph). The next day, the En1-het mice were separated into two groups. One group received buffer and the other group recombinant hEN1 (1 µg in 5 µl), injected at the L5 level. One and a half months later (4.5 months of age) En1-het mice injected with hEN1 have recovered normal strength, in contrast with non-injected mice or mice injected with buffer. Unpaired two-sided t-test used at three months of age. ****p<0.0001. For 4.5-month comparisons, 1-way ANOVA followed by Tukey corrected post-hoc comparisons. ***p<0.0005; ****p<0.0001. n=9 to 31


B . Based on the results shown in A , a new experiment was performed with a second injection 12 weeks after the first injection at 3 months of age . Again , the injection at three months of age restored strength and in mice receiving the second injection strength was maintained an additional ten weeks or more compared to mice receiving a single injection . The three strength graphs demonstrate a positive effect of the second injection with values intermediate between those measured in WT mice and En1 - Het mice with a single injection . The percentage of fully occupied endplates and the number of αMNs are back to wild type values in En1 - Het mice injected twice . Two - way ANOVA revealed significant main effects for grip strength ( F ( 2 , 81 ) = 25 . 47 , p < 0 . 0001 ) , inverted grid ( F ( 2 , 91 ) = 51 . 96 , p < 0 . 0001 ) and extensor score ( F ( 2 , 104 ) = 30 . 42 , p < 0 . 0001 ) . At all times groups were compared by Unpaired T - test with equal variances through 12 weeks . After , the groups were compared by Tukey corrected post - hoc comparisons ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 ) . For the endplate analysis and αMNs , groups were compared by 1 - way ANOVA followed by Tukey corrected post hoc comparisons ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 ) . n = 4 to 10 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Based on the results shown in A, a new experiment was performed with a second injection 12 weeks after the first injection at 3 months of age. Again, the injection at three months of age restored strength and in mice receiving the second injection strength was maintained an additional ten weeks or more compared to mice receiving a single injection. The three strength graphs demonstrate a positive effect of the second injection with values intermediate between those measured in WT mice and En1-Het mice with a single injection. The percentage of fully occupied endplates and the number of αMNs are back to wild type values in En1-Het mice injected twice. Two-way ANOVA revealed significant main effects for grip strength (F(2,81)=25.47, p<0.0001), inverted grid (F(2,91)=51.96, p<0.0001) and extensor score (F(2,104)=30.42, p<0.0001). At all times groups were compared by Unpaired T-test with equal variances through 12 weeks. After, the groups were compared by Tukey corrected post-hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). For the endplate analysis and αMNs, groups were compared by 1-way ANOVA followed by Tukey corrected post hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n= 4 to 10.


A . Top left panel shows that 24 hours after intrathecal injection ( 1 μg in 5µL ) at the L5 level of 2 - month - old mice , hEN1 ( red ) can be primarily visualized in ventral horn ChAT + cells ( green ) . Arrowheads show hEN1 internalized by MNs . Scale bar : 100 µm . Top right panels show the progressive accumulation and clearance of hEN1 in ventral horn MNs , with a peak between 6 and 24 hours . EN1 was revealed by the 86 / 8 antibody allowing for the visualization of exogenous EN1 only ( scale bar : 100µm ) . Bottom panels show the quantification of EN1 in γMNs and αMNs with the 86 / 8 ( two left panels ) and the LSBio ( two right panels ) antibodies . Since the LSBio sees both endogenous and exogenous EN1 , the increase is only 3 - fold , but qualitatively , the results are very similar , demonstrating rapid internalization and clearance of the exogenous protein and allowing one to calculate a half - life of 24 hours . 1 - way ANOVA followed by Tukey corrected post - hoc comparisons ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 ) . When not significant , p values are shown . n = 3 . Values are mean ± SD . Data information : The internalization experiments were performed once with each antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Top left panel shows that 24 hours after intrathecal injection (1 μg in 5µL) at the L5 level of 2-month-old mice, hEN1 (red) can be primarily visualized in ventral horn ChAT+ cells (green). Arrowheads show hEN1 internalized by MNs. Scale bar: 100 µm. Top right panels show the progressive accumulation and clearance of hEN1 in ventral horn MNs, with a peak between 6 and 24 hours. EN1 was revealed by the 86/8 antibody allowing for the visualization of exogenous EN1 only (scale bar: 100µm). Bottom panels show the quantification of EN1 in γMNs and αMNs with the 86/8 (two left panels) and the LSBio (two right panels) antibodies. Since the LSBio sees both endogenous and exogenous EN1, the increase is only 3-fold, but qualitatively, the results are very similar, demonstrating rapid internalization and clearance of the exogenous protein and allowing one to calculate a half-life of 24 hours. 1-way ANOVA followed by Tukey corrected post-hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005). When not significant, p values are shown. n=3. Values are mean ± SD. Data information: The internalization experiments were performed once with each antibody.


