( B ) Representative flow cytometric analyses of the relocation of B220 + CD11c + NK1 . 1 + cells in lungs that were photoconverted in TCM - stimulated KikGR mouse liver ( gated in red polygonal region ) . Percent is shown for total cells ( upper panel in B ) , and for gated KikGR red or KikGR green cells ( lower panels in B ) . Region where the photoconverted cells locate is magnified in the right . Yellow arrow shows the photoconversion direction . Three independent experiments . LTCM stands for TCM derived from LLC

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(B) Representative flow cytometric analyses of the relocation of B220+CD11c+NK1.1+ cells in lungs that were photoconverted in TCM-stimulated KikGR mouse liver (gated in red polygonal region). Percent is shown for total cells (upper panel in B), and for gated KikGR red or KikGR green cells (lower panels in B). Region where the photoconverted cells locate is magnified in the right. Yellow arrow shows the photoconversion direction. Three independent experiments. LTCM stands for TCM derived from LLC


( A ) Representative photos of IFNγ expression in B220 + CD11c + NK1 . 1 + cells derived from TCM - stimulating mouse liver . These cells were cultured with LiCM or LuCM that had been prepared by culture with tumour - bearing - liver or lungs , respectively . NoCM stands for CM without tissues , and it was used as control ( scale bar , 10 μm )

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(A) Representative photos of IFNγ expression in B220+CD11c+NK1.1+cells derived from TCM-stimulating mouse liver. These cells were cultured with LiCM or LuCM that had been prepared by culture with tumour-bearing- liver or lungs, respectively. NoCM stands for CM without tissues, and it was used as control (scale bar,10 μm)


( B ) Immunohistochemical quantifications of IFNγ in B220 + CD11c + NK1 . 1 + cells . Cells derived from liver and spleen were primed with LiCM or LuCM . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

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

(B) Immunohistochemical quantifications of IFNγ in B220+CD11c+NK1.1+ cells. Cells derived from liver and spleen were primed with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( C ) Flow cytometric analysis of living Zombie - tumour cells with B220 + CD11c + NK1 . 1 + cells that were primed by NoCM or LuCM . NoCM : N = 3 , LuCM : N = 4 . Shown are averages with SEM . Welch " s t - test . Rhodamin + tumour cells were counted as viable cells

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(C) Flow cytometric analysis of living Zombie- tumour cells with B220+CD11c+NK1.1+ cells that were primed by NoCM or LuCM. NoCM: N=3, LuCM: N=4. Shown are averages with SEM. Welch"s t-test. Rhodamin+ tumour cells were counted as viable cells


( D ) Representative immunohistochemical stainings of rhodamin + ( red ) Zombie + ( green ) tumour cells ( scale bar , 10 μm )

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(D) Representative immunohistochemical stainings of rhodamin+ (red) Zombie+ (green) tumour cells (scale bar, 10 μm)


( E ) Ratios of living tumour cells after co - culture with B220 + CD11c + NK1 . 1 + cells that had been primed with lung - or liver - CM . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

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(E) Ratios of living tumour cells after co-culture with B220+CD11c+NK1.1+cells that had been primed with lung- or liver-CM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( A ) Western blotting of fibrinogen in tumour - bearing and no tumour - bearing livers

(A) Western blotting of fibrinogen in tumour-bearing and no tumour-bearing livers


( C ) IP - Western blot analysis shows protein - protein interactions between fibrinogen and Vtn / TSP

(C) IP-Western blot analysis shows protein-protein interactions between fibrinogen and Vtn/TSP


( D ) Quantification of fibrinogen deposition areas after injection of CD45 + cells derived from the liver in tumour - bearing mice . Shown are averages ( N = 7 / group ) with SEM . 1 - way ANOVA

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(D) Quantification of fibrinogen deposition areas after injection of CD45+ cells derived from the liver in tumour-bearing mice. Shown are averages (N = 7/ group) with SEM. 1-way ANOVA


( E ) Quantifications of fibrinogen deposition areas in TCM - stimulating wild - type mouse lungs after injection of Vtn + HepELs . Vtn + cells were isolated from E0771 - bearing wild - type or FX - / - mouse liver by using anti - Vtn beads ( 1 x 106 cells / mouse ) . Shown are averages ( N = 5 / group , 3 fields / sample ) with SEM . Welch " s t - test

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(E) Quantifications of fibrinogen deposition areas in TCM-stimulating wild-type mouse lungs after injection of Vtn+ HepELs. Vtn+ cells were isolated from E0771-bearing wild-type or FX-/-mouse liver by using anti-Vtn beads (1 x 106 cells /mouse). Shown are averages (N=5/ group, 3 fields/ sample) with SEM. Welch"s t-test


( F ) Separation of Vtn + / CD45 + HepELs from tumour - bearing FX + / - and FX - / - mouse livers by a cell sorter ( Moflo Astrios ) . Sorted Vtn + CD45 + HepELs cells obtained from a littermate ( 3 animals each ) were pooled , and 8 x 103 Vtn + CD45 + HepELs cells were i . v . injected into a tumour - bearing mouse . Right graph shows fibrinogen depositions in the lung . Shown are averages ( N = 3 / group , 9 fields / sample ) with SEM . 1 - way

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(F) Separation of Vtn+/ CD45+ HepELs from tumour-bearing FX+/- and FX-/- mouse livers by a cell sorter (Moflo Astrios). Sorted Vtn+CD45+HepELs cells obtained from a littermate (3 animals each) were pooled, and 8 x 103 Vtn+CD45+HepELs cells were i.v. injected into a tumour-bearing mouse. Right graph shows fibrinogen depositions in the lung. Shown are averages (N = 3/ group, 9 fields/ sample) with SEM. 1-way ANOVA


( A ) Representative immunostaining of fibrinogen depositions in tumour - bearing mouse lungs received an injection of rhodamine - labelled B220 + CD11c + NK1 . 1 + HepELs derived from tumour - bearing - FX + / − or FX − / − mice ( scale bar , 20 μm )

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(A) Representative immunostaining of fibrinogen depositions in tumour-bearing mouse lungs received an injection of rhodamine-labelled B220+CD11c+NK1.1+HepELs derived from tumour-bearing- FX+/− or FX−/− mice (scale bar, 20 μm)


( B ) Immunohistochemical quantifications of fibrinogen depositions in tumour - bearing mouse lung after an injection of B220 + CD11c + NK1 . 1 + cells derived from tumour - bearing - FX + / − or FX − / − mouse liver . Sorted B220 + CD11c + NK1 . 1 + cells were prepared from three mice per each genotype , and were i . v . injected . Shown are averages ( N = 6 / group , 4 sections / mouse ) with SEM and Welch " s t - test

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(B) Immunohistochemical quantifications of fibrinogen depositions in tumour-bearing mouse lung after an injection of B220+CD11c+NK1.1+cells derived from tumour-bearing- FX+/− or FX−/− mouse liver. Sorted B220+CD11c+NK1.1+cells were prepared from three mice per each genotype, and were i.v. injected. Shown are averages (N = 6/group, 4 sections/mouse) with SEM and Welch"s t-test


( B ) Relative mRNA levels of FX , Vtn and TSP1 in liver B220 + CD11c + NK1 . 1 + cells stimulated with LiCM or LuCM . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

O O O O O O O O O O O O O O O O O O O O O O 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) Relative mRNA levels of FX, Vtn and TSP1 in liver B220+CD11c+NK1.1+cells stimulated with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( C ) Relative mRNA levels of FX in liver B220 + CD11c + NK1 . 1 + cells stimulated with various factors . Shown are averages ( N = 4 ) with SEM and one - way ANOVA

O O O O O O O O O O O O O O O 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) Relative mRNA levels of FX in liver B220+CD11c+NK1.1+cells stimulated with various factors. Shown are averages (N = 4) with SEM and one-way ANOVA


( D ) In vitro adhesion assay . Liver B220 + CD11c + NK1 . 1 + cells were seeded on a fibrinogen plate . Cells attached to the plate were counted . Twentyμg / mL of anti - Vtn Ab or isotype control IgG were used to block the adhesion . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

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(D) In vitro adhesion assay. Liver B220+CD11c+NK1.1+cells were seeded on a fibrinogen plate. Cells attached to the plate were counted. Twentyμg/mL of anti-Vtn Ab or isotype control IgG were used to block the adhesion. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( E ) Lung B220 + CD11c + NK1 . 1 + cells were seeded on ECM coated ( fibrinogen , fibronectin , or collagen I ) plates . Shown are averages ( N = 5 / group ) with SEM and one - way ANOVA

(E) Lung B220+CD11c+NK1.1+cells were seeded on ECM coated (fibrinogen, fibronectin, or collagen I) plates. Shown are averages (N = 5/group) with SEM and one-way ANOVA


( B ) Tumour cell homing in the lungs pre - treated with LiCM - primed B220 + CD11c + NK1 . 1 + cells from tumour - bearing WT or FX − / − mouse liver . Representative photos of homing of rhodamine - labelled tumour cells ( upper , scale bar , 20 μm ) . Number of homing tumour cells are shown ( lower ) . Shown are averages ( N = 60 sections , 5 / group , all field count / section , 12 sections / sample ) with SEM and Welch " s t - test

