D ) Lysate from HEK 293 cells expressing a GFP - tagged copy of either wild - type ( WT ) or F147 / 149 / 229 / 231L ( F4L ) TDP - 43 was added to a mixture of recombinant WT or F4L TDP - 43 ( 5 . 3 μM , 10 % Cy3 - labeled protein ) and A ( GU ) 6 ( 22 . 8 μM ) , A ( CA ) 18 ( 7 . 6 μM ) , A ( GU ) 18 ( 7 . 6 μM ) , or no RNA control at 250 mM NaCl . Droplets were imaged by brightfield and fluorescence microscopy . Representative images for 3 biological replicates using 2 protein preparations . Scale bars , 10 μm . E ) LLPS measured by turbidity in the same experimental conditions as panel D . Mean and SD of 4 biological replicates using 2 recombinant protein preparations . Analyzed by one - way ANOVA ( F ( 7 , 24 ) = 69 . 30 , P < 0 . 0001 ) . Sidak ' s multiple comparisons test was used to compare selected groups . * P < 0 . 05 , * * * * P < 0 . 0001 .

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D) Lysate from HEK 293 cells expressing a GFP-tagged copy of either wild-type (WT) or F147/149/229/231L (F4L) TDP-43 was added to a mixture of recombinant WT or F4L TDP-43 (5.3 μM, 10% Cy3-labeled protein) and A(GU)6 (22.8 μM), A(CA)18 (7.6 μM), A(GU)18 (7.6 μM), or no RNA control at 250 mM NaCl. Droplets were imaged by brightfield and fluorescence microscopy. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm. E) LLPS measured by turbidity in the same experimental conditions as panel D. Mean and SD of 4 biological replicates using 2 recombinant protein preparations. Analyzed by one-way ANOVA (F(7,24)=69.30, P<0.0001). Sidak's multiple comparisons test was used to compare selected groups. *P<0.05, ****P<0.0001.


A ) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP - 43 ( 4 μM , 10 % labeled protein ) was mixed with no RNA control or increasing A ( GU ) 18 RNA concentration at 250 mM salt . Representative images from 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm .

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A) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or increasing A(GU)18 RNA concentration at 250 mM salt. Representative images from 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.


D ) Phase separation of TDP - 43 ( 4 μM ) at increasing salt concentration without RNA or in the presence of increasing A ( GU ) 18 concentration . Scale bars , 10 μm . Inlay added to top right panel for increased visibility , scale bar 1 μm . E ) Phase diagram derived from panel D , denoting either no droplets or droplet formation for each condition .

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D) Phase separation of TDP-43 (4 μM) at increasing salt concentration without RNA or in the presence of increasing A(GU)18 concentration. Scale bars, 10 μm. Inlay added to top right panel for increased visibility, scale bar 1 μm. E) Phase diagram derived from panel D, denoting either no droplets or droplet formation for each condition.


A ) TDP - 43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP - 43 ( 4 μM , 10 % labeled protein ) was mixed with no RNA control or GU - repeat RNA oligonucleotides of increasing length at 250 mM salt . As indicated , RNA concentration varied to maintain a constant total number of TDP - 43 binding sites . Scale bars , 10 μm . B ) The plot shows droplet area quantified as a function of GU - repeat length . Mean and SD of > 300 droplets from 3 biological replicates using 3 different protein preparations . C ) TDP - 43 concentration in the light phase ( Cout ) as a function of GU ( blue ) or CA ( red ) - repeat length quantified by Bradford protein assay . Mean and SD from > 5 biological replicates using 2 different protein preparations . ( 250 mM NaCl , 4 µM TDP - 43 )

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A) TDP-43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or GU-repeat RNA oligonucleotides of increasing length at 250 mM salt. As indicated, RNA concentration varied to maintain a constant total number of TDP-43 binding sites. Scale bars, 10 μm. B) The plot shows droplet area quantified as a function of GU-repeat length. Mean and SD of >300 droplets from 3 biological replicates using 3 different protein preparations. C) TDP-43 concentration in the light phase (Cout) as a function of GU (blue) or CA (red)-repeat length quantified by Bradford protein assay. Mean and SD from >5 biological replicates using 2 different protein preparations. (250 mM NaCl, 4 µM TDP-43)


B ) Phase separation of full - length and TDP - 43 variants ( 4 μM ) mixed with A ( GU ) 30 RNA ( 0 . 8 μM ) or no RNA control at 250 mM NaCl observed by brightfield microscopy . Representative images for 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm .

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B) Phase separation of full-length and TDP-43 variants (4 μM) mixed with A(GU)30 RNA (0.8 μM) or no RNA control at 250 mM NaCl observed by brightfield microscopy. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.


A ) TDP - 43 ALS - linked amino acid substitutions K181E , A315T / E , A321G , Q331K , M337V , and A382T . Boxed in the C - terminal domain is the α - helical structure within the conserved region ( a . a . 320 - 343 ) . Droplets formed by TDP - 43 wild - type ( WT ) and the different mutants ( 4 μM ) observed by brightfield microscopy in the presence of no RNA control or A ( GU ) 18 RNA ( 3 . 9 μM ) at 250 mM NaCl . Mutations A321G and M337V , showing decreased liquidity of the condensates in the presence of A ( GU ) 18 , are boxed in red . Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V , 2 preparations of A321G , Q331K , K181E and one preparation of A315T / E and A382T . Scale bars , 10 μm .

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A) TDP-43 ALS-linked amino acid substitutions K181E, A315T/E, A321G, Q331K, M337V, and A382T. Boxed in the C-terminal domain is the α-helical structure within the conserved region (a.a. 320-343). Droplets formed by TDP-43 wild-type (WT) and the different mutants (4 μM) observed by brightfield microscopy in the presence of no RNA control or A(GU)18 RNA (3.9 μM) at 250 mM NaCl. Mutations A321G and M337V, showing decreased liquidity of the condensates in the presence of A(GU)18, are boxed in red. Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V, 2 preparations of A321G, Q331K, K181E and one preparation of A315T/E and A382T. Scale bars, 10 μm.


B ) Phase separation of WT ( 4 μM , 10 % Oregon green - labeled protein ) and M337V ( 4 μM , 10 % Cy - 3 - labeled protein ) observed by brightfield and fluorescence microscopy in the presence of A ( GU ) 18 ( 3 . 9 μM ) at 250 mM NaCl . The middle panel shows mixing of these WT ( 2 μM ) and M337V ( 2 μM ) samples . Representative images for 3 biological replicates using 2 protein preparations . Scale bars , 10 μm .

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B) Phase separation of WT (4 μM, 10% Oregon green-labeled protein) and M337V (4 μM, 10% Cy-3-labeled protein) observed by brightfield and fluorescence microscopy in the presence of A(GU)18 (3.9 μM) at 250 mM NaCl. The middle panel shows mixing of these WT (2 μM) and M337V (2 μM) samples. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.


A ) Binding of TDP - 43 to GU - rich RNA promotes liquid properties of TDP - 43 condensates , as shown by the fusion of small condensates into larger droplets . This fluidity is impaired by the loss of RNA binding affinity or disease - linked mutations that alter phase separation , such as M337V and A321G , which could explain increased TDP - 43 aggregation .

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A) Binding of TDP-43 to GU-rich RNA promotes liquid properties of TDP-43 condensates, as shown by the fusion of small condensates into larger droplets. This fluidity is impaired by the loss of RNA binding affinity or disease-linked mutations that alter phase separation, such as M337V and A321G, which could explain increased TDP-43 aggregation.


C , Survival assays against substrates of QacA showed loss of activity against monovalent cationic antibacterials ( TPP : Tetraphenylphosphonium , Et : Ethidium ) , while still retaining partial activity against divalent cations ( Dq : Dequalinium , Ch : Chlorhexidine ) . EV : pBAD empty vector . n = 3 for biological replicates . CFU indicates colony forming units . Image color was inverted from black to white background to enhance clarity .

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C, Survival assays against substrates of QacA showed loss of activity against monovalent cationic antibacterials (TPP: Tetraphenylphosphonium, Et: Ethidium), while still retaining partial activity against divalent cations (Dq: Dequalinium, Ch:Chlorhexidine). EV: pBAD empty vector. n = 3 for biological replicates. CFU indicates colony forming units. Image color was inverted from black to white background to enhance clarity.


E , Population shift - based titration of QacA - GFP against ICabs yielded an apparent Kd of 65 ± 4 nM for QacA - A4 complex and 100 ± 10 nM for QacA - B7 . n = 3 for independent replicates . Error bars denote S . E . M . and each datapoint represents mean value .

