a , Apg7 and Apg3 are necessary for attachment of Apg8 to PE . Δapg cell lysates were subjected to SDS - PAGE containing 6 M urea , and immunoblotted with anti - Apg8 antibody .

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a, Apg7 and Apg3 are necessary for attachment of Apg8 to PE. Δapg cell lysates were subjected to SDS-PAGE containing 6 M urea, and immunoblotted with anti-Apg8 antibody.


b , Autophagic activity in apg3 mutants . Wild - type , Δapg3 and apg3C243A cells were grown in rich medium ( open bar ) and subjected to nitrogen starvation for 4 h ( filled bar ) . The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity . The apg3C243A mutant showed defective autophagy .

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b, Autophagic activity in apg3 mutants. Wild-type, Δapg3 and apg3C243A cells were grown in rich medium (open bar) and subjected to nitrogen starvation for 4 h (filled bar). The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity. The apg3C243A mutant showed defective autophagy.


D , Affinity capture of proteins by OC43p and OC43 - SCR bound magnetic beads . Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies . OC43p - SCR - coated and uncoated beads ( Beads ) were used to control non - specific binding . Spinal cords were isolated from male ( M ) and female ( F ) rats ( n = 2 / group ) . Input protein lysates ( right side , in duplicates ) served as loading controls . Approximate molecular weights ( in kDa ) are indicated by arrows .

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D, Affinity capture of proteins by OC43p and OC43-SCR bound magnetic beads. Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies. OC43p-SCR-coated and uncoated beads (Beads) were used to control non-specific binding. Spinal cords were isolated from male (M) and female (F) rats (n=2/group). Input protein lysates (right side, in duplicates) served as loading controls. Approximate molecular weights (in kDa) are indicated by arrows.


a , b , Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells ( a ) ( n = 5 / 4 ) and for autophagy proteins in primary intestinal epithelial cell scrapings ( b ) ( n = 3 ) .

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a, b, Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells (a) (n = 5/4) and for autophagy proteins in primary intestinal epithelial cell scrapings (b) (n = 3).


c , Transmission electron microscopy ( TEM ) of crypts . Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr ; IEC hypomorphic Paneth cells .

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

c, Transmission electron microscopy (TEM) of crypts. Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr;IEC hypomorphic Paneth cells.


d , e , Crypt showing GFP - LC3 punctae ( d ) , quantified in e ( n = 10 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . Scale bar , 5 µm .

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d, e, Crypt showing GFP-LC3 punctae (d), quantified in e (n = 10; unpaired Student's t-test; mean ± s.e.m.). Scale bar, 5 µm.


f , g , Immunoblot of silenced MODE - K cells ( f ) and primary intestinal epithelial cell scrapings ( g ) for the PERK - eIF2α branch ( n = 3 ) .

O O O O O O O O O O O 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, g, Immunoblot of silenced MODE-K cells (f) and primary intestinal epithelial cell scrapings (g) for the PERK-eIF2α branch (n = 3).


j , GFP - LC3 punctae per crypt after treatment with tamoxifen , and vehicle or salubrinal ( n = 10 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) .

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j, GFP-LC3 punctae per crypt after treatment with tamoxifen, and vehicle or salubrinal (n = 10; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).


k , Enteritis histology score after salubrinal and tamoxifen co - treatment ( n = 12 / 14 / 13 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent three ( a , f , g ) or two ( c , e , h , i ) independent experiments . * P 0 . 05 , * * * P 0 . 001 .

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k, Enteritis histology score after salubrinal and tamoxifen co-treatment (n = 12/14/13; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P 0.05, ***P 0.001.


a , TEM of crypts of Lieberkühn ( n = 2 ) . Scale bars , 2 µm .

a, TEM of crypts of Lieberkühn (n = 2). Scale bars, 2 µm.


b , Representative haematoxylin and eosin stainings . Note transmural inflammation extending through muscularis propria ( white arrow ) into serosa ( black arrow ) in Atg7 / Xbp1Dgr ; IEC and Atg16l1 / Xbp1Dgr ; IEC mice scored in c and e . Scale bars , 50 µm , except for lower two panels ( 10 µm ) . c , Enteritis histology score ( n = 26 / 12 / 18 / 27 ; 10 - 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . e , Enteritis histology score ( n = 11 ; 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . NS , not significant .

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b, Representative haematoxylin and eosin stainings. Note transmural inflammation extending through muscularis propria (white arrow) into serosa (black arrow) in Atg7/Xbp1Dgr;IEC and Atg16l1/Xbp1Dgr;IEC mice scored in c and e. Scale bars, 50 µm, except for lower two panels (10 µm). c, Enteritis histology score (n = 26/12/18/27; 10-18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).e, Enteritis histology score (n = 11; 18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). NS, not significant.


d , CryptTEM in indicated genotypes ( n = 2 ) . Scale bars , 2 µm .

d, CryptTEM in indicated genotypes (n = 2). Scale bars, 2 µm.


f , Xbp1 mRNA splicing ( Xbp1u , unspliced ; Xbp1s , spliced ) of crypts ; densitometry in g ( n = 7 ; unpaired Student ' s t - test ; mean ± s . e . m . ) .

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f, Xbp1 mRNA splicing (Xbp1u, unspliced; Xbp1s, spliced) of crypts; densitometry in g (n = 7; unpaired Student's t-test; mean ± s.e.m.).


h , GRP78 ( green ) immunofluorescence , white arrows indicate GRP78 + crypts ( DAPI , blue ; n = 5 ) . Scale bars , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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

h, GRP78 (green) immunofluorescence, white arrows indicate GRP78+ crypts (DAPI, blue; n = 5). Scale bars, 10 µm. *P 0.05, **P 0.01, ***P 0.001.


a , shCtrl or shXbp1 MODE - K cells were co - silenced for Atg16l1 ( siAtg16l1 ) or with scrambled siRNA ( siCtrl ) , and analysed by flow cytometry for annexin V and propidium iodide ( PI ; n = 3 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI; n = 3; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).


b , Immunoblot of primary intestinal epithelial cell scrapings ( n = 3 ) .

b, Immunoblot of primary intestinal epithelial cell scrapings (n = 3).


c , Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE - K cells after TNF stimulation .

c, Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE-K cells after TNF stimulation.


d , TUNEL + intestinal epithelial cells ( IECs ) per 100 crypts after BAY11 - 7082 or vehicle treatment ( n = 3 / 4 / 4 ; one - way ANOVA with post - hoc Holm ' s - corrected unpaired Student ' s t - test ; mean ± s . e . m . ) . e , Enteritis histology score of mice treated with BAY11 - 7082 or vehicle ( n = 10 / 10 / 9 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) .

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d, TUNEL+ intestinal epithelial cells (IECs) per 100 crypts after BAY11-7082 or vehicle treatment (n = 3/4/4; one-way ANOVA with post-hoc Holm's-corrected unpaired Student's t-test; mean ± s.e.m.). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n = 10/10/9; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).


f , Immunoblot of intestinal epithelial cell scrapings for ( p - ) IRE1α ; after IRE1α ; immunoprecipitation ( IP ) . beta - actin , loading control of whole lysates .