B . Immunohistochemical staining shows the presence of high amounts of p62 / SQTSM1 in ChAT + cell bodies of 3 - month - old mice . Scale bar : 50 µm .

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

B. Immunohistochemical staining shows the presence of high amounts of p62/SQTSM1 in ChAT+ cell bodies of 3-month-old mice. Scale bar: 50 µm.


C . Intensity measurements demonstrates that the mean of SQTSM1 / p62 expression increases with age in WT γMNs ( left ) and αMNs ( right ) . However , comparing WT and En1 - Het shows a significant difference only at 3 months and not later . Unpaired two - sided t - test . ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 , * * * * p < 0 . 0001 ) . Data information : this experiment was done once . Values are mean ± SD . Between 151 and 1215 neurons and 3 to 5 mice were analyzed for each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Intensity measurements demonstrates that the mean of SQTSM1/p62 expression increases with age in WT γMNs (left) and αMNs (right). However, comparing WT and En1-Het shows a significant difference only at 3 months and not later. Unpaired two-sided t-test. (*p<0.05; **p<0.005; ***p<0.0005, ****p<0.0001). Data information: this experiment was done once. Values are mean ± SD. Between 151 and 1215 neurons and 3 to 5 mice were analyzed for each condition.


D . Mean intensity of SQTSM1 / p62 expression is increased in γMNs ( left ) and αMNs ( right ) in mice expressing scFvEN1 6 months after virus injection ( 7 - month - old mice ) demonstrating that EN1 extracellular neutralization increases SQTSM1 / p62 expression . Expression of the mutated antibody ( scFvMUT ) does not increase p62 expression . Unpaired two - sided t - test . ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 , * * * * p < 0 . 0001 ) . Data information : this experiment was performed once . Values are mean ± SD . Between 448 and 759 neurons and 5 mice were analyzed for each condition .

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D. Mean intensity of SQTSM1/p62 expression is increased in γMNs (left) and αMNs (right) in mice expressing scFvEN1 6 months after virus injection (7-month-old mice) demonstrating that EN1 extracellular neutralization increases SQTSM1/p62 expression. Expression of the mutated antibody (scFvMUT) does not increase p62 expression. Unpaired two-sided t-test. (*p<0.05; **p<0.005; ***p<0.0005, ****p<0.0001). Data information: this experiment was performed once. Values are mean ± SD. Between 448 and 759 neurons and 5 mice were analyzed for each condition.


A . Left panel : Injection and analysis protocol . Right panel : muscle strength analysis demonstrating that hEN1 injection at 1 month prevents muscular strength decrease observed in 3 - month - old En1 - Het mice . One - way ANOVA followed by two - sided t - test . ( * p < 0 . 05 , * * * * p < 0 . 0001 ) . N = 6 - 10 . Data information : This experiment was performed once . Values are mean ± SD .

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A. Left panel: Injection and analysis protocol. Right panel: muscle strength analysis demonstrating that hEN1 injection at 1 month prevents muscular strength decrease observed in 3-month-old En1-Het mice. One-way ANOVA followed by two-sided t-test. (*p<0.05, ****p<0.0001). N=6-10. Data information: This experiment was performed once. Values are mean ± SD.