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E, Population shift-based titration of QacA-GFP against ICabs yielded an apparent Kd of 65 ± 4 nM for QacA-A4 complex and 100 ± 10 nM for QacA-B7. n = 3 for independent replicates. Error bars denote S.E.M. and each datapoint represents mean value.


G , Colocalization of QacAWT or QacAD411N ( tagged with GFP ) on E . coli spheroplasts ( stained with DAPI for viability ) with ICabs ( labelled with NHS - Rhodamine ) was screened against ICab A4 ( left ) and ICab B7 ( right ) . n = 10 - 30 spheroplasts were imaged for each sample .

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G, Colocalization of QacAWT or QacAD411N (tagged with GFP) on E. coli spheroplasts (stained with DAPI for viability) with ICabs (labelled with NHS-Rhodamine) was screened against ICab A4 (left) and ICab B7 (right). n = 10-30 spheroplasts were imaged for each sample.


H , Representative reference - free 2D classes of QacAD411N embedded in UDM micelle , in complex with ICab A4 ( top ) and with ICabs A4 and B7 ( bottom ) , pointed with pink and green arrows respectively . Representative classes shown for QacA - A4 complex are from a set of 68 , 000 particles and QacA - A4 / B7 complex are from 218 , 040 particles .

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H, Representative reference-free 2D classes of QacAD411N embedded in UDM micelle, in complex with ICab A4 (top) and with ICabs A4 and B7 (bottom), pointed with pink and green arrows respectively. Representative classes shown for QacA-A4 complex are from a set of 68,000 particles and QacA-A4/B7 complex are from 218,040 particles.


A , CryoEM model of QacA ( blue ) in complex with ICab A4 ( pink ) and ICab B7 ( green )

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A, CryoEM model of QacA (blue) in complex with ICab A4 (pink) and ICab B7 (green)


C , Whole cell ethidium efflux assay with overexpressed EL7 deletion construct in the background of wild type and D411N mutant . n = 3 for independent replicates , one of which is shown here . Error bars represent S . E . M . from six technical replicates .

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C, Whole cell ethidium efflux assay with overexpressed EL7 deletion construct in the background of wild type and D411N mutant. n = 3 for independent replicates, one of which is shown here. Error bars represent S.E.M. from six technical replicates.


D , Survival assays of QacAΔEL7 and QacAD411N / ΔEL7 mutants in the presence of 10 μM Tetraphenylphosphonium ( TPP ) and 16 μM Chlorhexidine ( Ch ) . Data from one representative biological replicate is shown here . Image colors were inverted for improved clarity .

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D, Survival assays of QacAΔEL7 and QacAD411N/ΔEL7 mutants in the presence of 10 μM Tetraphenylphosphonium (TPP) and 16 μM Chlorhexidine (Ch). Data from one representative biological replicate is shown here. Image colors were inverted for improved clarity.


C Whole cell ethidium efflux assay done with E . coli JD838 cells expressing QacAWT or QacA5A . Empty vector ( EV ) was used as a control for the experiment . A graph of one independent replicate is shown with error bars representing S . E . M . of 6 technical replicates .

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C Whole cell ethidium efflux assay done with E.coli JD838 cells expressing QacAWT or QacA5A. Empty vector (EV) was used as a control for the experiment. A graph of one independent replicate is shown with error bars representing S.E.M. of 6 technical replicates.


D A single representative replicate of survival assays shown for the same cells spotted in 10 - fold dilution series on 12 μM TPP and 16 μM Ch . Image colors were inverted for clarity in the figure .

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D A single representative replicate of survival assays shown for the same cells spotted in 10-fold dilution series on 12 μM TPP and 16 μM Ch. Image colors were inverted for clarity in the figure.


G Survival assays of solely EL7 chimeric constructs compared against empty vector ( EV ) and QacAWT . One representative biological replicate is shown . Image color inverted for clarity .

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G Survival assays of solely EL7 chimeric constructs compared against empty vector (EV) and QacAWT. One representative biological replicate is shown. Image color inverted for clarity.


Whole cell - based ethidium efflux assay done with E . coli JD838 cells expressing QacAWT , its EL7 deletion construct ( QacAΔEL7 ) , and the chimeric mutants of LfrA and SmvA . All assays were done in at least 2 independent replicates . One representative replicate shown with data normalized to highest value in each trace . Error bars depict S . E . M . for six technical replicates .

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Whole cell-based ethidium efflux assay done with E.coli JD838 cells expressing QacAWT, its EL7 deletion construct (QacAΔEL7), and the chimeric mutants of LfrA and SmvA. All assays were done in at least 2 independent replicates. One representative replicate shown with data normalized to highest value in each trace. Error bars depict S.E.M. for six technical replicates.


I Whole cell - based ethidium efflux assay done with E . coli JD838 cells expressing QacAWT , its EL7 deletion construct ( QacAΔEL7 ) , and the chimeric mutants of LfrA and SmvA . All assays were done in at least 2 independent replicates . One representative replicate shown with data normalized to highest value in each trace . Error bars depict S . E . M . for six technical replicates .

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I Whole cell-based ethidium efflux assay done with E.coli JD838 cells expressing QacAWT, its EL7 deletion construct (QacAΔEL7), and the chimeric mutants of LfrA and SmvA. All assays were done in at least 2 independent replicates. One representative replicate shown with data normalized to highest value in each trace. Error bars depict S.E.M. for six technical replicates.


J Everted vesicles - based assays of chimeric and EL7 deletion constructs of QacA . Traces represent mean values of three replicates performed on vesicles made with a single batch of cells for each construct . Timepoints indicated as 1 - 3 above the traces indicate addition of 100 μM ATP , 1 mM TPP and 2 μM Nigericin respectively . One representative shown for data obtained from biological triplicates . Error bars represent SEM for technical triplicates from one of three biological replicates .

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J Everted vesicles-based assays of chimeric and EL7 deletion constructs of QacA. Traces represent mean values of three replicates performed on vesicles made with a single batch of cells for each construct. Timepoints indicated as 1-3 above the traces indicate addition of 100 μM ATP, 1 mM TPP and 2 μM Nigericin respectively. One representative shown for data obtained from biological triplicates. Error bars represent SEM for technical triplicates from one of three biological replicates.


( C ) Quantification of renewed ECs ( RFP - only labelled cells ) , left graph , and number of ISC and EB cells ( double RFP + GFP + cells ) , right graph , in posterior midguts after 7 , 14 , or 21 days of tracing in normal ( ' Variable ' : gut scored n = 9 , 8 , 4 ) and low demand ( ' Low ' : n = 6 , 4 , 7 ) culturing conditions . Asterisks denote significances from one - way ANOVA with Bonferroni ' s Multiple Comparison Test ( * * , P = 0 . 0016 ; * * * * , P < 0 . 0001 ) . Data information In the boxplots , the box extends from the 25th to 75th percentiles and whiskers show all points , min to max . The line in the middle of the box is plotted at the

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(C) Quantification of renewed ECs (RFP-only labelled cells), left graph, and number of ISC and EB cells (double RFP+ GFP+ cells), right graph, in posterior midguts after 7, 14, or 21 days of tracing in normal ('Variable': gut scored n= 9, 8, 4) and low demand ('Low': n= 6, 4, 7) culturing conditions. Asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (**, P = 0.0016; ****, P < 0.0001). Data information In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. The line in the middle of the box is plotted at the median


( D ) Quantification of mitosis ( phospho - histone H3 ( PH3 ) + cells ) in low demand midguts at the indicated time points . * * , P = 0 . 0015 ; n . s . not significant from one - way ANOVA test . Data information : In the boxplots , the box extends from the 25th to 75th percentiles and whiskers show all points , min to max . The line in the middle of the box is plotted at the

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(D) Quantification of mitosis (phospho-histone H3 (PH3)+ cells) in low demand midguts at the indicated time points. **, P = 0.0015; n.s. not significant from one-way ANOVA test. Data information: In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. The line in the middle of the box is plotted at the median


( E ) Representative adult midgut from control esgReDDM at day 14 after temperature shift in variable demand . ECs renewed are marked positively by persistent RFP labelling ( red - only cells ) . ( F and G ) Few ECs had been renewed in ' Low ' midguts after 14 ( F ) and 21 ( G ) days of tracing . Data information : Scale bars , 50µm .

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(E) Representative adult midgut from control esgReDDM at day 14 after temperature shift in variable demand. ECs renewed are marked positively by persistent RFP labelling (red-only cells). (F and G) Few ECs had been renewed in 'Low' midguts after 14 (F) and 21 (G) days of tracing. Data information: Scale bars, 50µm.


