( A ) Micrographs of triple mutant hAPP after 15min internalization ( red ) , BACE1 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main images . Scale bar , 5μm . ( B ) Quantification of the proportion of BACE1 that co - localized with hAPP after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % BACE co - localized with hAPP . N = 3 independent experiments , each performed in duplicate ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( C ) Quantification of the proportion of internalized hAPP that co - localized with BACE1 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized hAPP co - localized with BACE1 . N = 3 independent experiments , each performed in duplicate ; * , P < 0 . 05 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) .

O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD B-ORGANISM O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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) Micrographs of triple mutant hAPP after 15min internalization (red), BACE1 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main images. Scale bar, 5μm. (B) Quantification of the proportion of BACE1 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % BACE co-localized with hAPP. N=3 independent experiments, each performed in duplicate; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (C) Quantification of the proportion of internalized hAPP that co-localized with BACE1 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with BACE1. N=3 independent experiments, each performed in duplicate; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test).


( D ) Micrographs of p75NTR after 15min internalization ( red ) , Rab11 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main merged images . Scale bar , 5μm . ( E ) Quantification of the proportion of Rab11 that co - localized with p75NTR after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % Rab11 co - localized with p75NTR . n = 4 ; * , P < 0 . 05 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( F ) Quantification of the proportion of internalized p75NTR that co - localized with Rab11 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized p75NTR co - localized with Rab11 . n = 4 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) .

O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O 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) Micrographs of p75NTR after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (E) Quantification of the proportion of Rab11 that co-localized with p75NTR after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with p75NTR. n=4; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (F) Quantification of the proportion of internalized p75NTR that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized p75NTR co-localized with Rab11. n=4; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test).


( G ) Micrographs of triple mutant hAPP after 15min internalization ( red ) , Rab11 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main merged images . Scale bar , 5μm . ( H ) Quantification of the proportion of Rab11 that co - localized with hAPP after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % Rab11 co - localized with hAPP . n = 4 ; * , P < 0 . 05 vs . WT ( one - way ANOVA followed by post hoc test ) . ( I ) Quantification of the proportion of internalized hAPP that co - localized with Rab11 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized hAPP co - localized with Rab11 . n = 4 ; * , P < 0 . 05 vs . WT ( one - way ANOVA followed by post hoc test ) .

O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD B-ORGANISM O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Micrographs of triple mutant hAPP after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (H) Quantification of the proportion of Rab11 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with hAPP. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test). (I) Quantification of the proportion of internalized hAPP that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with Rab11. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test).


F smFISH of Arglu1 pre - mRNA ( magenta ) and Arglu1 sisRNA ( green ) . Merged dots ( white , arrowheads ) represent Arglu1 pre - mRNA transcript . G Quantification of Arglu1 pre - mRNA and Arglu1 sisRNA copy number from ( F ) . ( n = 50 cells ) Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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F smFISH of Arglu1 pre-mRNA (magenta) and Arglu1 sisRNA (green). Merged dots (white, arrowheads) represent Arglu1 pre-mRNA transcript. G Quantification of Arglu1 pre-mRNA and Arglu1 sisRNA copy number from (F). (n = 50 cells) Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.


I RT - PCR of Arglu1 sisRNA , spliced mRNA levels and actin as control after AMOs treatment .

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I RT-PCR of Arglu1 sisRNA, spliced mRNA levels and actin as control after AMOs treatment.


G Left : Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells . Right : Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis ( left ) . ( n = 3 biological replicates ) .

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G Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells. Right: Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).


M Left : RT - PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs . Right : Quantification of relative spliced AXIN1a / AXIN1b expression ratio normalized to actin from RT - PCR analysis ( left ) . ( n = 3 biological replicates ) .

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M Left: RT-PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs. Right: Quantification of relative spliced AXIN1a/AXIN1b expression ratio normalized to actin from RT-PCR analysis (left). (n = 3 biological replicates).


B Left : Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells . Right : Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis ( left ) . ( n = 3 biological replicates ) .

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B Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells. Right: Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).


D Left : smFISH of Arglu1 sisRNA ( green ) coupled with immunostaining of ARGLU1 protein ( red ) shown in z - stack . Right : Zoom in z - slice on the boxed area showing co - localization ( arrowheads ) of Arglu1 sisRNA with ARGLU1 protein . The intensity plot shows the signal quantification and co - localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow . E Left : smFISH of Arglu1 pre - mRNA ( green ) coupled with ARGLU1 protein ( red ) immunostaining shown in z - stack . Right : Zoom in z - slice on the boxed area showing co - localization ( arrowhead ) of Arglu1 pre - mRNA with ARGLU1 protein . The intensity plot shows the signal quantification and co - localization of Arglu1 pre - mRNA and ARGLU1 protein in the direction of the white arrow .

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D Left: smFISH of Arglu1 sisRNA (green) coupled with immunostaining of ARGLU1 protein (red) shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowheads) of Arglu1 sisRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow. E Left: smFISH of Arglu1 pre-mRNA (green) coupled with ARGLU1 protein (red) immunostaining shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowhead) of Arglu1 pre-mRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 pre-mRNA and ARGLU1 protein in the direction of the white arrow.