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(B) Tumour cell homing in the lungs pre-treated with LiCM-primed B220+CD11c+NK1.1+cells from tumour-bearing WT or FX−/− mouse liver. Representative photos of homing of rhodamine-labelled tumour cells (upper, scale bar, 20 μm). Number of homing tumour cells are shown (lower). Shown are averages (N = 60 sections, 5/group, all field count/section, 12 sections/sample) with SEM and Welch"s t-test


( C ) Tumour cell homing in the lungs pre - treated with activated FX - overexpressed B220 + CD11c + NK1 . 1 + cells ( FX - OE - HepELs ) from tumour - bearing lungs . Number of homing tumour cells after an injection of lung HepELs or FX - OE - HepELs are shown . Shown here are averages ( N = 24 sections , 4 / group , all field count / section , 6 sections / sample ) SEM and one - way ANOVA

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(C) Tumour cell homing in the lungs pre-treated with activated FX-overexpressed B220+CD11c+NK1.1+cells (FX-OE-HepELs) from tumour-bearing lungs. Number of homing tumour cells after an injection of lung HepELs or FX-OE-HepELs are shown. Shown here are averages (N = 24 sections, 4/group, all field count/section, 6 sections/sample) SEM and one-way ANOVA


A Immunohistochemistry was used to assess STAMP2 expression in normal and malignant human prostate specimens . Tissue microarrays ( TMAs ) with normal prostate ( n = 27 ) and early - stage ( Gleason grade < 7 ) ( n = 35 ) or late - stage prostatetumors ( Gleason grade ≥ 7 ) ( n = 149 ) were subjected to immunohistochemistry as described in Materials and Methods . Representative images are shown . B Quantification of cytosolicSTAMP2 staining in TMAs is shown in ( A ) . One - way analysis of variance ( ANOVA ) with a post hoc test , * P < 0 . 0001 . Error bars indicate SEM . C , D The details of cytosolic ( C ) and membrane ( D ) STAMP2 staining in the TMAs described in ( A ) .

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A Immunohistochemistry was used to assess STAMP2 expression in normal and malignant human prostate specimens. Tissue microarrays (TMAs) with normal prostate (n = 27) and early-stage (Gleason grade < 7) (n = 35) or late-stage prostatetumors (Gleason grade ≥ 7) (n = 149) were subjected to immunohistochemistry as described in Materials and Methods. Representative images are shown.B Quantification of cytosolicSTAMP2 staining in TMAs is shown in (A). One-way analysis of variance (ANOVA) with a post hoc test, *P < 0.0001. Error bars indicate SEM.C, D The details of cytosolic (C) and membrane (D) STAMP2 staining in the TMAs described in (A).


A LNCaP cells were transfected with either control siRNA or two independent STAMP2 siRNAs , and membrane fractions of the cells were prepared followed by Western blotting analysis . STAMP1 is used as a loading control .

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A LNCaP cells were transfected with either control siRNA or two independent STAMP2 siRNAs, and membrane fractions of the cells were prepared followed by Western blotting analysis. STAMP1 is used as a loading control.


B LNCaP cells were cultured in RPMI 1640 medium containing 10 % CT - FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs . The cells were then cultured for the indicated times , and cell growth was measured by Cell Counting Kit - 8 . n = 3 , * P < 0 . 0001 . Error bars indicate SD .

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B LNCaP cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs. The cells were then cultured for the indicated times, and cell growth was measured by Cell Counting Kit-8. n = 3, *P < 0.0001. Error bars indicate SD.


C LNCaP cells were cultured and transfected with the indicated siRNAs as described in ( B ) . After transfection , the cells were cultured for 10 days . The colonies formed were stained and photographed .

C LNCaP cells were cultured and transfected with the indicated siRNAs as described in (B). After transfection, the cells were cultured for 10 days. The colonies formed were stained and photographed.


D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881 . The cells were then collected , and membrane fractions were prepared followed by Western blot analysis .

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D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881. The cells were then collected, and membrane fractions were prepared followed by Western blot analysis.


E LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured for the indicated times , and the cell numbers were determined using a hemocytometer . n = 3 , * P = 0 . 047 ; * * P = 0 . 0046 . Error bars indicate SD .

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E LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured for the indicated times, and the cell numbers were determined using a hemocytometer. n = 3, *P = 0.047; **P = 0.0046. Error bars indicate SD.


F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods . The plates were then stained and photographed . G Quantification of data from ( F ) . n = 3 , * P = 0 . 022 . Error bars indicate SD .

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F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD.


H LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into male SCID mice . Tumor size was measured after 8 weeks . n = 9 , * P = 0 . 037 . Error bars indicate SEM .

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H LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into male SCID mice. Tumor size was measured after 8 weeks. n = 9, *P = 0.037. Error bars indicate SEM.


I VCaP cells were transfected with the indicated siRNA as described in ( B ) . After transfection , VCaP cells were cultured for 2 weeks . The colonies formed were stained and photographed . J Quantification of the data from ( I ) . n = 3 , * P < 0 . 0001 . Error bars indicate SD .

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I VCaP cells were transfected with the indicated siRNA as described in (B). After transfection, VCaP cells were cultured for 2 weeks. The colonies formed were stained and photographed.J Quantification of the data from (I). n = 3, *P < 0.0001. Error bars indicate SD.


A LNCaP cells were transfected with either control or STAMP2 - specific siRNA . Two days after transfection , cells were subjected to cell cycle analysis as described in Materials and Methods . Representative histograms showing PI - stained cells are shown . B The proportion of cells in each stage of the cell cycle from the experiment in ( A ) is presented . Student ' s t - test was performed to analyze the statistical significance , n = 3 . * P = 0 . 0002 ; * * P = 0 . 0056 ; * * * P = 0 . 0099 . Error bars indicate SD .

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A LNCaP cells were transfected with either control or STAMP2-specific siRNA. Two days after transfection, cells were subjected to cell cycle analysis as described in Materials and Methods. Representative histograms showing PI-stained cells are shown.B The proportion of cells in each stage of the cell cycle from the experiment in (A) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0002; **P = 0.0056; ***P = 0.0099. Error bars indicate SD.


C Two days after transfection with the indicated siRNAs , LNCaP cells were harvested and cell lysates were prepared and subjected to Western blot analysis with the indicated antisera . Representative blots of three independent experiments are shown .

C Two days after transfection with the indicated siRNAs, LNCaP cells were harvested and cell lysates were prepared and subjected to Western blot analysis with the indicated antisera. Representative blots of three independent experiments are shown.


D LNCaP cells were transfected with either control or STAMP2 - specific siRNA . Three days after transfection , cells were treated with either 50 ng / ml TRAIL or 20 μmol / l LY294002 ( LY ) for 24 h , or both agents for 6 h , and then subjected to TUNEL / FACS analysis . Representative histograms of TUNEL - stained cells are shown . FL1 - H refers to the gating of the cells for the TUNEL staining with fluorescence measurement . E The extent of apoptosis from the experiment in ( D ) is presented . Student ' s t - test was performed to analyze the statistical significance , n = 3 . * P = 0 . 027 ; * * P = 0 . 0058 ; * * * P = 0 . 0016 . Error bars indicate SD .

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

D LNCaP cells were transfected with either control or STAMP2-specific siRNA. Three days after transfection, cells were treated with either 50 ng/ml TRAIL or 20 μmol/l LY294002 (LY) for 24 h, or both agents for 6 h, and then subjected to TUNEL/FACS analysis. Representative histograms of TUNEL-stained cells are shown. FL1-H refers to the gating of the cells for the TUNEL staining with fluorescence measurement.E The extent of apoptosis from the experiment in (D) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.027; **P = 0.0058; ***P = 0.0016. Error bars indicate SD.


F LNCaP cells were transfected with either control or two independent STAMP2 - specific siRNAs , ST2 - 1 and ST2 - 2 . Three days after transfection , cells were treated in the same way as in ( D ) . Whole - cell lysates were prepared and subjected to Western blot analysis with the indicated antisera .

F LNCaP cells were transfected with either control or two independent STAMP2-specific siRNAs, ST2-1 and ST2-2. Three days after transfection, cells were treated in the same way as in (D). Whole-cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.


G LNCaP cells stably expressing either control or STAMP2 vector were treated in the same way as in ( D ) . Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies .

G LNCaP cells stably expressing either control or STAMP2 vector were treated in the same way as in (D). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies.


A STAMP2 expression was determined by IHC of a neoadjuvant hormone therapy ( NHT ) TMA containing samples from hormone naïve ( untreated ) ( n = 28 ) , NHT 1 - 6 months ( n = 31 ) , NHT 7 - 12 months ( n = 41 ) , and patients with PSA recurrence ( n = 21 ) as indicated . Representative images are presented . B Quantitative presentation of the data from ( A ) . Unt , untreated ; Recur , PSA recurrence . The Kruskal - Wallis test was used for significance analysis . * P = 0 . 0499 . Error bars indicate SEM .