( H - J ) Age - synchronized posterior midguts of control ( esgReDDM > , H ) and p35 overexpression in stem and progenitor cells ( esgReDDM > p35 , p35 I and J ) at 7 days after temperature shift . Arrows in ( H , I ) point to newly differentiated ECs . In ( J ) , tumour mass is found in the anterior midgut . Data information : Scale bars , 50µm .

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(H-J) Age-synchronized posterior midguts of control (esgReDDM>, H) and p35 overexpression in stem and progenitor cells (esgReDDM>p35, p35 I and J) at 7 days after temperature shift. Arrows in (H, I) point to newly differentiated ECs. In (J), tumour mass is found in the anterior midgut. Data information: Scale bars, 50µm.


( K ) , Quantification of mitosis PH3 + cells in posterior midgut ( pmg ) ( control : gut scored n = 13 and esg > p35 , p35 : n = 22 ] ) . Student t - test ( * * * * = P < 0 . 0001 ) . Data information : In the boxplots , the box extends from the 25th to 75th percentiles and whiskers show all points , min to max .

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(K), Quantification of mitosis PH3+ cells in posterior midgut (pmg) (control: gut scored n=13 and esg>p35, p35: n=22]). Student t-test (**** = P < 0.0001). Data information: In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max.


( L ) , Diap1 is monitored by the GFP . 4 . 3 . ( green ) reporter ( Djiane et al . , 2013 ; Zhang et al . , 2008 ) . Diap1 - GFP is detected in a subset of adult midgut esg + cells that are negative for the mitotic marker PH3 ( red , inset ) and for EE marker Prospero ( Pros , grey in inset ) . Diap1 - GFP is not detected in mature EC ( large nuclei cells , DAPI in blue ) . Data information : Scale bars , 50µm .

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(L), Diap1 is monitored by the GFP.4.3. (green) reporter (Djiane et al., 2013; Zhang et al., 2008). Diap1-GFP is detected in a subset of adult midgut esg+ cells that are negative for the mitotic marker PH3 (red, inset) and for EE marker Prospero (Pros, grey in inset). Diap1-GFP is not detected in mature EC (large nuclei cells, DAPI in blue). Data information: Scale bars, 50µm.


( M - O ) , Diap1 - GFP ( green ) co - localized almost 100 % with the EB marker GBE - Su ( H ) - lacZ ( red , M and O ) . Arrow points to a Diap1 - GFP - negative GBE - Su ( H ) - lacZ - positive cell . Data information : Scale bars , 50µm .

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(M-O), Diap1-GFP (green) co-localized almost 100% with the EB marker GBE-Su(H)-lacZ (red, M and O). Arrow points to a Diap1-GFP-negative GBE-Su(H)-lacZ-positive cell. Data information: Scale bars, 50µm.


( B ) Lineage tracing using the initiator caspase sensor act - DBS - S - QF ( 14 days of tracing ) combined with QUAS - FLP and act > FRT > lacZ are visualized by ßGalactosidase ( ßGal , green ) . ISCs are labelled by anti - Dl ( red , insets ) . Red arrowhead points to the ISC . Gut is counterstained with DAPI ( blue ) and DAPI ( grey , in the inset ) . Data information : Scale bars ,

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(B) Lineage tracing using the initiator caspase sensor act-DBS-S-QF (14 days of tracing) combined with QUAS-FLP and act>FRT>lacZ are visualized by ßGalactosidase (ßGal, green). ISCs are labelled by anti-Dl (red, insets). Red arrowhead points to the ISC. Gut is counterstained with DAPI (blue) and DAPI (grey, in the inset). Data information: Scale bars, 100µm


( C ) Control GBE - Su ( H ) ReDDM > and ( D ) GBE - Su ( H ) ReDDM > p35 , p35 midguts after 7 days of tracing . Data information : Scale bars ,

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(C) Control GBE-Su(H)ReDDM> and (D) GBE-Su(H)ReDDM>p35, p35 midguts after 7 days of tracing. Data information: Scale bars, 100µm


( E - E ' ' ) Adult midgut klu > mCD8 : : GFP , GBE - Su ( H ) - lacZ stained for ß - Gal ( red ) and GFP ( green ) . DAPI counterstaining in blue ( E ' and E ' ' ) . The arrows point to examples of klu - negative Su ( H ) - lacZ - positive cells . The green label ( asterisk , E ) is in the overlying muscle . Data information : Scale bars 50µm .

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(E-E'') Adult midgut klu>mCD8::GFP, GBE-Su(H)-lacZ stained for ß-Gal (red) and GFP (green). DAPI counterstaining in blue (E' and E''). The arrows point to examples of klu-negative Su(H)-lacZ-positive cells. The green label (asterisk, E) is in the overlying muscle. Data information: Scale bars 50µm.


( F - J ) Adult midguts of ( F ) control kluReDDM > ( G ) kluReDDM > Debcl - RNAi , ( H ) kluReDDM > Drice - RNAi , ( I ) kluReDDM > Diap1 - RNAi , ( J ) kluReDDM > Diap1 midguts after 7 days of tracing . The arrows point to examples of PH3 + labelled ISCs . Data information : Scale bars ,

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(F-J) Adult midguts of (F) control kluReDDM> (G) kluReDDM>Debcl-RNAi, (H) kluReDDM>Drice-RNAi, (I) kluReDDM>Diap1-RNAi, (J) kluReDDM>Diap1 midguts after 7 days of tracing. The arrows point to examples of PH3+ labelled ISCs. Data information: Scale bars, 100µm


( K ) Quantification of klu + cells in the indicated midguts at day 7 in low demand ( Low ) or standard ( Variable ) culturing conditions ( n = 7 , 5 , 9 , 7 ) . ata information : ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction .

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(K) Quantification of klu+ cells in the indicated midguts at day 7 in low demand (Low) or standard (Variable) culturing conditions (n=7, 5, 9, 7). ata information: ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.


( L ) Intestinal cell turnover measured by quantification of the number of newly renewed ECs ( RFP - only cells : gut scored n = 9 , 13 , 9 , 13 ) . Data information : Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction .

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(L) Intestinal cell turnover measured by quantification of the number of newly renewed ECs (RFP-only cells: gut scored n=9, 13, 9, 13). Data information: Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.


( M ) Quantification of apoptotic progenitor cells ( esg + ) measured as GFP + TUNEL + cells ( n = 9 , 9 , 9 , 9 ) . Data information : Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction .

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(M) Quantification of apoptotic progenitor cells (esg+) measured as GFP+ TUNEL+ cells (n= 9, 9, 9, 9). Data information: Box and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.


( N ) Quantification of klu + cells and renewed ECs in kluReDDM > Diap1 posterior midguts ( n = 8 ) relative to ' Low ' control in K and L , respectively . Data information : ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles . The line in the middle of the box is plotted at the median . P values ( * , P < 0 . 0261 ; * * , P < 0 . 005 ; * * * , P < 0 . 0021 ) calculated by one - way ANOVA with Bonferroni correction .

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(N) Quantification of klu+ cells and renewed ECs in kluReDDM>Diap1 posterior midguts (n=8) relative to 'Low' control in K and L, respectively. Data information: ox and whisker plot showing min to max values and the box is extending from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. P values (*, P<0.0261; **, P<0.005; ***, P<0.0021) calculated by one-way ANOVA with Bonferroni correction.


( A - E ) Confocal images of representative midguts of the indicated genotypes using kluReDDM for tracing and age - and EB - specific transgene expression at day 7 after temperature shift . ISC mitosis was monitored by using PH3 labelling ( white ) in all guts . ( B - E ) Accumulation and loss of klu + cells by loss ( B and E ) and gain ( C and D ) of N and EGFR signalling . In ( C ) , the accumulation of EC - committed EBs ( klu + cells ) in midguts with constitutively activated EGFR ( kluReDDM > EGFRact ) is accompanied with a non - autonomous increase in ISC mitosis . Inset in ( C ) shows labelling with Dl ( blue ) , revealing symmetric ISC mitoses Data information : Scale bar , 100µm for all images .

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(A-E) Confocal images of representative midguts of the indicated genotypes using kluReDDM for tracing and age- and EB-specific transgene expression at day 7 after temperature shift. ISC mitosis was monitored by using PH3 labelling (white) in all guts. (B-E) Accumulation and loss of klu+ cells by loss (B and E) and gain (C and D) of N and EGFR signalling. In (C), the accumulation of EC-committed EBs (klu+ cells) in midguts with constitutively activated EGFR (kluReDDM> EGFRact) is accompanied with a non-autonomous increase in ISC mitosis. Inset in (C) shows labelling with Dl (blue), revealing symmetric ISC mitoses Data information: Scale bar, 100µm for all images.