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f, Immunoblot of intestinal epithelial cell scrapings for (p-)IRE1α; after IRE1α; immunoprecipitation (IP). beta-actin, loading control of whole lysates.


g , Immunoblot of primary intestinal epithelial cell scrapings ( n = 4 ) .

g, Immunoblot of primary intestinal epithelial cell scrapings (n = 4).


h , i , Enteritis histology score of indicated genotypes ( h , n = 15 / 16 / 14 / 15 ; i , n = 5 / 10 / 12 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) .

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h, i, Enteritis histology score of indicated genotypes (h, n = 15/16/14/15; i, n = 5/10/12; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).


j , Enteritis histology score of specific pathogen - free ( SPF ) and germ free ( GF ) housed mice ( n = 10 / 9 / 7 / 7 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . k , Representative images of p - IκBα immunoreactivity under conditions as in j ( n = 4 ) . Scale bars , 20 µm . Results represent four ( f ) , three ( c ) or two ( a , b ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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

j, Enteritis histology score of specific pathogen-free (SPF) and germ free (GF) housed mice (n = 10/9/7/7; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). k, Representative images of p-IκBα immunoreactivity under conditions as in j (n = 4). Scale bars, 20 µm. Results represent four (f), three (c) or two (a, b) independent experiments. *P  0.05, **P  0.01, ***P  0.001.


a , Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days ( n = 3 ) . b , Enteritis histology score for experiment as in a ( n = 4 ; median shown ; Mann - Whitney U - test ) .

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a, Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days (n = 3). b, Enteritis histology score for experiment as in a (n = 4; median shown; Mann-Whitney U-test).


c , Representative images of EYFP - Rosa26 / D6 - cre + / - reporter mice and EYFP - Rosa26 ( controls ) . Co - localization of Defa6 Cre - driven EYFP expression ( yellow ) with lysozyme - expressing Paneth cells ( red ; n = 3 ) . DAPI , blue ; scale bars , 50 µm .

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c, Representative images of EYFP-Rosa26/D6-cre+/- reporter mice and EYFP-Rosa26 (controls). Co-localization of Defa6 Cre-driven EYFP expression (yellow) with lysozyme-expressing Paneth cells (red; n = 3). DAPI, blue; scale bars, 50 µm.


d , Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr ; PC and wild - type controls ( n = 2 ) .

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d, Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr;PC and wild-type controls (n = 2).


e , f , Representative confocal images of lysozyme ( green ) expressing Paneth cells ( e ) with quantification of crypts with indicated number of lysozyme + granulated dots in f ( n = 5 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . DAPI , blue ; scale bars , 10 µm .

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e, f, Representative confocal images of lysozyme (green) expressing Paneth cells (e) with quantification of crypts with indicated number of lysozyme+ granulated dots in f (n = 5; unpaired Student's t-test; mean ± s.e.m.). DAPI, blue; scale bars, 10 µm.


g , Immunohistochemistry for p - eIF2α ( n = 3 ) . Scale bars , 20 µm .

g, Immunohistochemistry for p-eIF2α (n = 3). Scale bars, 20 µm.


h , Representative haematoxylin and eosin images of Xbp1Dgr ; PC and wild - type mice scored in i . Scale bars , 50 µm . i , Enteritis scoring in Xbp1fl / fl ( WTfl ) , Defa6 - cre + ( WTcre ) and Defa6 - cre + ; Xbp1fl / fl ( Xbp1Dgr ; PC ) mice ( n = 21 / 26 / 29 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent two ( b , d ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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h, Representative haematoxylin and eosin images of Xbp1Dgr;PC and wild-type mice scored in i. Scale bars, 50 µm. i, Enteritis scoring in Xbp1fl/fl (WTfl), Defa6-cre+ (WTcre) and Defa6-cre+;Xbp1fl/fl (Xbp1Dgr;PC) mice (n = 21/26/29; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent two (b, d) independent experiments. *P 0.05, **P 0.01, ***P 0.001.


D . BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N . benthamiana leaves . Images were taken at 60 hpi . Scale bar = 20 μm .

D. BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N. benthamiana leaves. Images were taken at 60 hpi. Scale bar = 20 μm.


E . Co - IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM . N . benthamiana leaves were agroinfiltrated and treated with 2 mM 3 - MA at 48 hpi , and then for IP with anti - Flag beads 12 h later .

O O O O O O O O O O O O O O O O O O O O O O O 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. Co-IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM. N. benthamiana leaves were agroinfiltrated and treated with 2 mM 3-MA at 48 hpi, and then for IP with anti-Flag beads 12 h later.


Confocal microscopy of YFP - ATG8e - labeled autophagic bodies in root cells of Col - 0 / YFP - ATG8e and VISP1OE / YFP - ATG8e plants . 7 - day - old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark . Scale bar = 20 μm . Puncta numbers of YFP - ATG8e - labeled autophagic bodies per 10 cells in panel A . Data points represent means of three biological repeats . Error bars indicate SD . * * * p < 0 . 001 ( Student ' s t - test ) .

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Confocal microscopy of YFP-ATG8e-labeled autophagic bodies in root cells of Col-0/YFP-ATG8e and VISP1OE/YFP-ATG8e plants. 7-day-old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark. Scale bar = 20 μm.   Puncta numbers of YFP-ATG8e-labeled autophagic bodies per 10 cells in panel A. Data points represent means of three biological repeats. Error bars indicate SD. ***p < 0.001 (Student's t-test).


Western blotting analysis of accumulation of ATG8a and ATG8a - PE in leaves of 4 - week - old Col - 0 , VISP1OE , VISP1mUIM / OE and VISP1mARM / OE plants .

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Western blotting analysis of accumulation of ATG8a and ATG8a-PE in leaves of 4-week-old Col-0, VISP1OE, VISP1mUIM/OE and VISP1mARM/OE plants.


Representative TEM images of autophagic structures in inoculated leaves of Col - 0 and VISP1OE plants treated with mock buffer or CMV - 2blm infection at 4 dpi . The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling . Magenta arrowheads indicate autophagic bodies inside the vacuoles . Scale bar = 2 μm . Cp , chloroplast ; CW , cell wall ; V , vacuole . Autophagic structure numbers per 10 cells in panel F . Error bars indicate SD . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) .

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Representative TEM images of autophagic structures in inoculated leaves of Col-0 and VISP1OE plants treated with mock buffer or CMV-2blm infection at 4 dpi. The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling. Magenta arrowheads indicate autophagic bodies inside the vacuoles. Scale bar = 2 μm. Cp, chloroplast; CW, cell wall; V, vacuole.   Autophagic structure numbers per 10 cells in panel F. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).


Long - term symptoms of Col - 0 , VISP1OE1 , VISP1OE2 , rdr6 - 15 , and sgs3 - 1 plants inoculated with CMV - 2blm at 50 dpi . Scale bar = 2 cm .