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A STAMP2 expression was determined by IHC of a neoadjuvant hormone therapy (NHT) TMA containing samples from hormone naïve (untreated) (n = 28), NHT 1-6 months (n = 31), NHT 7-12 months (n = 41), and patients with PSA recurrence (n = 21) as indicated. Representative images are presented.B Quantitative presentation of the data from (A). Unt, untreated; Recur, PSA recurrence. The Kruskal-Wallis test was used for significance analysis. *P = 0.0499. Error bars indicate SEM.


C Boxplots of STAMP2 expression levels in normal prostate tissue , primary prostate cancer , and metastatic CRPC were analyzed in two independent GEO datasets ( GSE6919 and GSE35988 ) . The values of individual samples are presented as dots . Thick horizontal lines represent the median , with the box representing the upper and lower quartile . The whiskers represent the 5th and 95th percentiles . The statistical significance , indicated in the tables at the bottom , was determined by Student ' s t - test .

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C Boxplots of STAMP2 expression levels in normal prostate tissue, primary prostate cancer, and metastatic CRPC were analyzed in two independent GEO datasets (GSE6919 and GSE35988). The values of individual samples are presented as dots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles. The statistical significance, indicated in the tables at the bottom, was determined by Student's t-test.


D CWR22 xenografts were grown in nude mice , and tumors were collected at different times after castration . mRNA was extracted from the tumors and used for qPCR analysis of STAMP2 expression . The results are presented as boxplots . Thick horizontal lines represent the median , with the box representing the upper and lower quartile . The whiskers represent the 5th and 95th percentiles , and the outlier is presented as an open circle . The statistical significance was determined by one - way ANOVA with a post hoc test . n = 3 in group week 0 , 1 , 2 , and 4 ; n = 5 in refractory group . * P = 0 . 011 ; * * P = 0 . 049 . Error bars indicate SEM .

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D CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. mRNA was extracted from the tumors and used for qPCR analysis of STAMP2 expression. The results are presented as boxplots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles, and the outlier is presented as an open circle. The statistical significance was determined by one-way ANOVA with a post hoc test. n = 3 in group week 0, 1, 2, and 4; n = 5 in refractory group. *P = 0.011; **P = 0.049. Error bars indicate SEM.


A 22Rv1 cells were cultured in RPMI 1640 medium containing 10 % CT - FBS and treated with or without 1 nM R1881 for 24 h and were then transfected with the indicated siRNAs . The cells were then cultured for 10 days . The colonies formed were stained and photographed . B Quantification of data from ( A ) . Student ' s t - test was performed to analyze the statistical significance , n = 3 . * P = 0 . 0003 ; * * P < 0 . 0001 . Error bars indicate SD .

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A 22Rv1 cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h and were then transfected with the indicated siRNAs. The cells were then cultured for 10 days. The colonies formed were stained and photographed.B Quantification of data from (A). Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0003; **P < 0.0001. Error bars indicate SD.


C 22Rv1 cells from Supplementary Fig S2 were subcutaneously implanted into both flanks of male nude mice ( six mice per group ) . Tumor size was measured at the indicated time points . Student ' s t - test was performed to analyze the statistical significance , n = 10 . * P = 0 . 037 ; * * P = 0 . 039 . Error bars indicate SEM . D Representative tumors from ( C ) right after resection at the final time point are shown .

C 22Rv1 cells from Supplementary Fig S2 were subcutaneously implanted into both flanks of male nude mice (six mice per group). Tumor size was measured at the indicated time points. Student's t-test was performed to analyze the statistical significance, n = 10. *P = 0.037; **P = 0.039. Error bars indicate SEM.D Representative tumors from (C) right after resection at the final time point are shown.


A LNCaP cells were transfected with either control or STAMP2 - specific siRNA in the presence of 10 − 8 M R1881 . RNA was isolated , and qPCR was used to determine ATF4mRNA levels . Student ' s t - test was used to analyze the statistical significance , n = 3 . * P = 0 . 002 . Error bars indicate SD .

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A LNCaP cells were transfected with either control or STAMP2-specific siRNA in the presence of 10−8 M R1881. RNA was isolated, and qPCR was used to determine ATF4mRNA levels. Student's t-test was used to analyze the statistical significance, n = 3. *P = 0.002. Error bars indicate SD.


B Cell lysates from ( A ) were subjected to Western blot analysis .

B Cell lysates from (A) were subjected to Western blot analysis.


C , D ATF4 expression in LNCaP ( C ) and 22Rv1 ( D ) cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot analysis .

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C, D ATF4 expression in LNCaP (C) and 22Rv1 (D) cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot analysis.


E ATF4 expression in xenografted tumors of 22Rv1 cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR . Student ' s t - test was used to analyze the statistical significance , n = 4 . * P = 0 . 022 ; * * P = 0 . 015 ; * * * P = 0 . 044 . Error bars indicate SEM .

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E ATF4 expression in xenografted tumors of 22Rv1 cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR. Student's t-test was used to analyze the statistical significance, n = 4. *P = 0.022; **P = 0.015; ***P = 0.044. Error bars indicate SEM.


F CWR22 xenografts were grown in nude mice , and tumors were collected at different times after castration . The association between ATF4 and STAMP2 in the tumor samples was determined by Western blot analysis .

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F CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. The association between ATF4 and STAMP2 in the tumor samples was determined by Western blot analysis.


G The MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from the cBio Cancer Genomics Portal . Analysis was performed as described in Materials and Methods . The expression levels of STAMP2 and ATF4 , as well as two ATF4 target genes ( ASNS and SLC7A11 ) , are presented .

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G The MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from the cBio Cancer Genomics Portal. Analysis was performed as described in Materials and Methods. The expression levels of STAMP2 and ATF4, as well as two ATF4 target genes (ASNS and SLC7A11), are presented.


A LNCaP cells were transfected with either control or ATF4 siRNA . Two days after transfection , cells were harvested and whole - cell lysates were made and used in Western blot analysis .

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A LNCaP cells were transfected with either control or ATF4 siRNA. Two days after transfection, cells were harvested and whole-cell lysates were made and used in Western blot analysis.


B LNCaP cells were transfected with either control or ATF4 siRNA and were cultured for 2 weeks . The colonies formed were stained and photographed . C The area covered by the colonies on each plate in ( B ) was quantified and represented as percentage of the total area of the plate . * P = 0 . 0023 .

B LNCaP cells were transfected with either control or ATF4 siRNA and were cultured for 2 weeks. The colonies formed were stained and photographed.C The area covered by the colonies on each plate in (B) was quantified and represented as percentage of the total area of the plate. *P = 0.0023.


D Cell lysates of LNCaP cells stably expressing an empty vector ( Vector ) or a vector expressing ATF4 ( ATF4 ) were prepared and subjected to Western blot analysis with the indicated antisera .

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D Cell lysates of LNCaP cells stably expressing an empty vector (Vector) or a vector expressing ATF4 (ATF4) were prepared and subjected to Western blot analysis with the indicated antisera.


E After transfection with the indicated siRNAs , cells from ( D ) were cultured for 2 weeks . The colonies formed were stained and photographed . F The area of the colonies in ( E ) was quantitated using an image analysis system and represented as percentage of the total area of the plate . * P = 0 . 024 .

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E After transfection with the indicated siRNAs, cells from (D) were cultured for 2 weeks. The colonies formed were stained and photographed.F The area of the colonies in (E) was quantitated using an image analysis system and represented as percentage of the total area of the plate. *P = 0.024.


G LNCaP cells were transfected with either control ( Ctrl ) or ASNS siRNA . The cells were then cultured for 2 weeks . The colonies formed were stained and photographed . Knockdown of ASNS was confirmed by Western blot analysis shown at the top . H Quantification of the data shown in ( G ) . * P = 0 . 0087 .

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G LNCaP cells were transfected with either control (Ctrl) or ASNS siRNA. The cells were then cultured for 2 weeks. The colonies formed were stained and photographed. Knockdown of ASNS was confirmed by Western blot analysis shown at the top.H Quantification of the data shown in (G). *P = 0.0087.


A 293T cells were transfected with plasmids expressing either an empty vector ( Vec ) or vectors expressing HA - tagged STEAP , STAMP1 ( ST1 ) , STAMP2 ( ST2 ) , or STAMP3 ( ST3 ) . Whole - cell extracts were prepared and subjected to Western blot analysis using anti - HA antibody or GAPDH as a loading control .

A 293T cells were transfected with plasmids expressing either an empty vector (Vec) or vectors expressing HA-tagged STEAP, STAMP1 (ST1), STAMP2 (ST2), or STAMP3 (ST3). Whole-cell extracts were prepared and subjected to Western blot analysis using anti-HA antibody or GAPDH as a loading control.


B Relative ferrireductase activities were measured in the cells expressing the different constructs used in ( A ) as described in Materials and Methods . * P < 0 . 0001 .

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B Relative ferrireductase activities were measured in the cells expressing the different constructs used in (A) as described in Materials and Methods. *P < 0.0001.


C 293T cells were transfected with different plasmids expressing either an empty vector ( Vec ) or vectors expressing His - tagged wild - type STAMP2 ( WT ) or three STAMP2 mutants ( dGSR , H304L , or H397L ) as indicated . Whole - cell extracts were prepared and used in Western blot analysis with anti - His antibody or GAPDH antibody as a loading control .