( F ) Quantification of the number of klu + ( EBs ) in the indicated midguts ( n = 21 , 14 , 5 , 6 , 8 , 6 , 4 , 10 , 12 , 9 , 13 , 14 ) . Data information : Note that impaired cell renewal reduces the intestinal width . * * , P < 0 . 01 ; * * * * , P < 0 . 0001 , one - way ANOVA with Bonferroni correction .

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(F) Quantification of the number of klu+ (EBs) in the indicated midguts (n= 21, 14, 5, 6, 8, 6, 4, 10, 12, 9, 13, 14). Data information: Note that impaired cell renewal reduces the intestinal width. **, P< 0.01; ****, P< 0.0001, one-way ANOVA with Bonferroni correction.


( G ) Quantification of apoptotic esg + ( TUNEL + GFP + ) cells in the indicated midguts ( n = 28 , 4 , 7 , 7 , 16 , 8 , 8 , 7 , 10 , 7 , 20 , 24 ) . Data information : Note that impaired cell renewal reduces the intestinal width . * * , P < 0 . 01 ; * * * * , P < 0 . 0001 , one - way ANOVA with Bonferroni correction .

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(G) Quantification of apoptotic esg+ (TUNEL+ GFP+) cells in the indicated midguts (n= 28, 4, 7, 7, 16, 8, 8, 7, 10, 7, 20, 24). Data information: Note that impaired cell renewal reduces the intestinal width. **, P< 0.01; ****, P< 0.0001, one-way ANOVA with Bonferroni correction.


Confocal images of representative midguts of the indicated genotypes Rescue of the loss of EBs induced by gain of Notch ( H - RNAi ) by expressing Debcl - RNAi . Data information : Scale bar , 100µm for all images .

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Confocal images of representative midguts of the indicated genotypes Rescue of the loss of EBs induced by gain of Notch (H-RNAi) by expressing Debcl-RNAi. Data information: Scale bar, 100µm for all images.


Confocal images of representative midguts of the indicated genotypes escue of the loss of EBs induced by loss of EGFR ( EGFRDN ) by expressing Debcl - RNAi . ( J and K ) Impact of gain of expression ( J ) and loss by RNAi expression ( K ) of lz using kluReDDM White arrowheads point to examples of mitosis labelled by PH3 + . ( L and M ) Impact of gain of expression ( L ) or loss by RNAi expression ( M ) of klu ( M ) using kluReDDM . Data information : Scale bar , 100µm for all images .

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Confocal images of representative midguts of the indicated genotypes escue of the loss of EBs induced by loss of EGFR (EGFRDN) by expressing Debcl-RNAi. (J and K) Impact of gain of expression (J) and loss by RNAi expression (K) of lz using kluReDDM White arrowheads point to examples of mitosis labelled by PH3+. (L and M) Impact of gain of expression (L) or loss by RNAi expression (M) of klu (M) using kluReDDM. Data information: Scale bar, 100µm for all images.


( N - R ) Images of representative midguts of esgReDDM at day 7 after temperature shift of the indicated genotypes . PH3 labelling ( white ) detected rare mitoses in the mutant genotypes . Data information : Scale bar , 100µm for all images .

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(N-R) Images of representative midguts of esgReDDM at day 7 after temperature shift of the indicated genotypes. PH3 labelling (white) detected rare mitoses in the mutant genotypes. Data information: Scale bar, 100µm for all images.


( C ) All EBs show Diap1 - GFP expression but at varying levels , consistent with EB death fate being tuned by apoptotic input counteracting survival signals . Data information : ( n = 17 , 13 , 12 , 10 , 21 , 24 ) . P values were calculated using one - way ANOVA Bonferroni corrected ( * * * * , P < 0 , 0001 ) .

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(C) All EBs show Diap1-GFP expression but at varying levels, consistent with EB death fate being tuned by apoptotic input counteracting survival signals. Data information: (n= 17, 13, 12, 10, 21, 24). P values were calculated using one-way ANOVA Bonferroni corrected (****, P < 0,0001).


( D ) Quantification of the number of Diap1 - GFP + . Note that in p35 - expressing midguts reared in ' Low ' , GFP levels are not increased , consistent with the p35 protein suppressing apoptosis downstream of Diap1 . Data information ( n = 17 , 13 , 12 , 10 , 21 , 24 ) . P values were calculated using one - way ANOVA Bonferroni corrected ( * * * * , P < 0 , 0001 ) .

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(D) Quantification of the number of Diap1-GFP+. Note that in p35-expressing midguts reared in 'Low', GFP levels are not increased, consistent with the p35 protein suppressing apoptosis downstream of Diap1. Data information (n= 17, 13, 12, 10, 21, 24). P values were calculated using one-way ANOVA Bonferroni corrected (****, P < 0,0001).


( E ) Quantification of the number of newly renewed ECs in the indicated genotypes after 7 days Increase in EB numbers and EC turnover in the EGFRact midgut reflects both suppression of EB deaths and the non - autonomous stimulation of ISC in this background . Data information : ( n = 17 , 13 , 12 , 10 , 21 , 24 ) . P values were calculated using one - way ANOVA Bonferroni corrected ( * * * * , P < 0 , 0001 ) .

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(E) Quantification of the number of newly renewed ECs in the indicated genotypes after 7 days Increase in EB numbers and EC turnover in the EGFRact midgut reflects both suppression of EB deaths and the non-autonomous stimulation of ISC in this background. Data information: (n= 17, 13, 12, 10, 21, 24). P values were calculated using one-way ANOVA Bonferroni corrected (****, P < 0,0001).


( F ) Representative images acquired using the same laser intensity are shown for Diap1 - GFP in the indicated illustrative examples . Scale bar , 100µm .

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(F) Representative images acquired using the same laser intensity are shown for Diap1-GFP in the indicated illustrative examples. Scale bar, 100µm.


( A ) Immunostaining of ES cells ( Oct4 ) , NPCs ( day4 , Nestin ) , and neurons ( day 8 , Tuj1 ) . Scale bar : 200µm .

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(A) Immunostaining of ES cells (Oct4), NPCs (day4, Nestin), and neurons (day 8, Tuj1). Scale bar: 200µm.


( B ) Expression levels of candidate lncRNAs during three stages of differentiation , measured by RNA - seq . Gene models of lncRNAs appear in green , adjacent protein - coding genes appear in grey . Blue depicts transcripts from the ' + ' strand , orange represents transcripts from the ' - ' strand . All three RNA - seq tracks were scaled separately to the maximum coverage in each locus .

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(B) Expression levels of candidate lncRNAs during three stages of differentiation, measured by RNA-seq. Gene models of lncRNAs appear in green, adjacent protein-coding genes appear in grey. Blue depicts transcripts from the '+' strand, orange represents transcripts from the '-' strand. All three RNA-seq tracks were scaled separately to the maximum coverage in each locus.


( C ) qRT - PCR of Reno1 , Cox10as1 , and lnc - Nr2f1 in ES cells ( top ) and following eight days of neuronal differentiation ( bottom ) , following knockdown using two different shRNAs , normalized to sh - NT . Mean ± SEM is shown for 3 - 6 independent experiments , * P < 0 . 05 , * * P < 0 . 01 ( unpaired two sample t - test ) .

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(C) qRT-PCR of Reno1, Cox10as1, and lnc-Nr2f1 in ES cells (top) and following eight days of neuronal differentiation (bottom), following knockdown using two different shRNAs, normalized to sh-NT. Mean ± SEM is shown for 3-6 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).


( A ) Immunostaining using anti - TUJ1 antibody of ES - cell - derived neurons following infection with either non - targeting shRNA ( sh - NT ) , or two different shRNAs targeting Reno1 . Scale bar : 200µm . ( B ) Quantification of cell numbers and neurite lengths of ten images of non - overlapping fields for each shRNA . Mean ± SEM is shown , * * P < 0 . 01 ( unpaired two sample t - test ) .

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(A) Immunostaining using anti-TUJ1 antibody of ES-cell-derived neurons following infection with either non-targeting shRNA (sh-NT), or two different shRNAs targeting Reno1. Scale bar: 200µm. (B) Quantification of cell numbers and neurite lengths of ten images of non-overlapping fields for each shRNA. Mean ± SEM is shown, ** P<0.01 (unpaired two sample t-test).