B . Left panel : Increased p62 / SQTSM1 staining in 3 - month - old ChAT + MNs from control En1 - Het is abolished by hEN1 injection at 1 month . Right panel : quantification of p62 / SQTSM1 staining in γMNs and αMNs of control and hEN1 - injected 3 - month - old En1 - het mice . One - way ANOVA followed by two - sided t - test . ( * * p < 0 . 01 , * * * * p < 0 . 0001 ) . Between XXX neurons in YY mice were analyzed in this experiment . Data information : This experiment was performed once . Values are mean ± SD . Between 111 and 303 neurons and 4 - 5 mice were analyzed for each condition .

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B. Left panel: Increased p62/SQTSM1 staining in 3-month-old ChAT+ MNs from control En1-Het is abolished by hEN1 injection at 1 month. Right panel: quantification of p62/SQTSM1 staining in γMNs and αMNs of control and hEN1-injected 3-month-old En1-het mice. One-way ANOVA followed by two-sided t-test. (**p<0.01, ****p<0.0001). Between XXX neurons in YY mice were analyzed in this experiment. Data information: This experiment was performed once. Values are mean ± SD. Between 111 and 303 neurons and 4-5 mice were analyzed for each condition.


( A ) Quantitative analysis of Rnf114 mRNA by realtime - PCR in various mouse tissues and organs shows that Rnf114 was predominately expressed in oocytes .

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(A) Quantitative analysis of Rnf114 mRNA by realtime-PCR in various mouse tissues and organs shows that Rnf114 was predominately expressed in oocytes.


( B ) Immunohistochemistry analysis of RNF114 in mouse ovary shows that RNF114 was mainly expressed in the oocytes ( Scale bar = 100um ) .

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(B) Immunohistochemistry analysis of RNF114 in mouse ovary shows that RNF114 was mainly expressed in the oocytes (Scale bar = 100um).


( C ) Knockdown efficiency of Rnf114 in the 2 - cell stage embryos derived from Rnf114 siRNA # 1 or siRNA # 2 injected zygotes was verified by realtime - PCR and western blotting . NC indicates negative control injected with non - silencing siRNA . Three independent experiment replicates were performed for realtime - PCR , error bars represent s . e . m . ; * * : P < 0 . 01 , * * * : P < 0 . 01 in unpaired two - tailed t - test .

(C) Knockdown efficiency of Rnf114 in the 2-cell stage embryos derived from Rnf114 siRNA #1 or siRNA #2 injected zygotes was verified by realtime-PCR and western blotting. NC indicates negative control injected with non-silencing siRNA. Three independent experiment replicates were performed for realtime-PCR, error bars represent s.e.m.; **:P<0.01, ***:P<0.01 in unpaired two-tailed t-test.


( D ) RNF114 depletion impaired the early embryonic development . Rnf114 siRNA # 1 or siRNA # 2 injected zygotes were cultured and quantified ( siRNA1 # , n = 120 ; siRNA2 # , n = 128 ; three independent experiments ) ; NC indicates negative control ( n = 193 ; six independent experiments ) . The error bars represent s . d . , * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 in unpaired two - tailed t - test .

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(D) RNF114 depletion impaired the early embryonic development. Rnf114 siRNA #1 or siRNA #2 injected zygotes were cultured and quantified (siRNA1#, n = 120; siRNA2#, n = 128; three independent experiments); NC indicates negative control (n = 193; six independent experiments). The error bars represent s.d., *:P<0.05, **:P<0.01, ***:P<0.001 in unpaired two-tailed t-test.


( E ) Representative images to show early embryonic development defects . The embryos arrested at two - cell stage are indicated by arrows ( Scale bar = 100um ) .

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(E) Representative images to show early embryonic development defects. The embryos arrested at two-cell stage are indicated by arrows (Scale bar = 100um).


( A ) In vitro ubiquitination assay . Purified His - RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti - FLAG ( Ub ) antibody . Lanes 1 - 4 indicate negative controls . The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains .

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(A) In vitro ubiquitination assay. Purified His-RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti-FLAG (Ub) antibody. Lanes 1-4 indicate negative controls. The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains.


( B , C ) Purified wild - type and RING mutated His - RNF114 were stained by Coomassie Blue ( B ) . Purified wild - type and RING mutated His - RNF114 were subjected to ubiquitination assays together with E1 , Ubc4 , FLAG - Ub , and ATP . The results show that mutant RNF114 lacked ubiquitination activity ( C ) .