C 293T cells were transfected with different plasmids expressing either an empty vector (Vec) or vectors expressing His-tagged wild-type STAMP2 (WT) or three STAMP2 mutants (dGSR, H304L, or H397L) as indicated. Whole-cell extracts were prepared and used in Western blot analysis with anti-His antibody or GAPDH antibody as a loading control.


D Relative ferrireductase activities were measured in the cells from ( C ) as described in Materials and Methods . * P = 0 . 0003 .

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D Relative ferrireductase activities were measured in the cells from (C) as described in Materials and Methods. *P = 0.0003.


E 293T cell line with Dox - inducible STAMP2 expression or vector control was generated . Western blot analysis confirmed STAMP2 expression in a Dox - inducible manner .

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E 293T cell line with Dox-inducible STAMP2 expression or vector control was generated. Western blot analysis confirmed STAMP2 expression in a Dox-inducible manner.


F The cells from ( E ) were either left untreated or treated with increasing amounts of Dox , and ferrireductase activity was determined . * P < 0 . 0001 .

F The cells from (E) were either left untreated or treated with increasing amounts of Dox, and ferrireductase activity was determined. *P < 0.0001.


G The cells with Dox - inducible STAMP2 expression from ( E ) were either left untreated or treated with Dox for 48 h . Prior to ferrireductase activity measurement , the cells were treated with or without DPI as indicated for 1 h . * P < 0 . 0001 ; * * P = 0 . 0012 ; * * * P = 0 . 048 .

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G The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Prior to ferrireductase activity measurement, the cells were treated with or without DPI as indicated for 1 h. *P < 0.0001; **P = 0.0012; ***P = 0.048.


H The cells from ( E ) were treated with 100 ng / ml Dox for 2 days . Oxidative stress was then measured by NBT staining , and the stained cells were photographed . There were equal numbers of cells on the plates for − / + Dox as shown in Supplementary Fig S4 .

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H The cells from (E) were treated with 100 ng/ml Dox for 2 days. Oxidative stress was then measured by NBT staining, and the stained cells were photographed. There were equal numbers of cells on the plates for −/+ Dox as shown in Supplementary Fig S4.


I The cells with Dox - inducible STAMP2 expression from ( E ) were either left untreated or treated with Dox for 48 h . Then , the cells were treated with or without 10 μM DPI before being subjected to NBT staining . * P < 0 . 0001 .

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I The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Then, the cells were treated with or without 10 μM DPI before being subjected to NBT staining. *P < 0.0001.


A LNCaP cells stably expressing an empty vector ( LN - Vec ) or a vector expressing STAMP2 ( LN - ST2 ) were established by lentivirus delivery . Cell lysates were prepared and subjected to Western blot analysis with the indicated antisera .

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A LNCaP cells stably expressing an empty vector (LN-Vec) or a vector expressing STAMP2 (LN-ST2) were established by lentivirus delivery. Cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.


B Relative ferrireductase activities were measured in the cells described in ( A ) . * P = 0 .

B Relative ferrireductase activities were measured in the cells described in (A). *P = 0.0003


C NBT staining was performed in LNCaP cells stably expressing either an empty vector ( LN - Vec ) or a vector expressing wild - type STAMP2 ( LN - ST2 / WT ) or a STAMP2 mutant ( LN - ST2 / dGSR ) as indicated . The stained cells were then photographed and quantified . * P = 0 . 004 .

C NBT staining was performed in LNCaP cells stably expressing either an empty vector (LN-Vec) or a vector expressing wild-type STAMP2 (LN-ST2/WT) or a STAMP2 mutant (LN-ST2/dGSR) as indicated. The stained cells were then photographed and quantified. *P = 0.004.


D LNCaP cells were transfected with either scrambled siRNA ( siCtrl ) or siRNA against STAMP2 ( siST2 ) and were cultured in the presence of 10 nM R1881 or vehicle for 2 days . Intracellular ROS levels were then measured by CellROX reagent staining . * P = 0 . 002 . ns , not significant .

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D LNCaP cells were transfected with either scrambled siRNA (siCtrl) or siRNA against STAMP2 (siST2) and were cultured in the presence of 10 nM R1881 or vehicle for 2 days. Intracellular ROS levels were then measured by CellROX reagent staining. *P = 0.002. ns, not significant.


E LNCaP cells stably expressing an empty vector ( Vec ) , a vector expressing wild - type STAMP2 ( WT ) , or a STAMP2 mutant ( dGSR ) were cultured , and the NADPH / NADP + ratio was determined as described in Materials and Methods . * P = 0 . 015 .

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E LNCaP cells stably expressing an empty vector (Vec), a vector expressing wild-type STAMP2 (WT), or a STAMP2 mutant (dGSR) were cultured, and the NADPH/NADP+ ratio was determined as described in Materials and Methods. *P = 0.015.


F LNCaP cells were transfected with either control siRNA ( siCtrl ) or STAMP2 siRNA ( siST2 ) . Cells were then cultured for 2 days and harvested , and the NADPH / NADP + ratio was determined as above . * P = 0 . 026 .

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F LNCaP cells were transfected with either control siRNA (siCtrl) or STAMP2 siRNA (siST2). Cells were then cultured for 2 days and harvested, and the NADPH/NADP+ ratio was determined as above. *P = 0.026.


G LNCaP cells stably expressing wild - type STAMP2 were treated with or without DPI ( 1 μM ) for 4 h , harvested , and used in the NADPH / NADP + assay . * P = 0 . 018 .

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G LNCaP cells stably expressing wild-type STAMP2 were treated with or without DPI (1 μM) for 4 h, harvested, and used in the NADPH/NADP+ assay. *P = 0.018.


A LNCaP cells were implanted subcutaneously into nu / nu mice . Once tumors reached 5 mm in size , mice ( n = 5 per group ) were given nanoliposome - encapsulated control siRNA or STAMP2 siRNA as described in Materials and Methods . Tumor volumes were measured at the indicated time points . * P = 0 . 001 .

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A LNCaP cells were implanted subcutaneously into nu/nu mice. Once tumors reached 5 mm in size, mice (n = 5 per group) were given nanoliposome-encapsulated control siRNA or STAMP2 siRNA as described in Materials and Methods. Tumor volumes were measured at the indicated time points. *P = 0.001.


B Same experiment as in ( A ) , but VCaP cells were used instead . * P = 0 . 001 .

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B Same experiment as in (A), but VCaP cells were used instead. *P = 0.001.


D , Western blot showing AGO1x expression in three cell lines . For comparison , a parallel blot was probed with an antibody directed against canonical AGO1 . Tubulin served as loading control .

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D, Western blot showing AGO1x expression in three cell lines. For comparison, a parallel blot was probed with an antibody directed against canonical AGO1. Tubulin served as loading control.


E , F , Annotated MS / MS spectrum of peptides specific for the endogenous AGO1x , " QNAVTSLDR " , depicted in green ( E ) and " LSKPQELCHPNPEEAR " , depicted in blue ( F ) . The Mascot Ion Score ( text color corresponds to peptides marked in Fig 1C for reference ) as well as the annotated fragments ( blue = y - ions ; red = b - ions ) together with the corresponding amino acids are indicated .

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E,F, Annotated MS/MS spectrum of peptides specific for the endogenous AGO1x, "QNAVTSLDR", depicted in green (E) and "LSKPQELCHPNPEEAR", depicted in blue (F). The Mascot Ion Score (text color corresponds to peptides marked in Fig 1C for reference) as well as the annotated fragments (blue = y-ions; red = b-ions) together with the corresponding amino acids are indicated.


A , Representative immunofluorescence images showing the subcellular distribution of AGO1x ( red ) relative to nuclear and cytoplasmic markers . DAPI was used to mark the nucleus ( blue ) . The co - stained subcellular marker is indicated in each panel in green . SC 35 , Lsm4 , α - Tubulin , ERP72 , p54 ( NRB ) , and Nucleolin serve as markers for nuclear speckles , cytosol and nucleus , cytosol , endoplasmic reticulum , paraspeckle and nucleolus , respectively .

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A, Representative immunofluorescence images showing the subcellular distribution of AGO1x (red) relative to nuclear and cytoplasmic markers. DAPI was used to mark the nucleus (blue). The co-stained subcellular marker is indicated in each panel in green. SC 35, Lsm4, α-Tubulin, ERP72, p54 (NRB), and Nucleolin serve as markers for nuclear speckles, cytosol and nucleus, cytosol, endoplasmic reticulum, paraspeckle and nucleolus, respectively.


B , Mean ( + / - s . d . ) pixel intensities of AGO1x staining in nucleolus and nucleoplasm , computed from z - stack images of MDA - MB - 231 cells ( n = 20 ) . The P - value was determined using a paired two - tailed t - test .

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B, Mean (+/- s.d.) pixel intensities of AGO1x staining in nucleolus and nucleoplasm, computed from z-stack images of MDA-MB-231 cells (n=20). The P-value was determined using a paired two-tailed t-test.


C , Representative AGO1x and AGO1 blot from MDA - MB - 231 cell fractions . GAPDH and hnRNP C1 / C2 served as markers of purity of the individual fractions .

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C, Representative AGO1x and AGO1 blot from MDA-MB-231 cell fractions. GAPDH and hnRNP C1/C2 served as markers of purity of the individual fractions.