( C ) Mean expression levels of genes which were down - or upregulated following knockdown ( KD ) of Reno1 or lnc - Nr2f1 during an eight time point neuronal differentiation and maturation of mouse ES cells : Neurons1 corresponds to day in vitro ( DIV ) 1 ; Neurons2 - DIV7 ; Neurons3 - DIV16 ; Neurons4 - DIV21 ; and Neurons5 - DIV28 ( Hubbard et al . , 2013 ) .

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(C) Mean expression levels of genes which were down- or upregulated following knockdown (KD) of Reno1 or lnc-Nr2f1 during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013).


( D ) Transcriptional response of cell cycle and neuron - related genes . Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc - Nr2f1 compared to cells infected with sh - NT , following eight days of neuronal differentiation , and of day eight of differentiation compared to ES cells during differentiation of WT cells .

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(D) Transcriptional response of cell cycle and neuron-related genes. Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc-Nr2f1 compared to cells infected with sh-NT, following eight days of neuronal differentiation, and of day eight of differentiation compared to ES cells during differentiation of WT cells.


( A ) Reno1 locus in six vertebrate species . Shaded region indicates the Reno1 locus . Blue gene models are from RefSeq or Ensembl , red gene models are based on PLAR transcript reconstructions ( Hezroni et al . , 2015 ) . RNA - seq datasets from indicated tissues were taken from publicly - available datasets : BodyMap ( Li et al , 2017a ) ( mouse ) , Reference Epigenome ( human ) , FAANG ( sheep ) , SRP011985 ( opossum ) , SRP016501 ( chicken ) , DRP000627 ( coelacanth ) . RNA - seq data was scaled separately in each species to the highest coverage in the window , except for opossum and chicken , where a custom upper threshold was set as indicated .

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(A) Reno1 locus in six vertebrate species. Shaded region indicates the Reno1 locus. Blue gene models are from RefSeq or Ensembl, red gene models are based on PLAR transcript reconstructions (Hezroni et al., 2015). RNA-seq datasets from indicated tissues were taken from publicly-available datasets: BodyMap (Li et al, 2017a) (mouse), Reference Epigenome (human), FAANG (sheep), SRP011985 (opossum), SRP016501 (chicken), DRP000627 (coelacanth). RNA-seq data was scaled separately in each species to the highest coverage in the window, except for opossum and chicken, where a custom upper threshold was set as indicated.


( B ) The mouse Reno1 locus . Expression levels during neuronal differentiation of ES cells , in different cellular fractions of ES cells ( data from ( Engreitz et al , 2016 ) ) and motor neurons ( GSE90913 ) , and in forebrain during different stages of embryonic development ( data from the ENCODE project ) . Each set of tracks was normalized separately .

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(B) The mouse Reno1 locus. Expression levels during neuronal differentiation of ES cells, in different cellular fractions of ES cells (data from (Engreitz et al, 2016)) and motor neurons (GSE90913), and in forebrain during different stages of embryonic development (data from the ENCODE project). Each set of tracks was normalized separately.


( C ) RENO1 locus in human . Expression levels in dorsolateral prefrontal cortex across six age groups , and in different cellular fractions in fetal and adult cells . Each set of tracks was normalized separately .

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(C) RENO1 locus in human. Expression levels in dorsolateral prefrontal cortex across six age groups, and in different cellular fractions in fetal and adult cells. Each set of tracks was normalized separately.


( D ) Reno1 smFISH signal ( red ) and DAPI staining ( blue ) in WT and Reno1m / m cells at day eight of neuronal differentiation , imaged using 100x objective .

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(D) Reno1 smFISH signal (red) and DAPI staining (blue) in WT and Reno1m/m cells at day eight of neuronal differentiation, imaged using 100x objective.


( E ) Expression levels of Reno1 and Bahcc1 in 30 adult and embryonic tissues . Data from the ENCODE project , quantification was done using RSEM ( Li & Dewey , 2011 ) .

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(E) Expression levels of Reno1 and Bahcc1 in 30 adult and embryonic tissues. Data from the ENCODE project, quantification was done using RSEM (Li & Dewey, 2011).


( F ) Targeted Chromosome Conformation Capture ( 4C ) using the Reno1 promoter as bait . Top , smoothed trend lines and raw counts of the contact profile in ES cells ( red ) or differentiated neurons ( black ) ; bottom , domainogram showing mean contact per fragment end for a series of window sizes .

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(F) Targeted Chromosome Conformation Capture (4C) using the Reno1 promoter as bait. Top, smoothed trend lines and raw counts of the contact profile in ES cells (red) or differentiated neurons (black); bottom, domainogram showing mean contact per fragment end for a series of window sizes.


( B ) Expression levels of Reno1 ( both the spliced and unspliced isoforms ) and Bahcc1 following four days of differentiation of the indicated cells . Mean ± SEM is shown for 3 - 4 independent experiments , * P < 0 . 05 , * * P < 0 . 01 ( unpaired two sample t - test ) .

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(B) Expression levels of Reno1 (both the spliced and unspliced isoforms) and Bahcc1 following four days of differentiation of the indicated cells. Mean ± SEM is shown for 3-4 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).


( C ) Left : Oct4 staining of the indicated cells . Scale bar : 200 µm . Right : Cell count of the indicated ES cells grown in ES medium . Cells were harvested from one well of a 6 - well plate , and counted using Orflo MOXI Z Mini Automated Cell Counter . Mean ± SEM is shown for three independent experiments .

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(C) Left: Oct4 staining of the indicated cells. Scale bar: 200 µm. Right: Cell count of the indicated ES cells grown in ES medium. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments.


( D ) Left : Nestin staining of the indicated cells following four days of differentiation . Scale bar : 200 µm . Right : Cell count of the indicated cells following four days of differentiation . Cells were harvested from one well of a 6 - well plate , and counted using Orflo MOXI Z Mini Automated Cell Counter . Mean ± SEM is shown for three independent experiments . * * P < 0 . 01 ( unpaired two sample t - test ) .

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(D) Left: Nestin staining of the indicated cells following four days of differentiation. Scale bar: 200 µm. Right: Cell count of the indicated cells following four days of differentiation. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. ** P<0.01 (unpaired two sample t-test).


( E ) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation . Y axis : forward scatter ; X axis : PI fluorescence intensity .

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(E) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation. Y axis: forward scatter; X axis: PI fluorescence intensity.


( A ) . Heatmap showing log2 - transformed fold changes of Reno1 depleted cells compared to their respective controls . All genes which were significantly differentially expressed ( P < 0 . 05 ) in any of the treatments are shown . ( B ) Boxplots indicating the median , quartiles , and 5th and 95th percentiles of changes in expression levels of Reno1m / m cells compared to WT cells , for genes with significant ( P < 0 . 05 ) change in cells where Reno1 was perturbed with the indicated method . Number of genes in each group is indicated in the x axis . * * P < 0 . 01 ( two - sided Wilcoxon rank sum test ) .

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(A). Heatmap showing log2-transformed fold changes of Reno1 depleted cells compared to their respective controls. All genes which were significantly differentially expressed (P<0.05) in any of the treatments are shown. (B) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of Reno1m/m cells compared to WT cells, for genes with significant (P<0.05) change in cells where Reno1 was perturbed with the indicated method. Number of genes in each group is indicated in the x axis. ** P<0.01 (two-sided Wilcoxon rank sum test).


( C ) Mean expression levels of genes in genes which were significantly ( P < 0 . 05 ) dysregulated by at least two out of three Reno1 perturbations at day 2 in fig . 5A ( n = 168 down - regulated and 94 up - regulated ) , during an eight time point neuronal differentiation and maturation of mouse ES cells : Neurons1 corresponds to day in vitro ( DIV ) 1 ; Neurons2 - DIV7 ; Neurons3 - DIV16 ; Neurons4 - DIV21 ; and Neurons5 - DIV28 ( Hubbard et al . , 2013 ) .

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(C) Mean expression levels of genes in genes which were significantly (P<0.05) dysregulated by at least two out of three Reno1 perturbations at day 2 in fig. 5A (n= 168 down-regulated and 94 up-regulated), during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013).


( D ) Expression levels of Reno1 following Dox addition in WT and Reno1m / m cells . Mean ± SEM is shown for three independent experiments , * P < 0 . 05 ( unpaired two sample t - test ) .

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(D) Expression levels of Reno1 following Dox addition in WT and Reno1m/m cells. Mean ± SEM is shown for three independent experiments, * P<0.05 (unpaired two sample t-test).