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Long-term symptoms of Col-0, VISP1OE1, VISP1OE2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.


Northern blotting analyzing accumulation of genomic RNAs ( gRNAs ) in systemically leaves of Col - 0 , OE1 ( VISP1OE1 ) , OE2 ( VISP1OE2 ) , rdr6 - 15 , and sgs3 - 1 plants infected by CMV - 2blm at 14 dpi . Methylene blue - stained rRNA were used as loading controls . Northern blotting analyzing accumulation of RNA3 - derived vsiRNAs in the same samples as shown in panel D .

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

Northern blotting analyzing accumulation of genomic RNAs (gRNAs) in systemically leaves of Col-0, OE1 (VISP1OE1), OE2 (VISP1OE2), rdr6-15, and sgs3-1 plants infected by CMV-2blm at 14 dpi. Methylene blue-stained rRNA were used as loading controls.   Northern blotting analyzing accumulation of RNA3-derived   vsiRNAs in the same samples as shown in panel D.


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

Long-term systemic symptoms of Col-0, visp1-1, visp1-2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.


Northern blotting detecting accumulation of genomic RNAs and RNA3 - derived vsiRNAs in systemically infected leaves at 14 dpi . Relative accumulation ( RA ) of gRNA3 was calculated from band densities . Northern blotting analyzing accumulation of RNA3 - derived vsiRNAs in the same samples as shown in panel D . Bottom values represent relative accumulation ( RA ) of RNA3 - derived vsiRNAs .

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

Northern blotting detecting accumulation of genomic RNAs and RNA3-derived vsiRNAs in systemically infected leaves at 14 dpi. Relative accumulation (RA) of gRNA3 was calculated from band densities.   Northern blotting analyzing accumulation of RNA3-derived vsiRNAs in the same samples as shown in panel D. Bottom values represent relative accumulation (RA) of RNA3-derived vsiRNAs.


Co - immunoprecipitation analyses of the interactions between SGS3 , SGS3ΔCC , or SGS3CC and VISP1 or VISP1mARM . N . benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3 - MA at 48 hpi , and collected for IP with anti - Flag beads 12 h later .

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

Co-immunoprecipitation analyses of the interactions between SGS3, SGS3ΔCC, or SGS3CC and VISP1 or VISP1mARM. N. benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3-MA at 48 hpi, and collected for IP with anti-Flag beads 12 h later.


GST pull - down analysis of the in vitro interactions of SGS3CC with VISP1 , VISP1mARM or VISP1mUIM . GST - VISP1 , GST - VISP1mARM , GST - VISP1mUIM or GST were incubated with SGS3CC - His for IP with anti - GST beads , and detected by Western blotting with GST or His antibodies .

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

GST pull-down analysis of the in vitro interactions of SGS3CC with VISP1, VISP1mARM or VISP1mUIM. GST- VISP1, GST- VISP1mARM, GST- VISP1mUIM or GST were incubated with SGS3CC-His for IP with anti-GST beads, and detected by Western blotting with GST or His antibodies.


Confocal analysis of co - localization of CFP - NbATG8f - labelled autophagic bodies with the VISP1 - YN and SGS3 - YC bodies in N . benthamiana leaves . The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi . Scale bar = 20 μm .

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

Confocal analysis of co-localization of CFP-NbATG8f-labelled autophagic bodies with the VISP1-YN and SGS3-YC bodies in N. benthamiana leaves. The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi. Scale bar = 20 μm.


Confocal analysis of effect of VISP1 - Flag VISP1mARM - Flag , and VISP1mUIM - Flag on RDR6 - YN and SGS3 - YC formed bodies in N . benthamiana leaves at 60 hpi . EV : empty vector . Scale bar = 20 μm . Numbers of RDR6 - YN and SGS3 - YC formed bodies per 10 cells in E were used for quantification . Data points represent means of three biological repeats . Error bars indicate SD . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) .

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Confocal analysis of effect of VISP1-Flag VISP1mARM-Flag, and VISP1mUIM-Flag on RDR6-YN and SGS3-YC formed bodies in N. benthamiana leaves at 60 hpi. EV: empty vector. Scale bar = 20 μm.   Numbers of RDR6-YN and SGS3-YC formed bodies per 10 cells in E were used for quantification. Data points represent means of three biological repeats. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).


Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2 - week - old Col - 0 , VISP1OE , visp1 , and sgs3 - 1 Arabidopsis plants with anti - SGS3 antibodies . Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2 - week - old Col - 0 , VISP1OE , VISP1mUIM / OE , and VISP1mARM / OE Arabidopsis plants . Western blotting detecting accumulation of the endogenous SGS3 protein in 2 - week - old VISP1 - overexpressing Col - 0 , atg5 - 1 , and atg7 - 1 mutants .

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Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, visp1, and sgs3-1 Arabidopsis plants with anti-SGS3 antibodies.   Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, VISP1mUIM/OE, and VISP1mARM/OE Arabidopsis plants.   Western blotting detecting accumulation of the endogenous SGS3 protein in 2-week-old VISP1-overexpressing Col-0, atg5-1, and atg7-1 mutants.


Developmental phenotypes of rosette leaves of 5 - week - old Col - 0 , visp1 - 1 , visp1 - 2 , VISP1OE1 , VISP1OE2 , and sgs3 - 1 seedlings . Arrowheads indicate the 8th and 9th rosette leaves from the top leaves . Scale bar = 2 cm . The length / width ratios of the 8th and 9th rosette leaves of the seedlings shown in A . Every dot represents data from the length / width ratio of one leave ( n = 20 ) . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) .

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Developmental phenotypes of rosette leaves of 5-week-old Col-0, visp1-1, visp1-2, VISP1OE1, VISP1OE2, and sgs3-1 seedlings. Arrowheads indicate the 8th and 9th rosette leaves from the top leaves. Scale bar = 2 cm.   The length/width ratios of the 8th and 9th rosette leaves of the seedlings shown in A. Every dot represents data from the length/width ratio of one leave (n = 20). Letters indicate significant   differences (ANOVA, P < 0.05).


Northern blotting detecting accumulation of TAS1 - TasiR255 , TAS2 - TasiR1511 , TAS3 - TasiR5 ′ D8 , miR173 , and miR390 in leaves of Arabidopsis plants shown in panel A . Values represent relative accumulation ( RA ) of siRNAs and values in Col - 0 plants were set as 1 . rRNA served as loading controls .

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Northern blotting detecting accumulation of TAS1-TasiR255, TAS2-TasiR1511, TAS3-TasiR5′D8, miR173, and miR390 in leaves of Arabidopsis plants shown in panel A. Values represent relative accumulation (RA) of siRNAs and values in Col-0 plants were set as 1. rRNA served as loading controls.


( G ) t - SNE ( top ) and violin plots ( bottom ) showing the expression of selected signature genes in different cell clusters .

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(G) t-SNE (top) and violin plots (bottom) showing the expression of selected signature genes in different cell clusters.