A , Top : Scheme of the sites targeted by sgRNA in the generation of AGO1x mutant cell lines . Bottom : Western analysis of lysates from mutant MDA - MB - 231 cells showing the depletion of AGO1x and the comparable total AGO1 levels across the cell lines . Three independently collected sets of cells were lysed and analysed in parallel . After calculating the levels of AGO1x and AGO1 relative to GAPDH , values corresponding to individual lanes were normalized to the average over the replicates of the control line . These numbers are indicated below the blots . The P - values of the t - tests comparing the expression AGO1x in mutant ( W1A and W6A ) and control ( CTRL ) lines were W1A - CTRL : 0 . 0008 , W6A - CTRL : 0 . 0002 , and for AGO1 W1A - CTRL : 0 . 1227 , W6A - CTRL : 0 . 1173 .

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A, Top: Scheme of the sites targeted by sgRNA in the generation of AGO1x mutant cell lines. Bottom: Western analysis of lysates from mutant MDA-MB-231 cells showing the depletion of AGO1x and the comparable total AGO1 levels across the cell lines. Three independently collected sets of cells were lysed and analysed in parallel. After calculating the levels of AGO1x and AGO1 relative to GAPDH, values corresponding to individual lanes were normalized to the average over the replicates of the control line. These numbers are indicated below the blots. The P-values of the t-tests comparing the expression AGO1x in mutant (W1A and W6A) and control (CTRL) lines were W1A-CTRL: 0.0008, W6A-CTRL: 0.0002, and for AGO1 W1A-CTRL: 0.1227, W6A-CTRL: 0.1173.


B , Phase contrast images of control and mutant cell lines at 24 and 48 hr after plating equal numbers ( 10 ' 000 ) of cells in individual wells of a six well plate .

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B, Phase contrast images of control and mutant cell lines at 24 and 48 hr after plating equal numbers (10'000) of cells in individual wells of a six well plate.


C , Impedance - based mean ( + / - s . d . ) cell indices at the indicated time points after seeding equal numbers of CTRL ( n = 6 ) , W1A ( n = 5 ) and W6A ( n = 5 ) cells . From 6 hours on , there is a statistically significant difference between control and the two mutant cell lines ( P < 0 . 001 , two tailed t - test ) . D , Impedance - based mean ( + / - s . d . ) cell indices as a function of time for control ( n = 3 ) , W1A ( n = 4 ) and W6A ( n = 3 ) cells grown in electronically monitored Boyden chambers . From 12 hours on , there is a statistical significant difference between control and the two mutants ( P < 0 . 005 , two tailed t - test ) .

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C, Impedance-based mean (+/- s.d.) cell indices at the indicated time points after seeding equal numbers of CTRL (n=6), W1A (n=5) and W6A (n=5) cells. From 6 hours on, there is a statistically significant difference between control and the two mutant cell lines (P < 0.001, two tailed t-test). D, Impedance-based mean (+/- s.d.) cell indices as a function of time for control (n=3), W1A (n=4) and W6A (n=3) cells grown in electronically monitored Boyden chambers. From 12 hours on, there is a statistical significant difference between control and the two mutants (P < 0.005, two tailed t-test).


E , Mean ( + / - s . d . ) number of colonies obtained in a soft agar colony formation assay of control , W1A and W6A mutant cell lines ( all n = 4 , P - value determined using an unpaired two - tailed t - test ) . F , Mean ( + / - s . d . ) number of spheres obtained from a sphere formation assay of control , W1A and W6A mutant cell lines ( all n = 8 , P - values computed as in E ) .

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E, Mean (+/- s.d.) number of colonies obtained in a soft agar colony formation assay of control, W1A and W6A mutant cell lines (all n=4, P-value determined using an unpaired two-tailed t-test). F, Mean (+/- s.d.) number of spheres obtained from a sphere formation assay of control, W1A and W6A mutant cell lines (all n=8, P-values computed as in E).


G , AGO1x staining of tissue sections from breast cancers , classified as low - proliferating or high - proliferating based on Ki - 67 staining . The upper and lower panel represents two different tissue sections . H , Mean ( + / - s . d . ) percentage of AGO1x positive cells in breast tumors with low ( n = 7 ) and high ( n = 9 ) proliferation index , assessed by the expression level of Ki - 67 . P - value was determined using an unpaired two - tailed t - test .

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G, AGO1x staining of tissue sections from breast cancers, classified as low-proliferating or high-proliferating based on Ki-67 staining. The upper and lower panel represents two different tissue sections. H, Mean (+/- s.d.) percentage of AGO1x positive cells in breast tumors with low (n=7) and high (n=9) proliferation index, assessed by the expression level of Ki-67. P-value was determined using an unpaired two-tailed t-test.


A , Scatter plot of mean log2 mRNA fold - changes in the mutant cell lines versus control ( n = 3 ) . Shown are also the Pearson correlation coefficient and respective P - value . The dashed line indicates equal change in the two mutant lines .

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A, Scatter plot of mean log2 mRNA fold-changes in the mutant cell lines versus control (n=3). Shown are also the Pearson correlation coefficient and respective P-value. The dashed line indicates equal change in the two mutant lines.


C , Mean ( + / - s . d . ) activity of STAT1 / IRF1 / IRF2 / IRF3 / IRF9 transcription factor motifs estimated by ISMARA ( Balwierz et al , 2014 ) ( n = 3 ) . Shown are the P - values ( * * * P < 0 . 001 ) determined by Dunn ' s multiple comparisons test post hoc and the non - parametric Kruskal - Wallis test ( P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.d.) activity of STAT1/IRF1/IRF2/IRF3/IRF9 transcription factor motifs estimated by ISMARA (Balwierz et al, 2014) (n=3). Shown are the P-values (*** P < 0.001) determined by Dunn's multiple comparisons test post hoc and the non-parametric Kruskal-Wallis test (P < 0.0001).


D , Mean ( + / - s . e . m . ) log2 mRNA expression fold - changes ( inferred from RNA - Seq ) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control ( n = 3 ) . Multiple - testing - corrected P - values for Wald tests comparing fold - changes with respect to control are depicted above each bar ( * * 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

D, Mean (+/- s.e.m.) log2 mRNA expression fold-changes (inferred from RNA-Seq) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control (n=3). Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (** P < 0.01, *** P < 0.001).


E , Mean ( + / - s . d . ) expression fold - changes of the corresponding proteins ( if detected in the proteomics data ) in the two mutant cell lines ( W1A , n = 2 ; W6A , n = 1 ) compared to Control ( n = 2 ) . Multiple - testing - corrected P - values for unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * 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

E, Mean (+/- s.d.) expression fold-changes of the corresponding proteins (if detected in the proteomics data) in the two mutant cell lines (W1A, n=2; W6A, n=1) compared to Control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).


F , Representative result of the apoptosis assay using AnnexinV and propidium iodide ( PI ) staining in the control and the two mutant cell lines ( left ) . The percentage of cells in each quadrant is depicted for each cell line . Quantification of the mean ( + / - s . d . ) percentage of apoptotic cells ( Q1 + Q2 , AnnexinV + ) across the different cell lines ( n = 4 ) ( right ) . Shown is the P - value determined by unpaired two - tailed 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

F, Representative result of the apoptosis assay using AnnexinV and propidium iodide (PI) staining in the control and the two mutant cell lines (left). The percentage of cells in each quadrant is depicted for each cell line. Quantification of the mean (+/- s.d.) percentage of apoptotic cells (Q1+Q2, AnnexinV+) across the different cell lines (n=4) (right). Shown is the P-value determined by unpaired two-tailed t-test.


G , Impedance - based mean ( + / - s . d . ) cell index values at the indicated time points of growth after seeding equal numbers of control , W1A and W6A cells , upon treatment with DMSO or Ruxolitinib . For the DMSO treatment : Control ( n = 4 ) , W1A ( n = 3 ) and W6A ( n = 3 ) . For the Ruxolitinib treatment : Control ( n = 6 ) , W1A ( n = 3 ) and W6A ( n = 4 ) . From 6 hours after DMSO treatment , there is a statistically significant difference between control and the two mutants ( P < 0 . 05 , two tailed t - test ) , whereas no statistically significant difference is found after Ruxolitinib treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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-CONTROLLED_VAR O O

G, Impedance-based mean (+/- s.d.) cell index values at the indicated time points of growth after seeding equal numbers of control, W1A and W6A cells, upon treatment with DMSO or Ruxolitinib. For the DMSO treatment: Control (n=4), W1A (n=3) and W6A (n=3). For the Ruxolitinib treatment: Control (n=6), W1A (n=3) and W6A (n=4). From 6 hours after DMSO treatment, there is a statistically significant difference between control and the two mutants (P < 0.05, two tailed t-test), whereas no statistically significant difference is found after Ruxolitinib treatment.


B , Mean ( + / - s . e . m . ) log2 mRNA fold - changes of dsRNAs sensors ( from panel A ) in the two mutant cell lines compared to control ( n = 3 ) inferred from RNA - Seq . Multiple - testing - corrected P - values for Wald tests comparing fold - changes with respect to control are depicted above each bar ( * * * 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

B, Mean (+/- s.e.m.) log2 mRNA fold-changes of dsRNAs sensors (from panel A) in the two mutant cell lines compared to control (n=3) inferred from RNA-Seq. Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (*** P < 0.001).