F smFISH of Arglu1 sisRNA ( green ) and Arglu1 pre - mRNA ( magenta ) in 1 day Arglu1 sisRNA KD cells vs control cells . G , H Quantification of Arglu1 sisRNA and Arglu1 pre - mRNA copy number from ( F ) . ( n = 90 cells ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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F smFISH of Arglu1 sisRNA (green) and Arglu1 pre-mRNA (magenta) in 1 day Arglu1 sisRNA KD cells vs control cells. G, H Quantification of Arglu1 sisRNA and Arglu1 pre-mRNA copy number from (F). (n = 90 cells). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.


A Immunostaining of MED1 ( magenta ) and SC35 ( red ) with transiently expressed mCherry - ARGLU1 protein ( green ) in MCF - 7 cells . Zoom in of boxed area and the intensity plot shows the co - localization and signal quantification of mCherry - ARGLU1 protein , MED1 and SC35 in the direction of the white arrow .

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A Immunostaining of MED1 (magenta) and SC35 (red) with transiently expressed mCherry-ARGLU1 protein (green) in MCF-7 cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of mCherry-ARGLU1 protein, MED1 and SC35 in the direction of the white arrow.


B Confocal images of GFP - SRSF2 ( green , control ) and mCherry - ARGLU1 protein ( red ) at the pre - bleach , bleach , and post - bleach time points in FRAP analysis . The boxed area showing the speckle that was being bleached . C Fluorescence intensities of the nuclear speckle over time in GFP - SRSF2 ( green , control ) and mCherry - ARGLU1 protein ( red ) FRAP analysis . ( n = 3 separate nuclear speckles ) .

O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O

B Confocal images of GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) at the pre-bleach, bleach, and post-bleach time points in FRAP analysis. The boxed area showing the speckle that was being bleached. C Fluorescence intensities of the nuclear speckle over time in GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) FRAP analysis. (n = 3 separate nuclear speckles).


D Immunostaining of ARGLU1 protein ( green ) and SC35 ( red ) in 1 day Arglu1 sisRNA KD cells vs control cells . Zoom in of boxed area and the intensity plot shows the co - localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow .

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D Immunostaining of ARGLU1 protein (green) and SC35 (red) in 1 day Arglu1 sisRNA KD cells vs control cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow.


E Confocal images of 125 nM ARGLU1 - GFP protein ( green ) only and ARGLU1 - GFP protein added with 50 nM IVT RNA ( magenta ) , of either Arglu1 intron 2 with UCE ( Intron 2 WT ) or IVT Arglu1 intron without UCE ( Intron 2 ΔUCE ) . White arrowhead indicates ARGLU1 - GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1 - GFP droplet not incorporated with IVT Intron 2 WT . F Quantification of total ARGLU1 - GFP droplets from ( E ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . G Quantification of average ARGLU1 - GFP droplets size with / without incorporation of IVT intron 2 WT from ( E , ARGLU1 - GFP + Intron 2 WT ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O 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 Confocal images of 125 nM ARGLU1-GFP protein (green) only and ARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either Arglu1 intron 2 with UCE (Intron 2 WT) or IVT Arglu1 intron without UCE (Intron 2 ΔUCE). White arrowhead indicates ARGLU1-GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1-GFP droplet not incorporated with IVT Intron 2 WT. F Quantification of total ARGLU1-GFP droplets from (E). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. G Quantification of average ARGLU1-GFP droplets size with/without incorporation of IVT intron 2 WT from (E, ARGLU1-GFP + Intron 2 WT). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.


A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells . Samples with rabbit - IgG antibodies were used as control . ARGLU1 antibody # 1 , but not # 2 , worked for IP .

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A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells. Samples with rabbit-IgG antibodies were used as control. ARGLU1 antibody #1, but not #2, worked for IP.


F Left : Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control ( sibling control , MTD - GAL4 / CyO ) ovaries . Right : Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis . ( n = 3 biological replicates )

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

F Left: Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control (sibling control, MTD-GAL4/CyO) ovaries. Right: Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis. (n = 3 biological replicates)


G Left : Immunostaining of dARGLU1 protein ( green ) and VASA ( red ) in dArglu1 sisRNA KD germaria vs control ( sibling control , MTD - GAL4 / TM3 ) germaria . The asterisk ( * ) marks the anterior of the germarium . Right : Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

G Left: Immunostaining of dARGLU1 protein (green) and VASA (red) in dArglu1 sisRNA KD germaria vs control (sibling control, MTD-GAL4/TM3) germaria. The asterisk (*) marks the anterior of the germarium. Right: Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow.