( J ) Representative images of immunofluorescence of CCSC marker gene ( LGR5 ) and non - CCSC cluster signature genes in the xenografts harvested at different time points . Cluster T1 ( KRT19 ) , Cluster T2 ( HSPA1B ) , Cluster T3 ( Ki67 ) , Cluster T4 ( AGR2 ) , Cluster T5 ( ODC1 ) , and Cluster T6 ( ANXA1 ) were immunostained . Scale bar , 60 μm .

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(J) Representative images of immunofluorescence of CCSC marker gene (LGR5) and non-CCSC cluster signature genes in the xenografts harvested at different time points. Cluster T1 (KRT19), Cluster T2 (HSPA1B), Cluster T3 (Ki67), Cluster T4 (AGR2), Cluster T5 (ODC1), and Cluster T6 (ANXA1) were immunostained. Scale bar, 60 μm.


( K ) Quantification of ( J ) showing the dynamic change of cell clusters during the progression of CD133 + LGR5 + CCSC - formed xenografts . Error bars represent SEM of three xenografts . Three sections of each xenograft and 3 fields of each section were quantified .

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(K) Quantification of (J) showing the dynamic change of cell clusters during the progression of CD133+LGR5+ CCSC-formed xenografts. Error bars represent SEM of three xenografts. Three sections of each xenograft and 3 fields of each section were quantified.


( A ) Representative images of CD133 and LGR5 immunofluorescence in CCSC sphere cells bearing a Notch - GFP reporter . Scale bar , 60 μm . ( B ) Representative images of CD133 and LGR5 immunofluorescence in dividing CCSCs carrying a Notch - GFP reporter . Scale bar , 10 μm . ( C ) Representative time - lapse images of a Notch - GFP reporter during asymmetric division of CCSCs . Scale bar , 30 μm .

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(A) Representative images of CD133 and LGR5 immunofluorescence in CCSC sphere cells bearing a Notch-GFP reporter. Scale bar, 60 μm. (B) Representative images of CD133 and LGR5 immunofluorescence in dividing CCSCs carrying a Notch-GFP reporter. Scale bar, 10 μm. (C) Representative time-lapse images of a Notch-GFP reporter during asymmetric division of CCSCs. Scale bar, 30 μm.


( G ) t - SNE ( up ) and Violin plot ( bottom ) showing the expression of representative marker genes in each cluster .

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(G) t-SNE (up) and Violin plot (bottom) showing the expression of representative marker genes in each cluster.


( A ) Representative immunofluorescence images showing different cell clusters in CCSC spheres . Single CCSCs with high - Notch - activity ( Notch - GFP ) were seeded to grow into spheres and immunofluorescence was performed to identify cell clusters using representative marker genes . Scale bar , 60 μm . ( B ) Pair - cell assay showing the generation of the five cell clusters by CCSC asymmetric division . Scale bar , 60 μm .

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(A) Representative immunofluorescence images showing different cell clusters in CCSC spheres. Single CCSCs with high-Notch-activity (Notch-GFP) were seeded to grow into spheres and immunofluorescence was performed to identify cell clusters using representative marker genes. Scale bar, 60 μm. (B) Pair-cell assay showing the generation of the five cell clusters by CCSC asymmetric division. Scale bar, 60 μm.


( C ) Edu incorporation assay showing the proliferative potential of cluster C1 ( CCNE2 + ) and cluster C2 ( CCNB2 + ) cells in CCSC spheres . Scale bar , 60 μm . ( D ) Pair - cell assay showing that cluster C1 ( CCNE2 + ) and cluster C2 ( CCNB2 + ) cells immediately enter the second division . Scale bar , 100 μm .

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(C) Edu incorporation assay showing the proliferative potential of cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells in CCSC spheres. Scale bar, 60 μm. (D) Pair-cell assay showing that cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells immediately enter the second division. Scale bar, 100 μm.


( E and F ) Ki67 staining showing the proliferative potential of cluster C1 ( CCNE2 + ) and cluster C2 ( CCNB2 + ) cells in xenografts ( E ) and clinical CRC tissues ( F ) . Scale bar , 60 μm . Quantification of the immunostaining for ( F ) shown on the right . Error bars represent SEM of five fields in each section . P - value was calculated based on two - tailed Student ' s t - test . ns ( p > 0 . 05 ) , no significant .

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(E and F) Ki67 staining showing the proliferative potential of cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells in xenografts (E) and clinical CRC tissues (F). Scale bar, 60 μm. Quantification of the immunostaining for (F) shown on the right. Error bars represent SEM of five fields in each section. P-value was calculated based on two-tailed Student's t-test. ns (p>0.05), no significant.


( A and B ) RT - qPCR showing the enrichment of cluster C3 subpopulation ( CFAP54 + ) in CCSC1 ( A ) and CCSC2 ( B ) spheres treated with the chemotherapeutic agent 5FU or L - OHP . Error bars represent SD of three biological replicates . P - value was calculated based on two - tailed Student ' s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns , no significant .

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(A and B) RT-qPCR showing the enrichment of cluster C3 subpopulation (CFAP54+) in CCSC1 (A) and CCSC2 (B) spheres treated with the chemotherapeutic agent 5FU or L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on two-tailed Student's t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ns, no significant.


( C ) Representative RNA - FISH images and quantification showing the level of cluster C3 subpopulation CFAP54 + cell ( red ) levels in L - OHP treated xenografts . Scale bar , 40 μm . Three biological replicates were analyzed . Data represent the mean ± SD . P - value was calculated based on two - tailed Student ' s t test . * , p < 0 . 05 .

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(C) Representative RNA-FISH images and quantification showing the level of cluster C3 subpopulation CFAP54+ cell (red) levels in L-OHP treated xenografts. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. *, p<0.05.


( E ) Fluorescent images and quantification showing AP20187 - mediated ablation of cluster C4 ( PLAUR + ) cells from CCSC spheres carrying PLAUR - iCaspase9 - mCherry construct . Scale bar , 40 μm . Three biological replicates were analyzed . Data represent the mean ± SD . P - value was calculated based on two - tailed Student ' s t test . * * * , p < 0 . 001 .

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(E) Fluorescent images and quantification showing AP20187-mediated ablation of cluster C4 (PLAUR+) cells from CCSC spheres carrying PLAUR-iCaspase9-mCherry construct. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. ***, p<0.001.


( I ) Representative H & E images and quantification ( number of mice with invasive tumors ) showing the invasive potential of cluster C4 cells ( PLAUR + ) . CCSC sphere cells carrying PLAUR - iCaspase9 - mCherry construct were orthotopically injected into the mice . AP20187 was applied to ablate cluster C4 ( PLAUR + , red ) cells . Red arrows indicate invasive tumor . Scale bars , left , 100 μm ; right , 300 μm .

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(I) Representative H&E images and quantification (number of mice with invasive tumors) showing the invasive potential of cluster C4 cells (PLAUR+). CCSC sphere cells carrying PLAUR-iCaspase9-mCherry construct were orthotopically injected into the mice. AP20187 was applied to ablate cluster C4 (PLAUR+, red) cells. Red arrows indicate invasive tumor. Scale bars, left, 100 μm; right, 300 μm.