C , Mean ( + / - s . d . ) fold - changes of corresponding proteins ( see B ) , if detected in the proteomics data from the two mutant ( W1A , n = 2 ; W6A , n = 1 ) lines relative to control ( n = 2 ) . Multiple - testing - corrected P - values for unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

C, Mean (+/- s.d.) fold-changes of corresponding proteins (see B), if detected in the proteomics data from the two mutant (W1A, n=2; W6A, n=1) lines relative to control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).


D , Western blot detection of phosphorylated eIF2α ( Ser51 ) and total eIF2α levels in cell lysates of control and mutant MDA - MB - 231 cells . GAPDH and Tubulin serve as loading control for phosphorylated eIF2α and total eIF2α blots respectively . Numbers indicated below the blots represent the band intensities of target proteins normalized to the loading controls , with the value in the control line taken as baseline of 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Western blot detection of phosphorylated eIF2α (Ser51) and total eIF2α levels in cell lysates of control and mutant MDA-MB-231 cells. GAPDH and Tubulin serve as loading control for phosphorylated eIF2α and total eIF2α blots respectively. Numbers indicated below the blots represent the band intensities of target proteins normalized to the loading controls, with the value in the control line taken as baseline of 1.


E , Mean ( + / - s . d . ) fold - changes in phosphorylated peptide ( phosphorylation sites are indicated in brackets ) levels derived from a proteomic analysis of phosphopeptides in the two mutant lines ( W1A , n = 2 ; W6A , n = 2 ) compared to control ( n = 1 ) . Multiple - testing - corrected P - values for Wald tests ( transcriptomics ) or unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.d.) fold-changes in phosphorylated peptide (phosphorylation sites are indicated in brackets) levels derived from a proteomic analysis of phosphopeptides in the two mutant lines (W1A, n=2; W6A, n=2) compared to control (n=1). Multiple-testing-corrected P-values for Wald tests (transcriptomics) or unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (** P < 0.01).


F , Quantification of the relative abundance ( mean + / - s . e . m . ) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane , non - overlapping foci were counted from 100 cells of each type , and the numbers in three abundance bins ( < 1 , 1 - 5 , > 5 ) are shown .

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

F, Quantification of the relative abundance (mean+/- s.e.m.) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane, non-overlapping foci were counted from 100 cells of each type, and the numbers in three abundance bins (<1, 1-5, >5) are shown.


G , Representative immunofluorescence image of the control and mutant MDA - MB - 231 cells stained with AGO1x ( green ) and J2 antibody ( red ) and DAPI ( blue ) to mark the nucleus . Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR - induced mutations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative immunofluorescence image of the control and mutant MDA-MB-231 cells stained with AGO1x (green) and J2 antibody (red) and DAPI (blue) to mark the nucleus. Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR-induced mutations.


Concentration of recovered RNA ( mean + / - s . e . m . ) measured by Nanodrop ™ ( H ) Multiple testing corrected P - values from unpaired two - tailed t - tests are depicted above each comparison ( * P < 0 . 05 , * * * 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

Concentration of recovered RNA (mean +/- s.e.m.) measured by Nanodrop™ (H) Multiple testing corrected P-values from unpaired two-tailed t-tests are depicted above each comparison (* P < 0.05, *** P < 0.001).


relative abundance of various RNA species ( mean + / - s . d . ) in J2 antibody immunoprecipitates from three biological replicates of control and mutant cell lines ( I ) . Multiple testing corrected P - values from unpaired two - tailed t - tests are depicted above each comparison ( * P < 0 . 05 , * * * 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

relative abundance of various RNA species (mean +/- s.d.) in J2 antibody immunoprecipitates from three biological replicates of control and mutant cell lines (I). Multiple testing corrected P-values from unpaired two-tailed t-tests are depicted above each comparison (* P < 0.05, *** P < 0.001).


K , Boxplots showing the proportion of G / C nucleotides in all genes and in genes depleted / enriched in dsRNA - seq compared to total RNA - seq ( n = 3 ) . Shown are the P - values in the non - parametric Mann - Whitney U test ( * * * P < 0 . 001 ) . Boxes extend from the 25th to 75th percentiles ( inter - quartile range ( IQR ) ) , horizontal lines represent the median , whiskers indicate the lowest and highest datum within 1 . 5 * IQR from the lower and upper quartiles , respectively .

K, Boxplots showing the proportion of G/C nucleotides in all genes and in genes depleted/enriched in dsRNA-seq compared to total RNA-seq (n=3). Shown are the P-values in the non-parametric Mann-Whitney U test (*** P < 0.001). Boxes extend from the 25th to 75th percentiles (inter-quartile range (IQR)), horizontal lines represent the median, whiskers indicate the lowest and highest datum within 1.5*IQR from the lower and upper quartiles, respectively.


L , Left : Quantification of transcript abundance ( mean + / - s . e . m . ) by qRT - PCR in AGO1x ( black , n = 6 for putative targets , n = 3 for negative controls ) or IgG ( orange , n = 2 ) IP from control cells . n . d . , not detected . Multiple test corrected P - values for an unpaired two - tailed t - test are depicted above each comparison ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Right : Concentration of RNA ( mean + / - s . d . ) obtained with each type of antibody , with P - value from an unpaired 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

L, Left: Quantification of transcript abundance (mean +/- s.e.m.) by qRT-PCR in AGO1x (black, n=6 for putative targets, n=3 for negative controls) or IgG (orange, n=2) IP from control cells. n.d., not detected. Multiple test corrected P-values for an unpaired two-tailed t-test are depicted above each comparison (* P < 0.05, ** P < 0.01, *** P < 0.001). Right: Concentration of RNA (mean +/- s.d.) obtained with each type of antibody, with P-value from an unpaired two-tailed Student's t-test.


B , Western blot analysis to validate the interaction of AGO1x with PNPT1 and DHX9 in control MDA - MB - 231cells . AGO1x pulldowns were compared to IgG pulldowns to verify the relative levels of PNPT1 and DHX9 from four independent biological replicates . The immunoglobulin heavy chain band served as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Western blot analysis to validate the interaction of AGO1x with PNPT1 and DHX9 in control MDA-MB-231cells. AGO1x pulldowns were compared to IgG pulldowns to verify the relative levels of PNPT1 and DHX9 from four independent biological replicates. The immunoglobulin heavy chain band served as a loading control.


Immunoprecipitation of PNPT1 from control and mutant MDA - MB - 231 cells and western blotting to validate the specificity of PNPT1 antibody in comparison to IgG control ( C ) .

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

Immunoprecipitation of PNPT1 from control and mutant MDA-MB-231 cells and western blotting to validate the specificity of PNPT1 antibody in comparison to IgG control (C).


Validation of AGO1x - PNPT1 interaction by reciprocal pulldown . Presence of AGO1x and Fibrillarin in PNPT1 immunoprecipitates . Three independent biological replicates were used , and the eluted proteins were analyzed by western blotting ( 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

Validation of AGO1x-PNPT1 interaction by reciprocal pulldown. Presence of AGO1x and Fibrillarin in PNPT1 immunoprecipitates. Three independent biological replicates were used, and the eluted proteins were analyzed by western blotting (D).


Western blot analysis to verify the depletion of PNPT1 upon siRNA treatment ( F )

Western blot analysis to verify the depletion of PNPT1 upon siRNA treatment (F)


a representative immunofluorescence image of siPNPT1 / siControl - treated control and W6A mutant MDA - MB - 231 cells stained with J2 antibody ( red ) . DAPI was used to mark the nucleus ( blue ) ( G ) . The right - most panels show a magnification of the cells enclosed by the white boxes in the middle 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

a representative immunofluorescence image of siPNPT1/siControl-treated control and W6A mutant MDA-MB-231 cells stained with J2 antibody (red). DAPI was used to mark the nucleus (blue) (G). The right-most panels show a magnification of the cells enclosed by the white boxes in the middle panels.


A Anti - TK Western blot of ubiquitinated titin samples .

A Anti-TK Western blot of ubiquitinated titin samples.


( B - D ) Micrographs of H9C2 cells corresponding to ( B ) expressions of single species : MuRF1 - mCherry ( red ) , titin EGFP - A168 - TK ( green ) , Venus - Nbr1 ( yellow ) or Cerulean - p62 ( cyan ) ; ( C ) Co - expression of titin EGFP - A168 - TK with mCherry - MuRF1 . Neither Nbr1 nor p62 were co - transfected . The titin fragment and MuRF1 co - localize forming filamentous formations , which is in agreement with their known complexation ; ( D ) Co - expression of MuRF1 - mCherry ( red ) , titin EGFP - A168 - TK ( green ) and Venus - Nbr1 ( yellow ) or Cerulean - p62 ( cyan ) or respective truncated variants of the latter lacking the ubiquitin binding domain ( ∆ UBA ) . ( Throughout , cell nuclei are stained with DAPI ; blue ) . ( Scale bar is 10 μm in all cases ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Micrographs of H9C2 cells corresponding to (B) expressions of single species: MuRF1-mCherry (red), titin EGFP-A168-TK (green), Venus-Nbr1 (yellow) or Cerulean-p62 (cyan); (C) Co-expression of titin EGFP-A168-TK with mCherry-MuRF1. Neither Nbr1 nor p62 were co-transfected. The titin fragment and MuRF1 co-localize forming filamentous formations, which is in agreement with their known complexation; (D) Co-expression of MuRF1-mCherry (red), titin EGFP-A168-TK (green) and Venus-Nbr1 (yellow) or Cerulean-p62 (cyan) or respective truncated variants of the latter lacking the ubiquitin binding domain (∆UBA). (Throughout, cell nuclei are stained with DAPI; blue). (Scale bar is 10 μm in all cases).