Confocal images of 125 nM dARGLU1 - GFP protein ( green ) only and dARGLU1 - GFP protein added with 50 nM IVT RNA ( magenta ) , of either dArglu1 intron 2 until the 3 ' end of sisRNA ( dArglu1 intron 2 ) or IVT dArglu1 intron 2 starting after 3 ' end of sisRNA ( dArglu1 pre - mRNA ) . White arrowheads indicate dARGLU1 - GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1 - GFP droplets not incorporated with IVT dArglu1 intron 2 . J Quantification of total dARGLU1 - GFP droplets from ( I ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . K Quantification of average dARGLU1 - GFP droplets size with / without incorporation of IVT dArglu1 intron 2 from ( I , dARGLU1 - GFP + dArglu1 intron 2 ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images of 125 nM dARGLU1-GFP protein (green) only and dARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either dArglu1 intron 2 until the 3' end of sisRNA (dArglu1 intron 2) or IVT dArglu1 intron 2 starting after 3' end of sisRNA (dArglu1 pre-mRNA). White arrowheads indicate dARGLU1-GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1-GFP droplets not incorporated with IVT dArglu1 intron 2. J Quantification of total dARGLU1-GFP droplets from (I). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. K Quantification of average dARGLU1-GFP droplets size with/without incorporation of IVT dArglu1 intron 2 from (I, dARGLU1-GFP + dArglu1 intron 2). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. D


A Confocal images of 125 nM d / hARGLU1 - GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT ( magenta ) or dArglu1 intron 2 ( red ) . White arrowheads indicate free IVT RNA dots that are not co - localized with the d / hARGLU1 - GFP droplets . B Quantification of non co - localized RNA dots from ( A , white arrowheads ) . ( n = 16 biological replicates ) . C , D Quantification of total hARGLU1 - GFP droplets and the average droplet size with / without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from ( A ) . ( n = 3 biological replicates ) . E , F Quantification of total dARGLU1 - GFP droplets and the average droplet size with / without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from ( A ) . ( n = 3 biological replicates ) .

O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O 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 Confocal images of 125 nM d/hARGLU1-GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT (magenta) or dArglu1 intron 2 (red). White arrowheads indicate free IVT RNA dots that are not co-localized with the d/hARGLU1-GFP droplets. B Quantification of non co-localized RNA dots from (A, white arrowheads). (n = 16 biological replicates). C, D Quantification of total hARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates). E, F Quantification of total dARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).


Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein of the huCoV - 19 / WH01 ( B ) and Beta variants ( D )

O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM O O O O

Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein of the huCoV-19/WH01 (B) and Beta variants (D)


Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein the ability to neutralize these viruses in vitro ( C and E ) .

O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O

Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein the ability to neutralize these viruses in vitro (C and E).


Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime and boost antibodies to S protein ( huCoV - 19 / WH01 ) following priming with a recombinant S protein in adjuvant ( F )

O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-GENEPROD I-GENEPROD O O O O O

Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime and boost antibodies to S protein (huCoV-19/WH01) following priming with a recombinant S protein in adjuvant (F)


Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro ( G ) .

O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-ORGANISM O B-ORGANISM O O O O O O O

Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro (G).


Finally , three doses of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 induce antibodies that cross react with S proteins from the huCoV - 19 / WH01 , Beta , and Delta variants in mice ( H ) ,

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O B-ORGANISM O O B-ORGANISM O O O O

Finally, three doses of the universal SARS-CoV-2 DNA vaccine OC-2.4 induce antibodies that cross react with S proteins from the huCoV-19/WH01, Beta, and Delta variants in mice (H),


four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro ( I ) .

O O O O O O O O O B-SUBCELLULAR O O O O B-ORGANISM O B-EXP_ASSAY O O B-ORGANISM O B-ORGANISM O O O O O O O

four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro (I).


A - D ) T cell responses as determined by ELISpot in Balb / c mice immunized three times with either spike protein in QS21 adjuvant ( rS / QS21 ; A ) , 50µg universal SARS - CoV - 2 vaccine OC - 2 . 4 ( B ) , primed with rS / QS21 and boosted with two doses of OC - 2 . 4 ( C ) , or empty pVAX plasmid ( D ) , using in vivo electroporation . Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ producing ( spot forming ) cells ( SFCs ) per million splenocytes . Each color indicate an individual mice .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O B-ORGANISM O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O B-ORGANISM O

A-D) T cell responses as determined by ELISpot in Balb/c mice immunized three times with either spike protein in QS21 adjuvant (rS/QS21; A), 50µg universal SARS-CoV-2 vaccine OC-2.4 (B), primed with rS/QS21 and boosted with two doses of OC-2.4 (C), or empty pVAX plasmid (D), using in vivo electroporation. Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ producing (spot forming) cells (SFCs) per million splenocytes. Each color indicate an individual mice.


E - H ) Also shown is analysis of T cell responses in New Zeeland White rabbits , which were immunized four times with either vehicle only ( E ) , or 84µg ( F ) or 840 µg ( G ) universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 using in vivo electroporation . Peripheral blood mononuclear cells ( PBMCs ) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ SFCs per million PBMCs . The dotted line indicates the 50 SFC cut off . Also shown ( H ) is the cumulated IFNγ SFCs per million PBMCs to the RBD , M and N peptide pools added together for each group of immunized rabbits ( 84µg DNA , 840µg DNA , and vehicle only ) at each time point ( day ) . The numbers on the x - axis of graphs A ) to D ) indicates the number of the respective peptide pool ( WH1 variant ) covering a part of the indicated protein .