( K ) Left : Representative H & E images of liver tissue sections and quantification ( number of mice with liver metastasis ) showing the metastatic potential of cluster C4 cells ( PLAUR + ) . AP20187 was applied to ablate cluster C4 . Yellow arrows indicate liver metastasis . Scale bars , left , 2 mm ; right , 50 μm . Right : Quantification of liver metastasis in mice with intrasplenic injection . Error bars denote mean ± SEM of three mice per group . P - value was calculated based on Student ' s t - test . * * * , p < 0 . 001 .

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(K) Left: Representative H&E images of liver tissue sections and quantification (number of mice with liver metastasis) showing the metastatic potential of cluster C4 cells (PLAUR+). AP20187 was applied to ablate cluster C4. Yellow arrows indicate liver metastasis. Scale bars, left, 2 mm; right, 50 μm. Right: Quantification of liver metastasis in mice with intrasplenic injection. Error bars denote mean ± SEM of three mice per group. P-value was calculated based on Student's t-test. ***, p<0.001.


( C ) RT - qPCR analysis of the signature genes of each cluster in CCSC sphere cells with control ( EV ) or ATF6 knockdown ( shATF6 - 1 and shATF6 - 2 ) . Error bars represent SD of three biological replicates . P - value was calculated based on Student ' s t - test . * , p < 0 . 05 ; * * , P < 0 . 01 . Diagram without asterisk indicates no statistical difference .

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(C) RT-qPCR analysis of the signature genes of each cluster in CCSC sphere cells with control (EV) or ATF6 knockdown (shATF6-1 and shATF6-2). Error bars represent SD of three biological replicates. P-value was calculated based on Student's t-test. *, p<0.05; **, P<0.01. Diagram without asterisk indicates no statistical difference.


( D ) RT - qPCR analysis of signature genes of each cluster in control ( EV ) or ATF6 knockdown ( shATF6 - 1 and shATF6 - 2 ) under the treatment with L - OHP . Error bars represent SD of three biological replicates . P - value was calculated based on Student ' s t - test . * * * , p < 0 . 001 . Diagram without asterisk indicates no statistical difference .

(D) RT-qPCR analysis of signature genes of each cluster in control (EV) or ATF6 knockdown (shATF6-1 and shATF6-2) under the treatment with L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on Student's t-test. ***, p<0.001. Diagram without asterisk indicates no statistical difference.


( F ) Representative RNA - FISH of cluster C3 signature gene , CFAP54 ( up ) and immunohistochemistry of transcription factor ATF6 ( bottom ) showing the influence of ATF6 knockdown ( shATF6 - 1 and shATF6 - 2 ) on cluster C3 ( CFAP54 + , red ) population in xenografts . The mice were treated with vehicle or L - OHP after 14 days after CCSC sphere cells were implanted . Scale bars , upper , 40 μm ; lower , 80 μm .

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(F) Representative RNA-FISH of cluster C3 signature gene, CFAP54 (up) and immunohistochemistry of transcription factor ATF6 (bottom) showing the influence of ATF6 knockdown (shATF6-1 and shATF6-2) on cluster C3 (CFAP54+, red) population in xenografts. The mice were treated with vehicle or L-OHP after 14 days after CCSC sphere cells were implanted. Scale bars, upper, 40 μm; lower, 80 μm.


( H ) RT - qPCR analysis of signature genes of each cell clusters in CCSC sphere cells with control ( EV ) or FOXQ1 knockdown ( shFOXQ1 - 1 and shFOXQ1 - 2 ) . Error bars denote the SD between triplicates . P - value was calculated based on Student ' s t test . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

(H) RT-qPCR analysis of signature genes of each cell clusters in CCSC sphere cells with control (EV) or FOXQ1 knockdown (shFOXQ1-1 and shFOXQ1-2). Error bars denote the SD between triplicates. P-value was calculated based on Student's t test. *, p<0.05; **, p<0.01; ***, p<0.001.


( I ) Representative H & E and immunofluorescence images showing the influence of FOXQ1 knockdown ( shFOXQ1 - 1 and shFOXQ1 - 2 ) on cluster C4 ( PLAUR + , red ) population and tumor invasion . CCSC sphere cells carrying control ( EV ) vector or FOXQ1 shRNAs were orthotopically injected into mice ( n = 5 ) . Scale bars , left , 300 μm ; right , 100 μm . Arrows indicate the location of tumor invasion .

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(I) Representative H&E and immunofluorescence images showing the influence of FOXQ1 knockdown (shFOXQ1-1 and shFOXQ1-2) on cluster C4 (PLAUR+, red) population and tumor invasion. CCSC sphere cells carrying control (EV) vector or FOXQ1 shRNAs were orthotopically injected into mice (n=5). Scale bars, left, 300 μm; right, 100 μm. Arrows indicate the location of tumor invasion.


( G ) Representative immunohistochemistry images for the expression of PLAUR ( cluster C4 maker gene ) in primary CRC tumors and liver metastasis from CRC patients . Scale bar , 200 μm .

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(G) Representative immunohistochemistry images for the expression of PLAUR (cluster C4 maker gene) in primary CRC tumors and liver metastasis from CRC patients. Scale bar, 200 μm.


( H and I ) Representative immunohistochemistry images for ATF6 expression ( H ) and RNA - FISH for CFAP54 expression ( I ) in paired CRC tissues collected from CRC patients before chemotherapy or after chemotherapy . Scale bars , left , 200 μm ; right , 40 μm . Data represent the mean ± SEM . Three to five technical replicates were analyzed . P - value was calculated based on two - tailed Student ' s t test . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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(H and I) Representative immunohistochemistry images for ATF6 expression (H) and RNA-FISH for CFAP54 expression (I) in paired CRC tissues collected from CRC patients before chemotherapy or after chemotherapy. Scale bars, left, 200 μm; right, 40 μm. Data represent the mean ± SEM. Three to five technical replicates were analyzed. P-value was calculated based on two-tailed Student's t test. *, p<0.05; **, p<0.01; ***, p<0.001.


( J and K ) Kaplan - Meier survival curves for CRC patients ( n = 326 ) with different proportions of cluster C4 ( J ) and cluster C3 ( K ) . P value was calculated based on log - rank test .

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(J and K) Kaplan-Meier survival curves for CRC patients (n=326) with different proportions of cluster C4 (J) and cluster C3 (K). P value was calculated based on log-rank test.


B . Genome views of RNA read density profiles on representative loci enriched ( left ) or low ( right ) in FLAG - HP1γ RNAchIP ( IP ) relative to input RNA ( inp . ) in overlaid replicate samples from HP1γ cells stimulated or not with PMA . Orange arrow , position of the locus analyzed in D .

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B. Genome views of RNA read density profiles on representative loci enriched (left) or low (right) in FLAG-HP1γ RNAchIP (IP) relative to input RNA (inp.) in overlaid replicate samples from HP1γ cells stimulated or not with PMA. Orange arrow, position of the locus analyzed in D.