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(B, C) Purified wild-type and RING mutated His-RNF114 were stained by Coomassie Blue (B). Purified wild-type and RING mutated His-RNF114 were subjected to ubiquitination assays together with E1, Ubc4, FLAG-Ub, and ATP. The results show that mutant RNF114 lacked ubiquitination activity (C).


( D ) Screening for proteins ubiquitinated by RNF114 on a proteome array . Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray ® Human Protein Microarrays v5 . 0 . Ubiquitination reactions were performed on quintuplicate arrays using the indicated components , the five sub - array are respectively negative controls , E1 + E2 ( no E3 ) , WT RNF114 ( two arrays , duplicate ) , and mutated RNF114 .

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(D) Screening for proteins ubiquitinated by RNF114 on a proteome array. Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray® Human Protein Microarrays v5.0. Ubiquitination reactions were performed on quintuplicate arrays using the indicated components, the five sub-array are respectively negative controls, E1+E2 (no E3), WT RNF114 (two arrays, duplicate), and mutated RNF114.


( E ) A heat map comparing hits between E1 + E2 , E1 + E2 + WT RNF114 , and E1 + E2 + mutated RNF114 reactions . Candidate substrates of RNF114 selected for further characterization are indicated in red font ; HIP1 , indicated by blue font , was considered to be a false positive because it was also detected in the mutated RNF114 reaction . Black font indicates proteins with ubiquitin - binding domains such as UIM , UBA or CUE or proteins without clearly reported functions , which were excluded from further investigation .

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(E) A heat map comparing hits between E1+E2, E1+E2+WT RNF114, and E1+E2+mutated RNF114 reactions. Candidate substrates of RNF114 selected for further characterization are indicated in red font; HIP1, indicated by blue font, was considered to be a false positive because it was also detected in the mutated RNF114 reaction. Black font indicates proteins with ubiquitin-binding domains such as UIM, UBA or CUE or proteins without clearly reported functions, which were excluded from further investigation.


( A ) RNF114 substrate validation . HEK293 cells were co - transfected with constructs expressing FLAG - tagged substrates , wild - type or mutated His - RNF114 and EGFP , and western blot was performed with anti - FLAG and anti - His antibodies . The results show that the protein levels of CD74 , TAB1 , OPTN , TNIP1 , APPL1 , PSAT1 , and RALGPS1 were reduced in the presence of wild - type RNF114 compared to co - transfecting with mutant RNF114 .

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(A) RNF114 substrate validation. HEK293 cells were co-transfected with constructs expressing FLAG-tagged substrates, wild-type or mutated His-RNF114 and EGFP, and western blot was performed with anti-FLAG and anti-His antibodies. The results show that the protein levels of CD74, TAB1, OPTN, TNIP1, APPL1, PSAT1, and RALGPS1 were reduced in the presence of wild-type RNF114 compared to co-transfecting with mutant RNF114.


( B ) RNF114 - dependent in vitro ubiquitination of substrates . Substrate ubiquitination assays were performed with purified RNF114 , the reaction products were immunoblotted using anti - His antibody to detect the ubiquitin ( upper panel ) or anti - FLAG antibody detecting the substrate protein ( middle panel ) , and the lower panel showed the merged signals . The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74 , TAB1 , TNIP1 , PSAT1 or RALGPS1 .

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(B) RNF114-dependent in vitro ubiquitination of substrates. Substrate ubiquitination assays were performed with purified RNF114, the reaction products were immunoblotted using anti-His antibody to detect the ubiquitin (upper panel) or anti-FLAG antibody detecting the substrate protein (middle panel), and the lower panel showed the merged signals. The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74, TAB1, TNIP1, PSAT1 or RALGPS1.


( A ) Mouse zygotes were microinjected with sterile ddH2O ( control group ) or Ralgps1 , Cd74 , Tnip1 , Psat1 , or Tab1 mRNA . During 96 - 100 h culturing , the percentages of embryos at various stages were counted under microscopy . The results show that overexpression of Tab1 impaired the early embryonic development . Three independent experiment replicates were performed , error bars represent s . d . ; * : P < 0 . 05 , * * * : P < 0 . 001 in unpaired two - tailed t - test .