O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY B-ORGANISM O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O

E-H) Also shown is analysis of T cell responses in New Zeeland White rabbits, which were immunized four times with either vehicle only (E), or 84µg (F) or 840 µg (G) universal SARS-CoV-2 DNA vaccine OC-2.4 using in vivo electroporation. Peripheral blood mononuclear cells (PBMCs) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ SFCs per million PBMCs. The dotted line indicates the 50 SFC cut off. Also shown (H) is the cumulated IFNγ SFCs per million PBMCs to the RBD, M and N peptide pools added together for each group of immunized rabbits (84µg DNA, 840µg DNA, and vehicle only) at each time point (day). The numbers on the x-axis of graphs A) to D) indicates the number of the respective peptide pool (WH1 variant) covering a part of the indicated protein.


Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues ( b )

O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-TISSUE I-TISSUE O O O O

Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues (b)


Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss ( c ) .

O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss (c).


D ) Three doses of respective vaccine also protect against viral replication in the nose , lungs and spleen . Values have been given as cycle times ( CT ) , where lower values indicate a higher viral load . Statistical comparisons in the graph are shown with lines with one asterisc indicating p < 0 , 05 and two asterics p < 0 ,

O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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) Three doses of respective vaccine also protect against viral replication in the nose, lungs and spleen. Values have been given as cycle times (CT), where lower values indicate a higher viral load. Statistical comparisons in the graph are shown with lines with one asterisc indicating p<0,05 and two asterics p<0,01


B , RMD assays performed after DSBs have been generated by sgRNAs / Cas9 . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is indicated . U2OS cells carrying a single copy of the EGFP - SSA reporter ( 3427 - 17 ) were transfected with sgRNA / Cas9 plasmids , and the percentage of GFP - positive cells was determined ( left ) . U2OS cells transfected with the indicated reporters EGFP - SSA - 17 / 6 bp , EGFP - SSA - 0 . 3 kb , EGFP - SSA - 0 . 6 kb and EGFP - SSA - 1 . 0 kb were pooled as a population after selection of the drug marker on the reporters , followed by sgRNA / Cas9 cleavage ( right ) .

O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O 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-GENEPROD O O O O O

B, RMD assays performed after DSBs have been generated by sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. U2OS cells carrying a single copy of the EGFP-SSA reporter (3427-17) were transfected with sgRNA/Cas9 plasmids, and the percentage of GFP-positive cells was determined (left). U2OS cells transfected with the indicated reporters EGFP-SSA-17/6 bp, EGFP-SSA-0.3 kb, EGFP-SSA-0.6 kb and EGFP-SSA-1.0 kb were pooled as a population after selection of the drug marker on the reporters, followed by sgRNA/Cas9 cleavage (right).


C , RMD is strongly induced when a DSB is generated close to one repeat . The U2OS ( EGFP - SSA - 3427 - 17 ) cell line carrying a single copy of the reporter was transfected with sgRNA / Cas9 plasmids as indicated , and the percentage of GFP - positive cells was determined .

O O O O O O O O B-SUBCELLULAR O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O

C, RMD is strongly induced when a DSB is generated close to one repeat. The U2OS (EGFP-SSA-3427-17) cell line carrying a single copy of the reporter was transfected with sgRNA/Cas9 plasmids as indicated, and the percentage of GFP-positive cells was determined.


Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when POLD3 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control .

O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O

Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when POLD3 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.


Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when RAD52 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control .

Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD52 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.


G , Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when RAD51 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control .

O O O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O

G, Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD51 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.


B , RMD efficiency is decreased when the distance from a DSB to one repeat is increased . RMD assays were performed in U2OS ( EGFP - SSA - 3427 - 17 ) cells using the indicated sgRNAs / Cas9 . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is shown .

O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O

B, RMD efficiency is decreased when the distance from a DSB to one repeat is increased. RMD assays were performed in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is shown.


C and D , Knockdown of POLD3 impairs RMD when a DSB is close to one repeat . RMD frequency was determined in U2OS ( EGFP - SSA - 3427 - 17 ) cells using the indicated sgRNAs / Cas9 with or without depletion of POLD3 by lentivirus - expressed shRNA . Cells infected with a lentiviral empty vector were used as control . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is indicated . The knockdown level of POLD3 was shown by Western blot , and KU70 was used as the loading control .

O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O

C and D, Knockdown of POLD3 impairs RMD when a DSB is close to one repeat. RMD frequency was determined in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9 with or without depletion of POLD3 by lentivirus-expressed shRNA. Cells infected with a lentiviral empty vector were used as control. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.