C . Genome - wide scatter plot of IP and input normalized ( norm . ) RNA read counts per gene for the mean of the triplicates ; n = 21 , 754 ; r , Pearson ' s correlation coefficient between IP and input .

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C. Genome-wide scatter plot of IP and input normalized (norm.) RNA read counts per gene for the mean of the triplicates; n=21,754; r, Pearson's correlation coefficient between IP and input.


D . Relative quantities of RNA in the RNAchIP samples , detected by RT - qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B ( orange arrow ) . n = 3 independent experiments . Data information : Histograms represent mean and s . d . Dots represent individual data points . P - values indicate significantly higher difference between IP in HP1γ and in KO cells ( * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

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D. Relative quantities of RNA in the RNAchIP samples, detected by RT-qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B (orange arrow). n=3 independent experiments. Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).


E . Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis ( 10 , 013 and 2609 peaks ) for a representative replicate ( replicate 1 ) . Black arrows represent peak summit .

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E. Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis (10,013 and 2609 peaks) for a representative replicate (replicate 1). Black arrows represent peak summit.


F . Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database ( green bars ) . Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations ( random , gray bars ) . Data information : Histograms represent mean and s . d . Dots represent individual data points . P - values indicate significantly higher difference between IP in HP1γ and in KO cells ( * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

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F. Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database (green bars). Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations (random, gray bars). Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).


B . RNAchIP peaks are enriched in CACACA and GAGAGA motifs . Number of peaks containing at least one exact hexameric motif as indicated , compared to the average number of motifs in peaks whose genomic location was randomized among genes with ten permutations ( random , light bars , represent mean and s . d . ) .

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B. RNAchIP peaks are enriched in CACACA and GAGAGA motifs. Number of peaks containing at least one exact hexameric motif as indicated, compared to the average number of motifs in peaks whose genomic location was randomized among genes with ten permutations (random, light bars, represent mean and s.d.).


C , D . Distribution profiles of average RNAchIP signal in both IP ( warm colors ) and input ( blue colors ) over ± 2kb centered on intronic ( C ) and exonic ( D ) CACACA hexameric motifs oriented in the same orientation ( sense ) or in opposite orientation ( antisense ) relative to the overlapping annotated transcript . The antisense CACACA motifs were obtained by querying the transcript sequences with the TGTGTG motif .

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C, D. Distribution profiles of average RNAchIP signal in both IP (warm colors) and input (blue colors) over ±2kb centered on intronic (C) and exonic (D) CACACA hexameric motifs oriented in the same orientation (sense) or in opposite orientation (antisense) relative to the overlapping annotated transcript. The antisense CACACA motifs were obtained by querying the transcript sequences with the TGTGTG motif.


E . Left , representative example of a locus on the Ppp3ca gene surrounding an RNAchIP merged peak , showing the RNAchIP read density as in Fig 1B . Red and orange arrowheads correspond to oriented CACACA and GAGAGA motifs , respectively . Right , sequence of the RNA surrounding a CACACA motif highlighted in red , as well as a second a neighboring imperfect motif . The sequence was used to design a CACACA - containing RNA oligonucleotide probe ( CACACA ) , compared to a control probe ( no - CA ) devoid of any related motif .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left, representative example of a locus on the Ppp3ca gene surrounding an RNAchIP merged peak, showing the RNAchIP read density as in Fig 1B. Red and orange arrowheads correspond to oriented CACACA and GAGAGA motifs, respectively. Right, sequence of the RNA surrounding a CACACA motif highlighted in red, as well as a second a neighboring imperfect motif. The sequence was used to design a CACACA-containing RNA oligonucleotide probe (CACACA), compared to a control probe (no-CA) devoid of any related motif.


F , Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST proteins , and tested for its direct interaction with the Cy3 - labeled RNA oligonucleotide probes The probes in the gels , either free ( white arrowheads ) or as shifted protein / RNA complexes ( red arrowheads ) were detected by their Cy3 fluorescence . Representative of 3 independent experiments .

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

F, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


G , Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST proteins , and tested for its direct interaction with the Cy3 - labeled dsDNA oligonucleotide probes The probes in the gels , either free ( white arrowheads ) or as shifted protein / RNA complexes ( red arrowheads ) were detected by their Cy3 fluorescence . Representative of 3 independent experiments .

G, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled dsDNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


H . Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST proteins , and tested for its direct interaction with the Cy3 - labeled RNA oligonucleotide probes The probes in the gels , either free ( white arrowheads ) or as shifted protein / RNA complexes ( red arrowheads ) were detected by their Cy3 fluorescence . Representative of 3 independent experiments .

H. Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


A . Fold enrichment values on all repeat masker classes and on Refseq genes , on the basis of all uniquely aligned reads , shown as mean and s . d . , n = 6 biological replicates . Dashed line represents an IP / Input ratio of 1 , taken as a reference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fold enrichment values on all repeat masker classes and on Refseq genes, on the basis of all uniquely aligned reads, shown as mean and s.d., n=6 biological replicates. Dashed line represents an IP/Input ratio of 1, taken as a reference.


C . Profiles of average RNAchIP signal as in Fig 2B over ± 2kb centered on intragenic LINE , LTR , or SINE repeats annotated in Repeat masker database ( n = 34 , 000 ) . All repeats are oriented in the same orientation ( sense ) relative to the overlapping annotated transcript , unless otherwise specified ( antisense , shuffled ) , and as depicted by an oriented white box overlapping the transcript in 5 ' to 3 ' orientation ( black line with arrowhead ) . D . Profiles of average RNAchIP signal , as in ( C ) centered on the 5 major families of intragenic SINE repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Profiles of average RNAchIP signal as in Fig 2B over ±2kb centered on intragenic LINE, LTR, or SINE repeats annotated in Repeat masker database (n=34,000). All repeats are oriented in the same orientation (sense) relative to the overlapping annotated transcript, unless otherwise specified (antisense, shuffled), and as depicted by an oriented white box overlapping the transcript in 5' to 3' orientation (black line with arrowhead). D. Profiles of average RNAchIP signal, as in (C) centered on the 5 major families of intragenic SINE repeats.


E . Proportions of the families of CACACA - containing SINE repeats within each family , as a percentage of total intragenic sense SINEs .

O 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. Proportions of the families of CACACA-containing SINE repeats within each family, as a percentage of total intragenic sense SINEs.


F . Profiles of average RNAchIP signal , as in ( D ) centered on the subset of intragenic B4 SINEs containing a consensus CACACA motif .

O O O 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. Profiles of average RNAchIP signal, as in (D) centered on the subset of intragenic B4 SINEs containing a consensus CACACA motif.


G . Genomic position of a B4 SINE repeat and CACACA consensus motifs at the location of the RNAchIP

G. Genomic position of a B4 SINE repeat and CACACA consensus motifs at the location of the RNAchIP peak


H . North - western blot assay showing direct association between the indicated bacterially expressed , purified GST - fusion proteins and in vitro transcribed , biotinylated RNA probes based on the sequence of the B4 SINE depicted in ( G ) . Top panels , binding of a 256nt probe corresponding to the CACACA - containing B4 SINE sequence , was compared to an identical B4 SINE deleted of its CACACA by truncation of its 3 ' portion ( B4 SINE - Δ3 ' ) . RNA probes hybridized on the membranes were detected by their Cy3 fluorescence . Total GST - fusion protein loading was visualized by Ponceau S staining . Representative of two independent experiments .