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(A) Mouse zygotes were microinjected with sterile ddH2O (control group) or Ralgps1, Cd74, Tnip1, Psat1, or Tab1 mRNA. During 96-100 h culturing, the percentages of embryos at various stages were counted under microscopy. The results show that overexpression of Tab1 impaired the early embryonic development. Three independent experiment replicates were performed, error bars represent s.d.; *:P<0.05, ***:P<0.001 in unpaired two-tailed t-test.


( B ) TAB1 reduced with mouse early embryonic development , while abnormally accumulated at 2 - cell stage once treated with Rnf114 siRNAs . TAB1 was detected by western blotting with anti - TAB1 antibody . The relative level of TAB1 protein after Rnf114 siRNAs treatment were presented as mean ± s . e . m . ( * : P < 0 . 05 in unpaired two - tailed t - test , four independent experiment replicates ) .

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(B) TAB1 reduced with mouse early embryonic development, while abnormally accumulated at 2-cell stage once treated with Rnf114 siRNAs. TAB1 was detected by western blotting with anti-TAB1 antibody. The relative level of TAB1 protein after Rnf114 siRNAs treatment were presented as mean±s.e.m. (*:P<0.05 in unpaired two-tailed t-test, four independent experiment replicates).


( C ) Detection of endogenous TAB1 ubiquitination at 1 - cell or 2 - cell stages . Endogenous TAB1 proteins were immunoprecipated by anti - TAB1 antibody at either 1 - cell or 2 - cell stages , the immunoprecipates were detected by either anti - TAB1 antibody or anti - ubiquitin antibody . The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes , when the TAB1 proteins were at comparable level .

(C) Detection of endogenous TAB1 ubiquitination at 1-cell or 2-cell stages. Endogenous TAB1 proteins were immunoprecipated by anti-TAB1 antibody at either 1-cell or 2-cell stages, the immunoprecipates were detected by either anti-TAB1 antibody or anti-ubiquitin antibody. The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes, when the TAB1 proteins were at comparable level.


( D ) RNF114 - dependent TAB1 degradation during early embryo development . ( a - c ) Representive time lapse embryo images were shown for each treatment . Scale bar = 20um . ( a ' - c ' ) Quantification of the GFP fluorescent signals for respective treatments in ( a - c ) . n = 14 , 9 or 12 for the NC , Rnf114 siRNA1 # or Rnf114 siRNA2 # treatment group respectively . Gray traces : Tab1 - GFP fluorescent signals were normalized to the first time point ; Black trace : average signal of all embryos for each treatment .

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(D) RNF114-dependent TAB1 degradation during early embryo development. (a-c) Representive time lapse embryo images were shown for each treatment. Scale bar = 20um. (a'-c') Quantification of the GFP fluorescent signals for respective treatments in (a-c). n=14, 9 or 12 for the NC, Rnf114 siRNA1# or Rnf114 siRNA2# treatment group respectively. Gray traces: Tab1-GFP fluorescent signals were normalized to the first time point; Black trace: average signal of all embryos for each treatment.


( E ) Tab1 knockdown partly rescued the early embryo development defect observed with Rnf114 knockdown . Both Rnf114 and Tab1 siRNAs were injected into the GV oocytes , then the oocytes matured , and were fertilized by IVF , and the fertilized eggs were transferred into KSOM media , cultured for 48 - 50 h . The percentages of embryos arrested at 2 - cell stage were counted . Three independent experiment replicates were performed , error bars represent s . d . ; * * : P < 0 . 01 in unpaired two - tailed t - test .

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(E) Tab1 knockdown partly rescued the early embryo development defect observed with Rnf114 knockdown. Both Rnf114 and Tab1 siRNAs were injected into the GV oocytes, then the oocytes matured, and were fertilized by IVF, and the fertilized eggs were transferred into KSOM media, cultured for 48-50 h. The percentages of embryos arrested at 2-cell stage were counted. Three independent experiment replicates were performed, error bars represent s.d.; **:P<0.01 in unpaired two-tailed t-test.


( A ) NF - κB activation during MZT . Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two - cell stage and thereafter . Scale bar = 25um .

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(A) NF-κB activation during MZT. Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two-cell stage and thereafter. Scale bar = 25um.