A , Cyclin E overexpression results in increased RMD when the DSB is close to one repeat . The U2OS ( EGFP - SSA - 3427 - 3 ) cell line carrying Tet - on - cyclin E plasmid was used , and RMD frequency was determined with or without induction of cyclin E expression by doxycycline ( DOX , 0 . 5 µg / mL ) , which was added 24 hours before transfection of the indicated sgRNAs / Cas9 . Cyclin E expression levels were validated by Western blot , using KU70 as the loading control .

O O B-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O

A, Cyclin E overexpression results in increased RMD when the DSB is close to one repeat. The U2OS (EGFP-SSA-3427-3) cell line carrying Tet-on-cyclin E plasmid was used, and RMD frequency was determined with or without induction of cyclin E expression by doxycycline (DOX, 0.5 µg/mL), which was added 24 hours before transfection of the indicated sgRNAs/Cas9. Cyclin E expression levels were validated by Western blot, using KU70 as the loading control.


B , ATR is required for BIR / RMD upon cyclin E overexpression . RMD frequency was determined in U2OS ( EGFP - SSA - 3427 - 3 ) cells infected with lentivirus - expressed ATR shRNA or the control vector ( Ctrl ) with and without induction of cyclin E expression by DOX ( 0 . 5 µg / ml ) after R29 sgRNA / Cas9 cleavage . ATR knockdown was shown by Western blot with KU70 as the loading control .

O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O

B, ATR is required for BIR/RMD upon cyclin E overexpression. RMD frequency was determined in U2OS (EGFP-SSA-3427-3) cells infected with lentivirus-expressed ATR shRNA or the control vector (Ctrl) with and without induction of cyclin E expression by DOX (0.5 µg/ml) after R29 sgRNA/Cas9 cleavage. ATR knockdown was shown by Western blot with KU70 as the loading control.


D , RMD is stimulated when Cas9 - D10A is used to create a nick near the repeat . The repair frequencies of the U2OS ( MMEJ ) reporter cell line ( left ) and U2OS ( EGFP - SSA - 3427 - 17 ) cell line ( right ) were determined after expression of the indicated sgRNAs / Cas9 and sgRNAs / Cas9 - D10A . The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated ( right bottom ) .

O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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, RMD is stimulated when Cas9-D10A is used to create a nick near the repeat. The repair frequencies of the U2OS (MMEJ) reporter cell line (left) and U2OS (EGFP-SSA-3427-17) cell line (right) were determined after expression of the indicated sgRNAs/Cas9 and sgRNAs/Cas9-D10A. The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated (right bottom).


E , RMD induced by Cas9 - D10A is dependent on POLD3 . U2OS ( EGFP - SSA - 3427 - 17 ) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs / Cas9 - D10A . The distance between sgRNA / Cas9 - D10A cleavage sites and the left and right repeat sequences is indicated at the bottom . The knockdown level of POLD3 was shown by Western blot , and KU70 was used as the loading control .

O O O O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O

E, RMD induced by Cas9-D10A is dependent on POLD3. U2OS (EGFP-SSA-3427-17) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs/Cas9-D10A. The distance between sgRNA/Cas9-D10A cleavage sites and the left and right repeat sequences is indicated at the bottom. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.


F , p27 suppresses BIR / RMD and SSA / RMD . U2OS ( EGFP - SSA - 3427 - 3 ) cells carrying the Tet - on - cyclin E allele were infected with and without p27 lentiviral viruses . RMD was determined before and after induction of cyclin E overexpression by DOX ( 0 . 5 µg / mL ) added 24 hours before transfection with the indicated sgRNAs / Cas9 . Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6 .

O O B-GENEPROD O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O

F, p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP-SSA-3427-3) cells carrying the Tet-on-cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 µg/mL) added 24 hours before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.


B , Impact of the 5 ' DSB / repeat distance on RMD frequency when the 3 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

B, Impact of the 5' DSB/repeat distance on RMD frequency when the 3' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


C , Long - range RMD is dependent on POLD3 when one DSB is close to a repeat . mES cells carrying the RMD - GFP reporter were transfected with nontargeting siRNA ( Ctrl ) or a pool of POLD3 siRNAs ( siPOLD3 ) 24 hours before for the first time , and with transfection of sgRNAs / Cas9 the second time . The graphs show the frequency of GFP + cells normalized to the transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

C, Long-range RMD is dependent on POLD3 when one DSB is close to a repeat. mES cells carrying the RMD-GFP reporter were transfected with nontargeting siRNA (Ctrl) or a pool of POLD3 siRNAs (siPOLD3) 24 hours before for the first time, and with transfection of sgRNAs/Cas9 the second time. The graphs show the frequency of GFP+ cells normalized to the transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


D and E , Impact of the 3 ' DSB / repeat distance on RMD frequency when the 5 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

D and E, Impact of the 3' DSB/repeat distance on RMD frequency when the 5' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


F , RMD frequency of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 , with the frequency of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

F, RMD frequency of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9, with the frequency of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


G , The roles of POLD3 , RAD51 and RAD52 in RMD of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 after POLD3 ( left ) or RAD51 ( middle ) was depleted by siRNA or RAD52 was knocked out ( right ) . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

G, The roles of POLD3, RAD51 and RAD52 in RMD of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9 after POLD3 (left) or RAD51 (middle) was depleted by siRNA or RAD52 was knocked out (right). The distance between the sgRNA targeting sites and the repeats is indicated.