( E ) Cell count of WT and Reno1m / m cells following four days of differentiation , with or without Dox addition . Cells were harvested from one well of a 6 - well plate , and counted using Orflo MOXI Z Mini Automated Cell Counter . Mean ± SEM is shown for three independent experiments . * P < 0 . 05 ( unpaired two sample t - test ) .

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(E) Cell count of WT and Reno1m/m cells following four days of differentiation, with or without Dox addition. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. * P<0.05 (unpaired two sample t-test).


( F - G ) Boxplots indicating the median , quartiles , and 5th and 95th percentiles of changes in expression levels of WT ( F ) or Reno1m / m ( G ) cell treated with Dox for the first two days of differentiation compared to untreated cells , in genes which were significantly ( P < 0 . 05 ) downregulated ( n = 252 ) or upregulated ( n = 223 ) by at least two out of three Reno1 perturbations at day 2 in fig . 5A , compared to all genes ( n = 16 , 511 ) . * * P < 0 . 01 ( two - sided Wilcoxon rank sum test ) .

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(F-G) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of WT (F) or Reno1m/m (G) cell treated with Dox for the first two days of differentiation compared to untreated cells, in genes which were significantly (P<0.05) downregulated (n=252) or upregulated (n=223) by at least two out of three Reno1 perturbations at day 2 in fig. 5A, compared to all genes (n=16,511). ** P<0.01 (two-sided Wilcoxon rank sum test).


( A ) Boxplots indicating the median , quartiles and 1 . 5 time the interquartile range of ratios of ATAC - seq signal for the indicated group of peaks , comparing the indicated genotypes or treatments . Core promoter peaks are < 300 nt from a TSS ; Extended promoter are more than 300 nt but < 2 Kb for a TSS ; Gene body peaks overlap transcription units , and Intergenic do not . Each group of peaks was compared to the intergenic peaks . In addition , for each group of peaks , the peaks within 1 Mb of Reno1 where compared to peaks further away . P - values computed using two - sided Wilcoxon rank sum test , and shown only for cases where P < 0 . 05 . Number of data points ( from left to right ) : 39 ; 10 , 777 ; 11 ; 3 , 550 ; 17 ; 14 , 380 ; 18 ; 20 , 930 . ( B ) Changes in ATAC - seq signal for peaks assigned as falling within core or extended promoters , of genes in the indicated group ( up / down - regulated by at least 30 % and with adjusted P < 0 . 05 ) in cells with Reno1 ( left ) or Bahcc1 ( right ) KD . P - values computed using two sided Wilcoxon rank sum test .

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(A) Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ratios of ATAC-seq signal for the indicated group of peaks, comparing the indicated genotypes or treatments. Core promoter peaks are <300 nt from a TSS; Extended promoter are more than 300 nt but <2 Kb for a TSS; Gene body peaks overlap transcription units, and Intergenic do not. Each group of peaks was compared to the intergenic peaks. In addition, for each group of peaks, the peaks within 1 Mb of Reno1 where compared to peaks further away. P-values computed using two-sided Wilcoxon rank sum test, and shown only for cases where P<0.05. Number of data points (from left to right): 39; 10,777; 11; 3,550; 17; 14,380; 18; 20,930. (B) Changes in ATAC-seq signal for peaks assigned as falling within core or extended promoters, of genes in the indicated group (up/down-regulated by at least 30% and with adjusted P<0.05) in cells with Reno1 (left) or Bahcc1 (right) KD. P-values computed using two sided Wilcoxon rank sum test.


( C ) Top : Boxplots indicating the median , quartiles and 1 . 5 time the interquartile range of changes in ATAC - seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation , classified according to the underlying chromatin state in mouse ES cells ; Bottom : Boxplots indicating the median , quartiles and 1 . 5 time the interquartile range of ChIP - seq coverage of the indicated chromatin marks in ENCODE mouse ES data in the peak regions . Number of peaks in each group is indicated at the bottom .

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(C) Top: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of changes in ATAC-seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation, classified according to the underlying chromatin state in mouse ES cells; Bottom: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ChIP-seq coverage of the indicated chromatin marks in ENCODE mouse ES data in the peak regions. Number of peaks in each group is indicated at the bottom.


( D ) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells ( H3K4me3 is from ENCODE project and Fam60a from ( Streubel et al . , 2017 ) ) . Coefficient and P - value computed using Spearman ' s correlation . n = 49 , 722 peaks .

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(D) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells (H3K4me3 is from ENCODE project and Fam60a from (Streubel et al., 2017)). Coefficient and P-value computed using Spearman's correlation. n=49,722 peaks.


( A ) CRISPR - guided AID ∆ C protein could support CSR in Aicda - / - CH12F3 cells . Left , CRISPR - guided AID is schematically illustrated with each component listed . Right , CRISPR - guided AID initiated CSR levels to IgA . Blue arrows represent multiple CRISPR recognition sites . The number in the parentheses indicates CRISPR recognition sites in each S region . Three biological replicates are plotted by dots and mean with standard deviation ( SD ) . Data information : For panels A one - way ANOVA followed by Dunnett ' s multiple comparisons test was used for significance assessment . For all panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(A) CRISPR-guided AID∆C protein could support CSR in Aicda-/- CH12F3 cells. Left, CRISPR-guided AID is schematically illustrated with each component listed. Right, CRISPR-guided AID initiated CSR levels to IgA. Blue arrows represent multiple CRISPR recognition sites. The number in the parentheses indicates CRISPR recognition sites in each S region. Three biological replicates are plotted by dots and mean with standard deviation (SD). Data information: For panels A one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( B ) CRISPR - guided AID generated breaks at the S region in Aicda - / - CH12F3 cells were quantitatively detected by PEM - seq . Top , schematic illustration shows the bait SaCas9 site at Iγ3 region and CRISPR - guided AID variants targeting sites at S regions . Bottom , percentages of translocation junctions between Iγ3 and Sμ or Sα regions are plotted as mean with SD of three biological replicates in the bar plot . Data information : For panels B , one - way ANOVA followed by Dunnett ' s multiple comparisons test was used for significance assessment . For all panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(B) CRISPR-guided AID generated breaks at the S region in Aicda-/- CH12F3 cells were quantitatively detected by PEM-seq. Top, schematic illustration shows the bait SaCas9 site at Iγ3 region and CRISPR-guided AID variants targeting sites at S regions. Bottom, percentages of translocation junctions between Iγ3 and Sμ or Sα regions are plotted as mean with SD of three biological replicates in the bar plot. Data information: For panels B, one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( C ) AID - AIDE58Q dimer proteins were expressed in AID - deficient CSR - activated B cells , AID dimer is schematically illustrated on top and CSR levels to IgG1 are shown . Three biological replicates are plotted by dots and mean with SD is shown . Data information : For panels C , one - way ANOVA followed by Dunnett ' s multiple comparisons test was used for significance assessment . For all panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(C) AID-AIDE58Q dimer proteins were expressed in AID-deficient CSR-activated B cells, AID dimer is schematically illustrated on top and CSR levels to IgG1 are shown. Three biological replicates are plotted by dots and mean with SD is shown. Data information: For panels C, one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( D ) CSR levels to IgA in Aicda - / - CH12F3 - miniS cells with indicated AID variants or CRISPR - guided AID variants . Levels of CSR to IgA are plotted and mean with SD of three to six biological replicates . Data information : Unpaired two - tailed Student ' s t - test was performed for panel D . For all panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(D) CSR levels to IgA in Aicda-/- CH12F3-miniS cells with indicated AID variants or CRISPR-guided AID variants. Levels of CSR to IgA are plotted and mean with SD of three to six biological replicates. Data information: Unpaired two-tailed Student's t-test was performed for panel D. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( E ) Mutation profiles of miniS region in Ung - / - Msh2 - / - Aicda - / - miniS cell lines complemented with indicated AID . Mutation frequency at each nucleotide along the 756 - bp miniS region is plotted as a bar graph with green error bars , representing mean with standard error of the mean ( SEM ) of three biological replicates . Positions of AID - preferred AGCT and other RGYW motifs are marked with orange and yellow respectively under each plot .

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(E) Mutation profiles of miniS region in Ung-/-Msh2-/-Aicda-/- miniS cell lines complemented with indicated AID. Mutation frequency at each nucleotide along the 756-bp miniS region is plotted as a bar graph with green error bars, representing mean with standard error of the mean (SEM) of three biological replicates. Positions of AID-preferred AGCT and other RGYW motifs are marked with orange and yellow respectively under each plot.