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

H. North-western blot assay showing direct association between the indicated bacterially expressed, purified GST-fusion proteins and in vitro transcribed, biotinylated RNA probes based on the sequence of the B4 SINE depicted in (G). Top panels, binding of a 256nt probe corresponding to the CACACA-containing B4 SINE sequence, was compared to an identical B4 SINE deleted of its CACACA by truncation of its 3' portion (B4 SINE-Δ3'). RNA probes hybridized on the membranes were detected by their Cy3 fluorescence. Total GST-fusion protein loading was visualized by Ponceau S staining. Representative of two independent experiments.


A . Box plot showing the distribution of fold enrichment values per genes sorted by exon number , n = 66 , 247 , 389 , and 574 genes with 1 , 2 , 3 , and 4 exons , respectively . Dashed line represents an IP / Input ratio of 1 , taken as a reference . B . Reads from input and IP counted in unstimulated and PMA stimulated triplicates give significant enrichment values of 1 . 4 and 0 . 7 on introns and exons , respectively , shown as mean and s . d . , n = 3 biological replicates . Dots represent individual data points . C . Box plot of reads belonging to spliced transcripts versus all reads in input or RNAchIP samples pooled between + and - PMA , calculated as a ratio between split reads and total reads , n = 3 biological replicates . Dots represent individual data points . Data information : All box boundaries represent 25th and 75th percentiles ; center line represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . All P - values are * P < 0 . 01 , * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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

A. Box plot showing the distribution of fold enrichment values per genes sorted by exon number, n= 66, 247, 389, and 574 genes with 1, 2, 3, and 4 exons, respectively. Dashed line represents an IP/Input ratio of 1, taken as a reference. B. Reads from input and IP counted in unstimulated and PMA stimulated triplicates give significant enrichment values of 1.4 and 0.7 on introns and exons, respectively, shown as mean and s.d., n=3 biological replicates. Dots represent individual data points. C. Box plot of reads belonging to spliced transcripts versus all reads in input or RNAchIP samples pooled between + and -PMA, calculated as a ratio between split reads and total reads, n=3 biological replicates. Dots represent individual data points. Data information: All box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test.


D , E . Average distribution profiles of the depicted hexameric motifs ( D ) , or SINE and LINE repeats ( E ) on RefSeq genes , computed over exons ( black box ) and on ± 0 . 7kb of surrounding introns ( left panels ) , or over entire gene bodies including introns ( pre - mRNA ; middle panels ) , or as metagenes over exons only ( Metagene mRNA ; right panels ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E. Average distribution profiles of the depicted hexameric motifs (D), or SINE and LINE repeats (E) on RefSeq genes, computed over exons (black box) and on ±0.7kb of surrounding introns (left panels), or over entire gene bodies including introns (pre-mRNA; middle panels), or as metagenes over exons only (Metagene mRNA; right panels).


F . Box plot showing the distribution of fold enrichment values on series of introns of comparable size ( 4kb in average , ranging from 3 to 5kb ) sorted by number of sense CACACA motifs per intron ( 2 + , two or more motifs ) , n = 5782 , 899 , 960 , and 3234 introns for all introns , 0 , 1 , and 2 + CACACA categories , respectively . R2 , Linear regression coefficient between 0 , 1 , and 2 + . Dashed line represents an IP / Input ratio of 1 , taken as a reference . Data information : All box boundaries in Fig 4 represent 25th and 75th percentiles ; center line represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . All P - values are * P < 0 . 01 , * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box plot showing the distribution of fold enrichment values on series of introns of comparable size (4kb in average, ranging from 3 to 5kb) sorted by number of sense CACACA motifs per intron (2+, two or more motifs), n= 5782, 899, 960, and 3234 introns for all introns, 0, 1, and 2+ CACACA categories, respectively. R2, Linear regression coefficient between 0, 1, and 2+. Dashed line represents an IP/Input ratio of 1, taken as a reference. Data information: All box boundaries in Fig 4 represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test.


G , H . Average read profiles on two groups of RefSeq genes , in the size - range 4 to 15kb , containing the highest ( G ) or lowest ( H ) CACACA motif density .

O O O O O O O O O O O O O O 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, H. Average read profiles on two groups of RefSeq genes, in the size-range 4 to 15kb, containing the highest (G) or lowest (H) CACACA motif density.


C . Box plot depicting the length of exon junctions found differentially regulated by MAJIQ between HP1γ ( green ) and KO ( orange ) in three categories as in Fig EV5A , all classified differential HP1γ / KO junctions , annotated alternative exon junctions , non - annotated de novo cryptic junctions detected in KO only ; n , differential events . Box boundaries represent 25th and 75th percentiles ; center line represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . Data information : All graphes represent mean and s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box plot depicting the length of exon junctions found differentially regulated by MAJIQ between HP1γ (green) and KO (orange) in three categories as in Fig EV5A, all classified differential HP1γ/KO junctions, annotated alternative exon junctions, non-annotated de novo cryptic junctions detected in KO only; n, differential events. Box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed Student's t-test).


D . RNAchIP read density per kb on the indicated introns for input and IP in samples from both untreated and PMA stimulated cells , n = 6 biological replicates , or 3 - times randomized intervals , n = 18 samples . Dots represent individual data points . HP1γ / KO introns are all 272 intronic intervals between exon junctions differentially detected in HP1γ and KO . Random introns are a matching library of random intronic intervals . E . RNAchIP read density per kb on the genes corresponding to the introns tested in ( D ) , and analyzed as in ( D ) , n = 6 biological replicates , or 3 - times randomized intervals , n = 18 samples . Data information : All graphes represent mean and s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RNAchIP read density per kb on the indicated introns for input and IP in samples from both untreated and PMA stimulated cells, n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Dots represent individual data points. HP1γ/KO introns are all 272 intronic intervals between exon junctions differentially detected in HP1γ and KO. Random introns are a matching library of random intronic intervals. E. RNAchIP read density per kb on the genes corresponding to the introns tested in (D), and analyzed as in (D), n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed Student's t-test).


F . Reads from input and IP counted on the indicated features in combined unstimulated and PMA stimulated triplicate samples , n = 6 biological replicates . Dots represent individual data points . Data information : All graphes represent mean and s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; one - tailed Student ' s t - test ) .

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

F. Reads from input and IP counted on the indicated features in combined unstimulated and PMA stimulated triplicate samples, n=6 biological replicates. Dots represent individual data points. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; one-tailed Student's t-test).


G . Average distribution profiles of sense SINE repeats on indicated exons and on ± 0 . 7kb of surrounding introns .