SSA / RMD and BIR / SSA are both dependent on ATM . SSA / RMD and BIR / SSA frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA /

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SSA/RMD and BIR/SSA are both dependent on ATM. SSA/RMD and BIR/SSA frequencies in U2OS (EGFP-SSA-3427-17) cells were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9


BIR / SSA are both dependent on ATM . BIR / RMD in mES cells carrying the RMD - GFP reporter were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA / Cas9 or in ATM KO mES cells carrying the RMD - GFP reporter ( right ) .

O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O O O O

BIR/SSA are both dependent on ATM. BIR/RMD in mES cells carrying the RMD-GFP reporter were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 or in ATM KO mES cells carrying the RMD-GFP reporter (right).


Dependence of RMD on H2AX . SSA / RMD and BIR / RMD frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined using the indicated sgRNA / Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by

O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O B-GENEPROD O O O O O O

Dependence of RMD on H2AX. SSA/RMD and BIR/RMD frequencies in U2OS (EGFP-SSA-3427-17) cells were determined using the indicated sgRNA/Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by qPCR


D , Dependence of RMD on H2AX . BIR / RMD in mES cells carrying the RMD - GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO

O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O

D, Dependence of RMD on H2AX. BIR/RMD in mES cells carrying the RMD-GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO cells


A , Frequency of long - range RMD is increased by an additional DSB downstream of the 3 ' repeat . Illustrations show the GFP - RMD reporter , with sgRNAs / Cas9 cleavage sites indicated as red arrows ( left ) . The frequency of BIR / SSA in mES cells carrying the RMD - GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs / Cas9 ( right ) .

O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O

A, Frequency of long-range RMD is increased by an additional DSB downstream of the 3' repeat. Illustrations show the GFP-RMD reporter, with sgRNAs/Cas9 cleavage sites indicated as red arrows (left). The frequency of BIR/SSA in mES cells carrying the RMD-GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs/Cas9 (right).


B and C , The effects of RAD52 , POLD3 , ATM and H2AX inactivation on BIR / RMD with cleavage at L17 / D10k . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter was determined using the sgRNAs / Cas9 at L17 / D10k in RAD52 KO cells or after POLD3 depletion by siRNA ( B ) , or after treatment with ATM inhibitor ( ku55933 , 15mM ) or H2AX depletion by siRNA ( C ) .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O

B and C, The effects of RAD52, POLD3, ATM and H2AX inactivation on BIR/RMD with cleavage at L17/D10k. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter was determined using the sgRNAs/Cas9 at L17/D10k in RAD52 KO cells or after POLD3 depletion by siRNA (B), or after treatment with ATM inhibitor (ku55933, 15mM) or H2AX depletion by siRNA (C).


A , Long - range BIR / RMD with identical repeats or divergent repeats . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter with identical repeats ( 0 % ) or repeats with 1 % and 3 % divergence was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k .

O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O 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, Long-range BIR/RMD with identical repeats or divergent repeats. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter with identical repeats (0%) or repeats with 1% and 3% divergence was determined after cleavage at L17/R3.3k or L17/R28.4k.


B and C , The effect of MSH2 on BIR / RMD when repeat sequences are divergent . The frequency of BIR / RMD was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k in mES cells carrying the RMD - GFP reporter with 1 % and 3 % divergent repeats , and with and without MSH2 KO ( B ) or with MSH2 KO and POLD3 depletion by siRNAs ( C ) . The MSH2 KO was validated by Western blot , using KU70 as the loading control as shown in ( B ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , two - tailed non - paired t - test .

O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C, The effect of MSH2 on BIR/RMD when repeat sequences are divergent. The frequency of BIR/RMD was determined after cleavage at L17/R3.3k or L17/R28.4k in mES cells carrying the RMD-GFP reporter with 1% and 3% divergent repeats, and with and without MSH2 KO (B) or with MSH2 KO and POLD3 depletion by siRNAs (C). The MSH2 KO was validated by Western blot, using KU70 as the loading control as shown in (B). Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, two-tailed non-paired t-test.


B . Western blot analysis of RAB6A / A ' and RAB6B protein levels in WT , Emx1 - Cre ; RAB6loxP / loxP ( RAB6A KO ) , RAB6B - / - ( RAB6B KO ) and Emx1 - Cre ; RAB6AloxP / loxP ; RAB6B - / - ( RAB6A / B dKO ) E15 . 5 cortical extracts .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-TISSUE O O

B. Western blot analysis of RAB6A/A' and RAB6B protein levels in WT, Emx1-Cre; RAB6loxP/loxP (RAB6A KO), RAB6B-/- (RAB6B KO) and Emx1-Cre; RAB6AloxP/loxP; RAB6B-/- (RAB6A/B dKO) E15.5 cortical extracts.