A : Brightfield and fluorescence images of a NTH / GFP zygote at 4h post - fertilization , showing dispersed extranuclear fluorescence . Hoechst DNA staining ( blue ) marks nucleus . Scale bar = 5μm . B : Image of an NTH / GFP ookinete , showing fluorescent spots co - localizing with pigment clusters ( arrows ) . Hoechst DNA staining ( blue ) marks nucleus . Scale bar = 5μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Brightfield and fluorescence images of a NTH/GFP zygote at 4h post-fertilization, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. B: Image of an NTH/GFP ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm.


D : Brightfield and fluorescence images of a zygote at 4h post - fertilization simultaneously expressing NTH : : GFP ( green ) and LAP3 : : mCherry ( red ) . Scale bar = 5μm . E : An ookinete simultaneously expressing NTH : : GFP ( green ) and LAP3 : : mCherry ( red ) , showing co - localization in the crystalloid . Scale bar = 5μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Brightfield and fluorescence images of a zygote at 4h post-fertilization simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red). Scale bar = 5μm. E: An ookinete simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red), showing co-localization in the crystalloid. Scale bar = 5μm.


F : Western blot of purified NTH / GFP ookinete samples with ( + ) and without ( - ) prior in vivo crosslinking . Mr markers ( kDa ) are indicated on the right - hand side .

F: Western blot of purified NTH/GFP ookinete samples with (+) and without (-) prior in vivo crosslinking. Mr markers (kDa) are indicated on the right-hand side.


A : Brightfield images of a NTH - KO and NTH / GFP ookinete , showing normal ookinete morphology , but lack of pigment clusters associated with crystalloids ( arrows ) in the NTH null mutant . Scale bar = 5μm .

O O O O O O O O O O O O O O O O O O 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: Brightfield images of a NTH-KO and NTH/GFP ookinete, showing normal ookinete morphology, but lack of pigment clusters associated with crystalloids (arrows) in the NTH null mutant. Scale bar = 5μm.


B : Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH / GFP , NTH - KO and NTH ∆ PP . Horizontal lines mark mean values ( n = 20 ) ; ns , not significantly different ( Mann - Whitney ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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: Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20); ns, not significantly different (Mann-Whitney).


C : Brightfield and fluorescence images of oocysts of parasite lines NTH / GFP and NTH - KO at 15 days post - infection , showing lack of sporulation in the NTH null mutant . Hoechst DNA staining ( artificially depicted red ) labels parasite nuclei . The large nuclei of the epithelial midgut cells also show . Scale bar = 20μm .

C: Brightfield and fluorescence images of oocysts of parasite lines NTH/GFP and NTH-KO at 15 days post-infection, showing lack of sporulation in the NTH null mutant. Hoechst DNA staining (artificially depicted red) labels parasite nuclei. The large nuclei of the epithelial midgut cells also show. Scale bar = 20μm.


D : Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post - infection with parasite lines NTH / GFP , NTH - KO and NTH ∆ PP . Horizontal lines mark mean values ( n = 20 ) . * * mark statistically significant differences ( P < 0 . 0001 , Mann - Whitney ) ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post-infection with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20). ** mark statistically significant differences (P<0.0001, Mann-Whitney); ns, not significant.


E : Images of ookinetes of parasite lines NTH ∆ PP showing dispersed NTH ∆ PP : : GFP fluorescence ; LAP3 / mCherry showing focal LAP3 : : mCherry fluorescence in crystalloids ; Cross ( carrying modified alleles encoding NTH ∆ PP : : GFP and LAP3 : : mCherry ) , showing dispersed LAP3 : : mCherry fluorescence . Arrow marks pigment cluster associated with crystalloids . Scale bar = 5μm .

E: Images of ookinetes of parasite lines NTH∆PP showing dispersed NTH∆PP::GFP fluorescence; LAP3/mCherry showing focal LAP3::mCherry fluorescence in crystalloids; Cross (carrying modified alleles encoding NTH∆PP::GFP and LAP3::mCherry), showing dispersed LAP3::mCherry fluorescence. Arrow marks pigment cluster associated with crystalloids. Scale bar = 5μm.


G : PCR diagnostic for the presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH ∆ PP x LAP3 / mCherry genetic cross ( lane NTH ∆ PP ) , amplifying an approximately 4 . 4kb fragment . NTH / GFP parasites are used as a PCR control , amplifying approximately 4 . 6kb . Parental parasites provide a negative control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: PCR diagnostic for the presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH∆PP x LAP3/mCherry genetic cross (lane NTH∆PP), amplifying an approximately 4.4kb fragment. NTH/GFP parasites are used as a PCR control, amplifying approximately 4.6kb. Parental parasites provide a negative control.


H : Image of a NTH / ND500LK ookinete , showing fluorescent spots co - localizing with pigment clusters ( arrows ) indicative of normal crystalloid formation . Scale bar = 5μm .

O O O O O O O O O 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: Image of a NTH/ND500LK ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows) indicative of normal crystalloid formation. Scale bar = 5μm.


A : Brightfield and fluorescence image of an unsporulated NTH / GFP oocyst at 15 days post - infection . Scale bar = 20μm . B : Image of a sporulated NTH / GFP oocyst at 15 days post - infection . Scale bar = 20μm .

O O O O O O O O O O O O O O O O O O O O O O O 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: Brightfield and fluorescence image of an unsporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm. B: Image of a sporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm.


C : Image of a NTH / GFP sporozoite , showing NTH : : GFP localisation in a tubular structure . Scale bar = 10μm .

O O 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: Image of a NTH/GFP sporozoite, showing NTH::GFP localisation in a tubular structure. Scale bar = 10μm.


D : Image of a sporulated oocyst simultaneously expressing NTH : : GFP ( green ) and ACP : : mCherry ( red ) , showing co - localization in the apicoplast . Scale bar = 20μm and 10μm ( inset ) .

O O O O O O O O O O O O O O O O O O O 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: Image of a sporulated oocyst simultaneously expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast. Scale bar = 20μm and 10μm (inset).


E : Image of sporozoites expressing NTH : : GFP ( green ) and ACP : : mCherry ( red ) , showing co - localization in the apicoplast ( arrows ) . Sporozoites expressing only ACP : : mCherry or NTH : : GFP are also present . Scale bar = 10μm .

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E: Image of sporozoites expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast (arrows). Sporozoites expressing only ACP::mCherry or NTH::GFP are also present. Scale bar = 10μm.


F : Image of group of NTH / GFP sporozoites stained with MitoTracker Red CMXRos , showing distinct subcellular positions of apicoplasts ( green ) and mitochondria ( red ) . Scale bar = 10μm .

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F: Image of group of NTH/GFP sporozoites stained with MitoTracker Red CMXRos, showing distinct subcellular positions of apicoplasts (green) and mitochondria (red). Scale bar = 10μm.


( D ) C / EBP β and AEP immunofluorescent staining of colons from age - matched healthy control and AD patients . White arrows indicate that C / EBP β and AEP were co - localized in AD patient ' s colon . ( E ) AEP and cleaved APP C586 fragments immunofluorescent staining of colons from age - matched healthy control and AD patient . White arrows indicate that AEP and cleaved APP C586 were co - localized in AD patient ' s colon . ( F ) AEP and cleaved Tau N368 fragments immunofluorescent staining of colons from age - matched healthy control and AD patient . Scale bar : 20 μm . White arrows indicate that APP C586 and AEP were co - localized in AD patient ' s colon .

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(D) C/EBP β and AEP immunofluorescent staining of colons from age-matched healthy control and AD patients. White arrows indicate that C/EBP β and AEP were co-localized in AD patient's colon. (E) AEP and cleaved APP C586 fragments immunofluorescent staining of colons from age-matched healthy control and AD patient. White arrows indicate that AEP and cleaved APP C586 were co-localized in AD patient's colon. (F) AEP and cleaved Tau N368 fragments immunofluorescent staining of colons from age-matched healthy control and AD patient. Scale bar: 20 μm. White arrows indicate that APP C586 and AEP were co-localized in AD patient's colon.


( A ) Immunoblot showing p - C / EBP β , C / EBP β , AEP , APP , and Tau expression and proteolytic processing in mouse colon . ( B ) Immunoblot showing p - C / EBP β , C / EBP β , AEP , APP , and Tau expression and proteolytic processing in mouse brain .

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(A) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse colon. (B) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse brain.