( F ) Mutation profiles of miniS region in Ung - / - Msh2 - / - Aicda - / - miniS cell lines expressing CRISPR - guided AID variants . Mutation frequency at each nucleotide is plotted as mean with SEM of three biological replicates in the bar graph with green error bar . Plots are labeled as in ( B ) . The sgRNA targeting position is indicated with blue lines at the bottom .

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(F) Mutation profiles of miniS region in Ung-/-Msh2-/-Aicda-/- miniS cell lines expressing CRISPR-guided AID variants. Mutation frequency at each nucleotide is plotted as mean with SEM of three biological replicates in the bar graph with green error bar. Plots are labeled as in (B). The sgRNA targeting position is indicated with blue lines at the bottom.


( A ) Representative images showing the localization of AID ( green ) , AIDR190X ( green ) , and endogenous IgH loci ( red ) in nucleofected Aicda - / - CH12F3 cells . The IgH loci are visualized using Sμ - gRNA and indicated by white arrows . An illustration of CRISPR - Sirius IgH locus visualization is shown on top . Scale bar , 5 µm .

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(A) Representative images showing the localization of AID (green), AIDR190X (green), and endogenous IgH loci (red) in nucleofected Aicda-/- CH12F3 cells. The IgH loci are visualized using Sμ-gRNA and indicated by white arrows. An illustration of CRISPR-Sirius IgH locus visualization is shown on top. Scale bar, 5 µm.


( B ) OptoDroplet formation in CH12F3 cells . Schematic illustration of light - induced optoDroplet system is depicted on top . AID variants were tagged to the N terminus of the optoDroplet construct and blue light was employed to promote the formation of puncta three times followed by imaging each time . Representative images after the third blue light activation in Aicda - / - CH12F3 cells with the indicated AID variants are shown at the bottom . White dashed line and green line depict the shapes of nucleus and nucleolus based on Hoechst staining or GFP - FBL , respectively . Scale bar , 5 µm .

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(B) OptoDroplet formation in CH12F3 cells. Schematic illustration of light-induced optoDroplet system is depicted on top. AID variants were tagged to the N terminus of the optoDroplet construct and blue light was employed to promote the formation of puncta three times followed by imaging each time. Representative images after the third blue light activation in Aicda-/- CH12F3 cells with the indicated AID variants are shown at the bottom. White dashed line and green line depict the shapes of nucleus and nucleolus based on Hoechst staining or GFP-FBL, respectively. Scale bar, 5 µm.


( C ) Representative images showing optoDroplet formation of indicated AID variants in HEK293T cells . CRM1 inhibitor ( CRM1i ) was used for selective trapping of AID in nuclei . Scale bar , 5 µm .

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(C) Representative images showing optoDroplet formation of indicated AID variants in HEK293T cells. CRM1 inhibitor (CRM1i) was used for selective trapping of AID in nuclei. Scale bar, 5 µm.


( D ) The fluorescence intensity of indicated AID variants in each HEK293T cell nucleus was measured and normalized relative to the fluorescence intensity of the whole cell . Data are shown as box plots . Values between lower quartile and upper quartile are represented by box ranges , a horizontal line within the box represents the median , and whisker extends from the minimum value to the maximum value . " + " indicates the mean level . Each dot indicates one cell , and more than 20 cells represent two biological replicate samples were counted . One - way ANOVA followed by Dunnett ' s multiple comparisons test was performed . * * * * , p < 0 . 0001 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(D) The fluorescence intensity of indicated AID variants in each HEK293T cell nucleus was measured and normalized relative to the fluorescence intensity of the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell, and more than 20 cells represent two biological replicate samples were counted. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ****, p<0.0001; *, p<0.05; ns, p>0.05.


( E ) Intensity recovery lines for optoAID ∆ C droplets in FRAP experiment in HEK293T cells . Bleaching occurs at t = 0 s . The background - subtracted fluorescence intensities are normalized by pre - bleach values . The mean with SD of 10 pairs of optoAIDR190X and 10 pairs of optoAIDcry is shown , respectively .

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(E) Intensity recovery lines for optoAID∆C droplets in FRAP experiment in HEK293T cells. Bleaching occurs at t = 0 s. The background-subtracted fluorescence intensities are normalized by pre-bleach values. The mean with SD of 10 pairs of optoAIDR190X and 10 pairs of optoAIDcry is shown, respectively.


( F ) Formation of puncta in primary B cells complemented with AID - FUS - GFP or AID - FUSm - GFP . FUSm contains 13 mutations on either Y or R residue . Left : Representative images showing cellular localization of AID - FUS - GFP and AID - FUSm - GFP with and without CRM1 inhibitor ( CRM1i ) treatment in Aicda - / - primary B cells . Scale bar , 5 µm . Right : Quantification of the number of cells with condensed puncta ( 3 biological replicates ) . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 .

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

(F) Formation of puncta in primary B cells complemented with AID-FUS-GFP or AID-FUSm-GFP. FUSm contains 13 mutations on either Y or R residue. Left: Representative images showing cellular localization of AID-FUS-GFP and AID-FUSm-GFP with and without CRM1 inhibitor (CRM1i) treatment in Aicda-/- primary B cells. Scale bar, 5 µm. Right: Quantification of the number of cells with condensed puncta (3 biological replicates). Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( G ) CSR levels to IgG1 in activated Aicda - / - primary B cells complemented with the indicated protein . Left , representative flow cytometry plots measuring CSR to IgG1 . Co - expressed GFP was used to indicate the infected fractions . Right , the mean with SD of three biological replicates is shown in the bar plot . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 .

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(G) CSR levels to IgG1 in activated Aicda-/- primary B cells complemented with the indicated protein. Left, representative flow cytometry plots measuring CSR to IgG1. Co-expressed GFP was used to indicate the infected fractions. Right, the mean with SD of three biological replicates is shown in the bar plot. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( A ) Representative immunofluorescence images of endogenous AID in the presence of ectopically - expressed EV , AID , AIDR190X , or AIDcry in cytokine - stimulated CH12F3 cells . EV , empty vector control . An antibody against AID C terminus ( 185 - 198 aa ) detected both endogenous and ectopically - expressed full - length AID . The nucleolus is indicated by co - nucleofected mCherry - FBL . The white dashed line depicts the shape of the nucleus based on Hoechst staining . Arrows indicate the location of puncta formed by endogenous AID . The images were taken by the N - SIM microscope and processed via N - SIM software . A middle slice of the images was shown . Scale bar , 5 µm .

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

(A) Representative immunofluorescence images of endogenous AID in the presence of ectopically-expressed EV, AID, AIDR190X, or AIDcry in cytokine-stimulated CH12F3 cells. EV, empty vector control. An antibody against AID C terminus (185-198 aa) detected both endogenous and ectopically-expressed full-length AID. The nucleolus is indicated by co-nucleofected mCherry-FBL. The white dashed line depicts the shape of the nucleus based on Hoechst staining. Arrows indicate the location of puncta formed by endogenous AID. The images were taken by the N-SIM microscope and processed via N-SIM software. A middle slice of the images was shown. Scale bar, 5 µm.


( B ) Co - condensation of AID and AIDR190X in the nucleus of CH12F3 cells in the early G1 phase . Top : experimental layout . GFP - tagged wild - type AID ( green ) and mCherry - tagged AID variants ( red ) were co - nucleofected in Aicda - / - CH12F3 cells . The resulting cells were arrested with nocodazole for 12 hours after transfection . Six hours later , nocodazole was removed from the system for 1 . 5 hours before imaging . Bottom left : representative images showing subcellular localization of indicated AID variants . The white dashed line depicts the shape of the nucleus based on Hoechst staining . The white arrow indicates the location of the co - condensed puncta . Scale bar , 5 µm . Bottom right : plot profiles of the images . Cyto : cytoplasm , Nuc : nucleus .

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

(B) Co-condensation of AID and AIDR190X in the nucleus of CH12F3 cells in the early G1 phase. Top: experimental layout. GFP-tagged wild-type AID (green) and mCherry-tagged AID variants (red) were co-nucleofected in Aicda-/- CH12F3 cells. The resulting cells were arrested with nocodazole for 12 hours after transfection. Six hours later, nocodazole was removed from the system for 1.5 hours before imaging. Bottom left: representative images showing subcellular localization of indicated AID variants. The white dashed line depicts the shape of the nucleus based on Hoechst staining. The white arrow indicates the location of the co-condensed puncta. Scale bar, 5 µm. Bottom right: plot profiles of the images. Cyto: cytoplasm, Nuc: nucleus.