A . PAX6 staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . B . Percentage of PAX6 + cells located in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 4282 cells from N = 6 brains . RAB6A / B dKO : 1241 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD .

O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O

A. PAX6 staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. B. Percentage of PAX6+ cells located in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 4282 cells from N=6 brains. RAB6A/B dKO: 1241 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.


C . Phospho - Vimentin ( p - Vim ) staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . D . Percentage p - VIM + cells dividing ectopically , in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 1713 cells from N = 6 brains . RAB6A / B dKO : 506 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD .

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O

C. Phospho-Vimentin (p-Vim) staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. D. Percentage p-VIM+ cells dividing ectopically, in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 1713 cells from N=6 brains. RAB6A/B dKO: 506 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.


G . Electroporation of RAB6AloxP / loxP ; RAB6B - / - E14 . 5 embryos with mCherry ( control ) or mCherry + CRE ( RAB6A / B dKO ) and live imaging of delamination events at E17 . 5 . H . Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17 . 5 . mCherry : N = 69 cells from 5 movies . mCherry + CRE : N = 52 cells from 4 movies . Fisher ' s exact test , * p ≤ 0 . 05 . Data information : All error bars indicate SD .

O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Electroporation of RAB6AloxP/loxP; RAB6B-/- E14.5 embryos with mCherry (control) or mCherry + CRE (RAB6A/B dKO) and live imaging of delamination events at E17.5. H. Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17.5. mCherry: N=69 cells from 5 movies. mCherry + CRE: N=52 cells from 4 movies. Fisher's exact test, *p ≤ 0.05. Data information: All error bars indicate SD.


A . Localization of the Golgi apparatus ( ManII - GFP ) and the centrosome ( 𝛾 - tubulin ) in E15 . 5 mCherry - electroporated radial glial cell . The Golgi apparatus is localized basally , away from the centrosome . Scale bar = 5µm . B . Average distance between the apical - most part of the Golgi apparatus and the apical surface in aRG cells . N = 224 cells from 3 independent brains .

O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-TISSUE O

A. Localization of the Golgi apparatus (ManII-GFP) and the centrosome (𝛾-tubulin) in E15.5 mCherry-electroporated radial glial cell. The Golgi apparatus is localized basally, away from the centrosome. Scale bar = 5µm. B. Average distance between the apical-most part of the Golgi apparatus and the apical surface in aRG cells. N=224 cells from 3 independent brains.


E . Live imaging of GFP - RAB6A in aRG cells at E15 . 5 allows tracking of individual RAB6A + vesicles in situ , from the basal Golgi apparatus towards the apical surface . Scale bar = 5µm . Distance = 5µm , time = 30 seconds . F . RAB6A + vesicle directionality in apical processes of aRG cells over one - minute movies . G . Relative time spent by RAB6A + vesicles in apical , basal or static phases . H . Velocity of apically and basally moving RAB6A + vesicles . Data information : ( F , G , H ) N = 388 vesicles from 30 cells . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Live imaging of GFP-RAB6A in aRG cells at E15.5 allows tracking of individual RAB6A+ vesicles in situ, from the basal Golgi apparatus towards the apical surface. Scale bar = 5µm. Distance = 5µm, time = 30 seconds. F. RAB6A+ vesicle directionality in apical processes of aRG cells over one-minute movies. G. Relative time spent by RAB6A+ vesicles in apical, basal or static phases. H. Velocity of apically and basally moving RAB6A+ vesicles. Data information: (F, G, H) N= 388 vesicles from 30 cells. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.


I Live imaging of GFP - RAB6A in control , dynarrestin - treated and CC1 - p150 - expressing aRG cells at E15 . 5 . Scale bars = 5µm . Distance = 5µm , time = 30 seconds . J . Number of RAB6A + vesicles in the apical process of DMSO and dynarrestin - treated mouse aRG cells . K . Relative time spent by RAB6A + vesicles in apical movement phase , in DMSO and dynarrestin - treated mouse aRG cells . L . Number of RAB6A + vesicles in the apical process of mCherry and CC1 - p150 - expressing aRG cells . M . Relative time spent by RAB6A + vesicles in apical movement phase , in mCherry and CC1 - p150 - expressing aRG cells . DMSO treatment slightly affected RAB6A dynamics , as compared to mCherry control . Data information : ( J , K , L , M ) 216 vesicles from N = 11 cells for DMSO , 145 vesicles from N = 25 cells for dynarrestin , 173 vesicles from N = 17 cells for mCherry control , 71 vesicles from N = 15 cells for CC1 - p150 . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Live imaging of GFP-RAB6A in control, dynarrestin-treated and CC1-p150-expressing aRG cells at E15.5. Scale bars = 5µm. Distance = 5µm, time = 30 seconds. J. Number of RAB6A+ vesicles in the apical process of DMSO and dynarrestin-treated mouse aRG cells. K. Relative time spent by RAB6A+ vesicles in apical movement phase, in DMSO and dynarrestin-treated mouse aRG cells. L. Number of RAB6A+ vesicles in the apical process of mCherry and CC1-p150-expressing aRG cells. M. Relative time spent by RAB6A+ vesicles in apical movement phase, in mCherry and CC1-p150-expressing aRG cells. DMSO treatment slightly affected RAB6A dynamics, as compared to mCherry control. Data information: (J, K, L, M) 216 vesicles from N=11 cells for DMSO, 145 vesicles from N=25 cells for dynarrestin, 173 vesicles from N=17 cells for mCherry control, 71 vesicles from N=15 cells for CC1-p150. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.