( B ) Quantitative analysis of AEP positive cells and C / EBPβ positive cells , respectively . The density of both AEP positive cells and C / EBPβ positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * P = 0 . 0390 ( * P < 0 . 05 ) , * * * P = 0 . 0002 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( C ) Quantitative analysis of AT8 positive cells ( left ) and TauN368 positive cells ( right ) , respectively . The density of both AT8 positive cells and TauN368 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . n = 5 in each group , data are shown as mean ± SEM . * P = 0 . 0194 ( * P < 0 . 05 ) , * * * P = 0 . 0001 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( D ) Quantitative analysis of Aβ positive cells ( left ) and APPC586 positive cells ( right ) , respectively . The fluorescence intensity of Aβ positive cells and the density APPC586 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * P = 0 . 0107 ( * P < 0 . 05 ) , * * P = 0 . 0048 ( 3xTg vs 3xTg AEP KO ) , * * P = 0 . 0044 ( 3xTg vs WT ) ( * * P < 0 . 01 ) , * * * P = 0 . 0006 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( E ) Quantitative analysis of AT8 positive cells ( upper ) and T22 positive cells ( lower ) , respectively . The fluorescence intensity of both AT8 positive cells and T22 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * * P = 0 . 0014 ( 3xTg vs 3xTg C / EBPβ + / - ) , * * P = 0 . 0030 ( 3xTg vs 3xTg AEP KO ) ( * * P < 0 . 01 ) , * * * P = 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative analysis of AEP positive cells and C/EBPβ positive cells, respectively. The density of both AEP positive cells and C/EBPβ positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P =0.0390 (*P < 0.05), ***P =0.0002 (***P < 0.001), ****P < 0.0001, one-way ANOVA. (C) Quantitative analysis of AT8 positive cells (left) and TauN368 positive cells (right), respectively. The density of both AT8 positive cells and TauN368 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. n=5 in each group, data are shown as mean± SEM. *P =0.0194 (*P< 0.05), ***P =0.0001 (***P < 0.001), ****P < 0.0001, one-way ANOVA. (D) Quantitative analysis of Aβ positive cells (left) and APPC586 positive cells (right), respectively. The fluorescence intensity of Aβ positive cells and the density APPC586 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P=0.0107 (*P < 0.05), **P=0.0048 (3xTg vs 3xTg AEP KO), **P=0.0044 (3xTg vs WT) (**P< 0.01), ***P=0.0006 (***P< 0.001), ****P < 0.0001, one-way ANOVA. (E) Quantitative analysis of AT8 positive cells (upper) and T22 positive cells (lower), respectively. The fluorescence intensity of both AT8 positive cells and T22 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. **P=0.0014 (3xTg vs 3xTg C/EBPβ+/-), **P=0.0030 (3xTg vs 3xTg AEP KO) (**P<0.01), ***P=0.001, ****P<0.0001, one-way ANOVA.


( G ) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining . ( Scale bar : 5 μm ) . ( H ) Quantitative analysis of the synaptic densities in DSS - treated mice . Representative data of five samples , Data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA . ( I ) Quantitative analysis of the spine density . Representative data of five samples , Data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA .

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(G) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (H) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA. (I) Quantitative analysis of the spine density. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA.


( E ) Gastrointestinal permeability barrier defect as determined by FITC - dextran translocation in pre - vagotomy DSS - treated , DSS - treated and vehicle - treated 3xTg mice . Data represent the mean ± SEM ; representative data of three samples ; * P = 0 . 0126 ( * P < 0 . 001 ) , * * * P = 0 . 0002 ( * * * P < 0 . 001 ) compared with control , one - way ANOVA .

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(E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P=0.0126 (*P < 0.001), ***P=0.0002 (***P < 0.001) compared with control, one-way ANOVA.


( F ) Pro - inflammatory cytokines IL - 6 , IL1 - β , and TNFα concentrations in colon lysates of vehicle - treated 3xTg mice , DSS - treated single side vagotomy 3xTg and DSS - treated 3xTg , respectively . Representative data of three samples ; data are shown as mean ± SEM . * P = 0 . 0372 ( * P < 0 . 05 ) , * * P = 0 . 0050 ( * * P < 0 . 01 ) , * * * P = 0 . 0008 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 compared with control , two - way ANOVA .

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(F) Pro-inflammatory cytokines IL-6, IL1-β, and TNFα concentrations in colon lysates of vehicle-treated 3xTg mice, DSS-treated single side vagotomy 3xTg and DSS-treated 3xTg, respectively. Representative data of three samples; data are shown as mean ± SEM. *P=0.0372 (*P < 0.05), **P=0.0050 (**P < 0.01), ***P=0.0008 (***P <0.001), ****P<0.0001 compared with control, two-way ANOVA.


( H ) Representative electron microscopy of the synaptic structures in hippocampus . Red stars indicate the synapses . ( Scale bar : 1 μm ) . ( I ) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining . ( Scale bar : 5 μm ) . ( J ) Quantitative analysis of the synaptic densities in DSS - treated mice . Representative data of five samples , data are shown as mean ± SEM . * * * * P < 0 . 0001 , unpaired t tests . ( K ) Quantitative analysis of the spine density . Representative data of five samples , data are shown as mean ± SEM . * * * P = 0 . 0001 ( * * * P < 0 . 001 ) , unpair t tests .

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(H) Representative electron microscopy of the synaptic structures in hippocampus. Red stars indicate the synapses. (Scale bar: 1 μm). (I) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (J) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, unpaired t tests. (K) Quantitative analysis of the spine density. Representative data of five samples, data are shown as mean ± SEM. ***P =0.0001 (***P < 0.001), unpair t tests.


( A & B ) Cued and Contextual Fear - conditioning tests . Data represent the mean ± SEM of n = 6 - 8 mice per group ; * * P = 0 . 0067 ( 3xTg + DSS vs 3xTg ctrl ) , * * P = 0 . 0059 ( 3xTg + DSS vs 3xTg R - vagotomy + DSS ) ( * * P < 0 . 01 ) , * * * P = 0 . 0009 ( * * * P < 0 . 001 ) , two - way ANOVA ( A ) ; * * P = 0 . 0086 ( 3xTg + DSS vs 3xTg ctrl ) , * * P = 0 . 0085 ( 3xTg + DSS vs 3xTg R - vagotomy + DSS ) ( * * P < 0 . 01 ) , * * * P = 0 . 0005 ( * * * P < 0 . 001 ) , one - way ANOVA ( B ) .

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(A&B) Cued and Contextual Fear-conditioning tests. Data represent the mean ± SEM of n = 6-8 mice per group; **P=0.0067 (3xTg+DSS vs 3xTg ctrl), **P=0.0059 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0009 (***P < 0.001), two-way ANOVA (A); **P=0.0086 (3xTg+DSS vs 3xTg ctrl), **P=0.0085 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0005 (***P < 0.001), one-way ANOVA (B).


( D ) Survival rate curve of the experimental mice during the experimental paradigm .

(D) Survival rate curve of the experimental mice during the experimental paradigm.


( B ) Immunofluorescent staining showed ATTO 550 - labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection . Scale bar : 20 μm .

(B) Immunofluorescent staining showed ATTO 550-labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection. Scale bar: 20 μm.


( C ) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic - injected with PBS , AD brain extracts or 5xFAD brain extracts , using human specific Aβ antibody ( BAN50 ) . Black arrows indicate the human Aβ in mouse vagus nerve . ( D ) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic - injected with PBS or AD brain extracts , using human Tau specific antibody ( HT7 ) . Black arrows indicate the human Tau in mouse vagus nerve .

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(C) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic-injected with PBS, AD brain extracts or 5xFAD brain extracts, using human specific Aβ antibody (BAN50). Black arrows indicate the human Aβ in mouse vagus nerve. (D) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic-injected with PBS or AD brain extracts, using human Tau specific antibody (HT7). Black arrows indicate the human Tau in mouse vagus nerve.


( A ) Immunofluorescent staining of AEP and p - C / EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs . Scale bar : 20 μm . White arrows indicate that AEP and p - C / EBP β signals ( green ) were colocalized with ATTO 550 signals ( red ) , respectively .

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(A) Immunofluorescent staining of AEP and p-C/EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs. Scale bar: 20 μm. White arrows indicate that AEP and p-C/EBP β signals (green) were colocalized with ATTO 550 signals (red), respectively.


( A ) Immunofluorescent staining of AEP ( red ) and C / EBP β ( green ) in CA1 region from hippocampus of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . Scale bar : 20μm . ( B ) Quantitative analysis of AEP positive cells and C / EBP β positive cells , respectively . The density of both AEP and C / EBP β positive cells were significantly increased by colonic - injection of AD brain extracts and decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± SEM . * * P = 0 . 016 ( * * P < 0 . 01 ) , * * * * P < 0 . 0001 , one - way ANOVA .

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(A) Immunofluorescent staining of AEP (red) and C/EBP β (green) in CA1 region from hippocampus of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. Scale bar: 20μm. (B) Quantitative analysis of AEP positive cells and C/EBP β positive cells, respectively. The density of both AEP and C/EBP β positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. **P =0.016 (**P < 0.01), ****P<0.0001, one-way ANOVA.