( C ) Quantification of the relative fluorescence intensity of AID - GFP in the diluted phase of cells shown in ( B ) . The fluorescence intensity of AID - GFP in the diluted nuclear phase was normalized by the total intensity from the whole cell . Data are shown as box plots . Values between lower quartile and upper quartile are represented by box ranges , a horizontal line within the box represents the median , and whisker extends from the minimum value to the maximum value . " + " indicates the mean level . Each dot indicates one cell ( n = 25 ) , and representative result from three biological replicates were shown . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 .

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(C) Quantification of the relative fluorescence intensity of AID-GFP in the diluted phase of cells shown in (B). The fluorescence intensity of AID-GFP in the diluted nuclear phase was normalized by the total intensity from the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell (n = 25), and representative result from three biological replicates were shown. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( D ) Dynamics of trapping of wild - type AID in optoAIDR190X condensates . Top , representative fluorescence images at different time points . Dashed circles indicate the co - condensed puncta formation of AID - GFP . Scale bar , 5 µm . Bottom , a summary of 12 observed cells is shown as mean ± SD in a bar graph . Percentages of optoAIDR190X puncta with trapped wild - type AID are plotted for the second and third blue - light activations , and the colored dot indicates one cell .

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(D) Dynamics of trapping of wild-type AID in optoAIDR190X condensates. Top, representative fluorescence images at different time points. Dashed circles indicate the co-condensed puncta formation of AID-GFP. Scale bar, 5 µm. Bottom, a summary of 12 observed cells is shown as mean ±SD in a bar graph. Percentages of optoAIDR190X puncta with trapped wild-type AID are plotted for the second and third blue-light activations, and the colored dot indicates one cell.


( B ) The optoDroplet formation of indicated AID mutants along with light stimulation . Top , the procedure of optoDroplet assay is illustrated with indicated light stimulation ( blue arrow ) and image acquisition ( black arrow ) time points . Bottom , line plots show quantification of cells with optoDroplet formation as mean ± SD at each acquisition time point . The total acquired cell numbers of each genotype are indicated in parentheses . Significance assessment between AIDR190X and AIDR190X - 4R was shown at each time point . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 .

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(B) The optoDroplet formation of indicated AID mutants along with light stimulation. Top, the procedure of optoDroplet assay is illustrated with indicated light stimulation (blue arrow) and image acquisition (black arrow) time points. Bottom, line plots show quantification of cells with optoDroplet formation as mean ± SD at each acquisition time point. The total acquired cell numbers of each genotype are indicated in parentheses. Significance assessment between AIDR190X and AIDR190X-4R was shown at each time point. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( C ) CSR levels of ectopically - expressed AID variants in AID - proficient CH12F3 cells . Left , representative flow cytometry plots of CSR to IgA . Co - expressed GFP indicates the infected cell fractions . Right , the CSR mean with SD of three biological replicates is shown in the bar plot . ( D ) CSR levels of ectopically - expressed AID variants in CSR - activated B cells . Left , representative flow cytometry plots of CSR to IgG1 . Right , the bar plot shows the CSR mean with SD of four biological replicates . Data information : One - way ANOVA followed by Dunnett ' s multiple comparisons test was performed for ( C ) and ( D ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 .

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

(C) CSR levels of ectopically-expressed AID variants in AID-proficient CH12F3 cells. Left, representative flow cytometry plots of CSR to IgA. Co-expressed GFP indicates the infected cell fractions. Right, the CSR mean with SD of three biological replicates is shown in the bar plot. (D) CSR levels of ectopically-expressed AID variants in CSR-activated B cells. Left, representative flow cytometry plots of CSR to IgG1. Right, the bar plot shows the CSR mean with SD of four biological replicates. Data information: One-way ANOVA followed by Dunnett's multiple comparisons test was performed for (C) and (D). ***, p<0.001; **, p<0.01; *, p<0.05.


( A ) Left , representative images of MBP - GFP - AID in the presence of 10 % PEG8000 . Scale bar , 10 µm and 2 µm , respectively . Right , the schematic protein sequences .

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(A) Left, representative images of MBP-GFP-AID in the presence of 10% PEG8000. Scale bar, 10 µm and 2 µm, respectively. Right, the schematic protein sequences.


( B ) Left , representative images of MBP - GFP - AIDcry + CTT or MBP - GFP - AIDcry in the presence 10 % PEG8000 at the indicated protein concentrations . Scale bar , 10 µm . Right , sizes of MBP - GFP - AIDcry + CTT or MBP - GFP - AIDcry protein condensates are plotted as box plots with three technical replicates . The box represents values between the lower quartile and the upper quartile , a horizontal line within the box represents the median , and whisker extends from the minimum value to the maximum value . All points are listed and mean is indicated by " + " . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 .

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(B) Left, representative images of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry in the presence 10% PEG8000 at the indicated protein concentrations. Scale bar, 10 µm. Right, sizes of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry protein condensates are plotted as box plots with three technical replicates. The box represents values between the lower quartile and the upper quartile, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. All points are listed and mean is indicated by "+". Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( A ) AID deamination assay was performed in vitro . Left , a schematic diagram of deamination activity assay . Right , the deamination reaction with indicated protein combinations was processed and visualized . " + " : 0 . 25 μM of indicated protein ; " + + " : 0 . 5 μM of indicated protein ; " - " : not added .

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(A) AID deamination assay was performed in vitro. Left, a schematic diagram of deamination activity assay. Right, the deamination reaction with indicated protein combinations was processed and visualized. "+": 0.25 μM of indicated protein; "++": 0.5 μM of indicated protein; "-": not added.


( B ) CSR to IgA induced by AID variants in CH12F3 cells . Left , representative flow cytometry plots . Right , the CSR mean with SD of three biological replicates is shown in the bar plot . One - way ANOVA followed by Dunnett ' s multiple comparisons test was performed . * * * , p < 0 . 001 ; * * , p < 0 . 01 .

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(B) CSR to IgA induced by AID variants in CH12F3 cells. Left, representative flow cytometry plots. Right, the CSR mean with SD of three biological replicates is shown in the bar plot. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ***, p<0.001; **, p<0.01.


( C ) Representative images of the co - condensation of MBP - mCherry - AIDcry and MBP - GFP - AID . The protein concentrations and molecular ratios are indicated . Scale bar , 10 µm .

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(C) Representative images of the co-condensation of MBP-mCherry-AIDcry and MBP-GFP-AID. The protein concentrations and molecular ratios are indicated. Scale bar, 10 µm.


( A ) Microcomputed tomography ( micro - CT ) images of the proximal femur from 12 - week - old male WT and Sigmar1 gKO mice . Scale bars , 1 mm . ( B ) Quantification of bone volume per tissue volume ( BV / TV ) , trabecular number ( Tb . N ) , trabecular separation ( Tb . Sp ) , trabecular thickness ( Tb . Th ) , cortical region BV / TV ( Ct . BV / TV ) and cortical thickness ( Ct . Th , mm ) ( n = 5 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - tailed Student ' s t - test ( B was used for statistical analysis .

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(A) Microcomputed tomography (micro-CT) images of the proximal femur from 12-week-old male WT and Sigmar1 gKO mice. Scale bars, 1 mm. (B) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), cortical region BV/TV (Ct. BV/TV) and cortical thickness (Ct. Th, mm) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test (B was used for statistical analysis.


( C ) Coronal images of the fifth lumbar spine . Scale Bars , 1 mm . ( D ) Quantification of trabecular bone parameters of lumbar spine ( n = 5 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - tailed Student ' s t - test D , was used for statistical analysis .

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(C) Coronal images of the fifth lumbar spine. Scale Bars, 1 mm. (D) Quantification of trabecular bone parameters of lumbar spine (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test D, was used for statistical analysis.


( E ) TRAP staining of tibias from male WT and Sigmar1 gKO mice . Scale bars , 50 μm . ( F and G ) Quantification of osteoclast number per bone surface ( N . Oc / BS ) and percentage of osteoclast surface per bone surface ( Oc . S / BS ) ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - tailed Student ' s t - test F - G , was used for statistical analysis .

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(E) TRAP staining of tibias from male WT and Sigmar1 gKO mice. Scale bars, 50 μm. (F and G) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test F-G, was used for statistical analysis.


( H and I ) Representative images and quantitative analysis of calcein double labeling . Scale bars , 20 μm . ( n = 6 biological replicates ) Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - tailed Student ' s t - test , I was used for statistical analysis .

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(H and I) Representative images and quantitative analysis of calcein double labeling. Scale bars, 20 μm. (n = 6 biological replicates) Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test , I was used for statistical analysis.