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

G. Average distribution profiles of sense SINE repeats on indicated exons and on ±0.7kb of surrounding introns.


B . RT - PCR analysis of the RNAs produced in the splicing assay with the Prl3 wt or Prl3 CAmut constructs as depicted in A transfected into HP1γ or KO cell lines . The top 210bp and bottom 120bp amplicons correspond to the included form ( beta globin with Prl exon , more included in lane1 , blue arrow , compared to lane 2 ) , and the skipped form ( beta globin exons only ) of RNA , respectively . Orange arrowheads , position of the HBGEX - F and HBGEX - R primers used in PCR analysis . C . Histogram showing the loss of inclusion of the Prl3 exon upon depletion of HP1γ ( in KO ) relative to inclusion in the presence of HP1γ with the Prl3 wt ( red ) or Prl3 CA mut ( grey ) reporters . Based on the quantitation of 4 experiments as the one depicted in B . Values represent the fold change relative to HP1γ as a reference ( dashed line ) . Graph represents mean and s . d . ( * * * P < 0 . 001 ; two - tailed Student ' s t - test ; n = 4 ) .

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

B. RT-PCR analysis of the RNAs produced in the splicing assay with the Prl3 wt or Prl3 CAmut constructs as depicted in A transfected into HP1γ or KO cell lines. The top 210bp and bottom 120bp amplicons correspond to the included form (beta globin with Prl exon, more included in lane1, blue arrow, compared to lane 2), and the skipped form (beta globin exons only) of RNA, respectively. Orange arrowheads, position of the HBGEX-F and HBGEX-R primers used in PCR analysis. C. Histogram showing the loss of inclusion of the Prl3 exon upon depletion of HP1γ (in KO) relative to inclusion in the presence of HP1γ with the Prl3 wt (red) or Prl3 CA mut (grey) reporters. Based on the quantitation of 4 experiments as the one depicted in B. Values represent the fold change relative to HP1γ as a reference (dashed line). Graph represents mean and s.d. (*** P < 0.001; two-tailed Student's t-test; n=4).


Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin .

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

Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.


Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin .

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

Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.


Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPO , erythropoietin .

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

Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPO, erythropoietin.


Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice .

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

Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting B , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA ,

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting B, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting E ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA ,

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting E). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290


APP / PS1 - 21 mice ( 5 m ) treated with rhEPO ( 10000 IU / kg / d ) or ARA290 ( 0 . 03 mg / kg / d ) ( i . p . , once a day , for 14 days ) ( n = 10 ) , and Aβ plaques in cortex and hippocampus ( n = 5 ) were detected by IHC ( I ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290 ; IHC , Immunohistochemical staining .

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

APP/PS1-21 mice (5 m) treated with rhEPO (10000 IU/kg/d) or ARA290 (0.03 mg/kg/d) (i.p., once a day, for 14 days) (n=10), and Aβ plaques in cortex and hippocampus (n = 5) were detected by IHC (I). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290; IHC, Immunohistochemical staining.


Proteins were extracted from spleens of 5 - or 11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , and soluble Aβ levels were measured by ELISA ( n = 5 ) ( J Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ELISA ,

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

Proteins were extracted from spleens of 5- or 11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), and soluble Aβ levels were measured by ELISA (n = 5) (J Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ELISA,


11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , Aβ plaques in the cortex and hippocampus of mice of 11 - month - old ( M ) ( n = 5 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining .

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

11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), Aβ plaques in the cortex and hippocampus of mice of 11-month-old (M) (n = 5) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.


R Aβ plaques in the cortex and hippocampus ( n = 4 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining .

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

R Aβ plaques in the cortex and hippocampus (n = 4) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.


A , Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


B Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Both non - cognitive and cognitive behavioral impairments of APP / PS1 - 21 mice ( 11 m ) were analyzed by nest construction assay , social interaction assay , NORT , FCT ( n = 10 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . NORT , novel object recognition test ; FCT , fear conditioning test .

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

Both non-cognitive and cognitive behavioral impairments of APP/PS1-21 mice (11 m) were analyzed by nest construction assay, social interaction assay, NORT, FCT (n = 10). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. NORT, novel object recognition test; FCT, fear conditioning test.


Splenic macrophages from APP / PS1 - 21 ( 5 - or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Splenic macrophages from APP/PS1-21 (5- or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Splenic macrophages from APP / PS1 - 21 11 - month - old ) or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) B ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Splenic macrophages from APP/PS1-21 11-month-old) or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) B). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies ( n = 3 ) ( G ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies (n = 3) (G). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( I ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin .

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

Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (I). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.


PMs were treated with Aβ42 ( 0 , 5 , 7 . 5 or 10 μM ) for 12 hr , and cell lysates were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( K ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

PMs were treated with Aβ42 (0, 5, 7.5 or 10 μM) for 12 hr, and cell lysates were detected by immunoblotting with the indicated antibodies (n = 3) (K). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


PMs were treated with Aβ42 ( 0 , 5 , 10 μM ) for 12 hr , and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( M ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin .

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

PMs were treated with Aβ42 (0, 5, 10 μM) for 12 hr, and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (M). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.


E Aβ plaques in the cortex and hippocampus ( n = 5 ) were detected by IHC . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Aβ plaques in the cortex and hippocampus (n = 5) were detected by IHC. Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


G Expression of MAP - 2 or the apoptotic neurons of hippocampus were detected by IF staining and then statistically quantified ( n = 5 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IF , Immunofluorescent ; MAP - 2 , microtubule - associated protein 2 ; NeuN , neuronal nuclei ; TUNEL , Terminal dUTP Nick End Labeling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Expression of MAP-2 or the apoptotic neurons of hippocampus were detected by IF staining and then statistically quantified (n = 5). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IF, Immunofluorescent; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclei; TUNEL, Terminal dUTP Nick End Labeling.


B . Pull - down assay of Sec62 domain with different biotin - labeled Sec63 C - terminal peptides . Sec63 - C2P denotes di - phosphorylated Sec63 - C2 .

O O O O O O O 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. Pull-down assay of Sec62 domain with different biotin-labeled Sec63 C-terminal peptides. Sec63-C2P denotes di-phosphorylated Sec63-C2.


C . ITC - thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63 - C2P .

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

C. ITC-thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63-C2P.


F . Pull - down assay of the Sec62 domain and a mutant in the interaction interface ( Sec62R51E ) with biotin - labeled Sec63 - C2P .

O O O O 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. Pull-down assay of the Sec62 domain and a mutant in the interaction interface (Sec62R51E) with biotin-labeled Sec63-C2P.


( a ) Representative photographs of Bax / Bak − / − MEFs treated with etoposide . Bax / Bak − / − MEFs were treated with 20 μM etoposide in the presence of PI ( 2 μM ) for the indicated times , and were analysed under a phase - contrast and fluorescence ( PI ) microscope .

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

(a) Representative photographs of Bax/Bak−/− MEFs treated with etoposide. Bax/Bak−/− MEFs were treated with 20 μM etoposide in the presence of PI (2 μM) for the indicated times, and were analysed under a phase-contrast and fluorescence (PI) microscope.