C . RUSH assay for CRB3 - GFP in control ( mcherry ) and dynactin - inhibited radial glial cells ( CC1 - p150 - dsRed ) , electroporated at E . 15 . 5 and imaged at E16 . 5 . Scale bar = 5µm . D . CRB3 localization at the apical surface upon release . E . Percentage of cells with 100 % of CRB3 signal at the apical surface upon release . Data information : ( D , E ) mCherry ( control ) : N = 3 brains per timepoint ( 361 cells total ) . CC1 - p150 : N = 3 brains per timepoint ( 2 brains for T0 ) ( 268 cells total ) . Fisher ' s exact test and Benjamini - Hochberg procedure , * * * p ≤ 0 . 001 . All error bars indicate SD .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O 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. RUSH assay for CRB3-GFP in control (mcherry) and dynactin-inhibited radial glial cells (CC1-p150-dsRed), electroporated at E.15.5 and imaged at E16.5. Scale bar = 5µm. D. CRB3 localization at the apical surface upon release. E. Percentage of cells with 100% of CRB3 signal at the apical surface upon release. Data information: (D, E) mCherry (control): N= 3 brains per timepoint (361 cells total). CC1-p150: N=3 brains per timepoint (2 brains for T0) (268 cells total). Fisher's exact test and Benjamini-Hochberg procedure, *** p ≤ 0.001. All error bars indicate SD.


A . PAX6 and phospho - Histone 3 ( p - H3 ) staining in WT and LIS1 KO E12 . 5 brains . Cortices were subdivided into 5 bins of equal size along the radial axis . Scale bar = 25µm . B . Percentage of PH3 + / PAX6 + cells located above the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1192 cells from N = 6 brains . LIS1 KO : 589 cells from N = 3 brains . Data information : ( B , Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O B-TISSUE O O 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. PAX6 and phospho-Histone 3 (p-H3) staining in WT and LIS1 KO E12.5 brains. Cortices were subdivided into 5 bins of equal size along the radial axis. Scale bar = 25µm. B. Percentage of PH3+/PAX6+ cells located above the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1192 cells from N=6 brains. LIS1 KO: 589 cells from N=3 brains. Data information: (B, Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


C . Phospho - Vimentin ( p - VIM ) staining in WT and LIS1 KO E12 . 5 brains . Scale bar = 25µm . D . Percentage p - VIM + cells dividing ectopically , away from the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1056 cells from N = 6 brains . LIS1 KO : 879 cells from N = 3 brains . Data information : D ) Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O B-TISSUE O 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. Phospho-Vimentin (p-VIM) staining in WT and LIS1 KO E12.5 brains. Scale bar = 25µm. D. Percentage p-VIM+ cells dividing ectopically, away from the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1056 cells from N=6 brains. LIS1 KO: 879 cells from N=3 brains. Data information: D) Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


A . CRB3 and PALS1 staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm .

O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O

A. CRB3 and PALS1 staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.


B . Quantification of CRB3 staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 13 brains ) , LIS1 KO ( E12 . 5 , N = 4 brains ) ) and RAB6A / B dKO ( E15 . 5 , N = 4 brains ) . Data information : ( B , Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of CRB3 staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=13 brains), LIS1 KO (E12.5, N=4 brains)) and RAB6A/B dKO (E15.5, N=4 brains). Data information: (B, Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.


C . N - Cadherin staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O

C. N-Cadherin staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.


D . Quantification of N - Cadh staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 6 brains ) , LIS1 KO ( E12 . 5 , N = 3 brains ) and RAB6A / B dKO ( E15 . 5 , N = 3 brains ) . Data information : D ) Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of N-Cadh staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=6 brains), LIS1 KO (E12.5, N=3 brains) and RAB6A/B dKO (E15.5, N=3 brains). Data information: D) Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.


( 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

O O O O O B-EXP_ASSAY B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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

O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O O

(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 .

O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O

(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 .

O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(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 ,

O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(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 ,

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-GENEPROD O B-GENEPROD B-TISSUE O O O O O O O O O O O O

(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 .

O O O O O O O O B-TISSUE B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD 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-TISSUE O O O O O O O O

(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 ,

O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY B-CELL_TYPE O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-GENEPROD O O O B-EXP_ASSAY B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O B-GENEPROD O O O O O O O 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-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(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 .

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O O 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-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O 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) 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 .

O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O 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-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O

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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O 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-GENEPROD O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O

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 .

O O O O O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O O O O O O O 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-EXP_ASSAY O

(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 ) .

O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-TISSUE O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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) 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).