C , Representative coronal of thalamus , immunolabeled for c - Fos , following retrograde labeling from excitatory ( C , top ) or inhibitory ( C , bottom ) neurons in ACC . arrows in high magnification indicate colocalization of c - Fos td - Tomato ( C , right top bottom ) . Scale bars represent 200 µm , 10 × magnification ( left top bottom ) , 25 µm ( right top bottom ) , 20 × magnification .

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

C, Representative coronal images of the thalamus, immunolabeled for c-Fos, following retrograde labeling from excitatory (C, top) or inhibitory (C, bottom) neurons in the ACC. White arrows in high magnification images indicate colocalization of c-Fos and td-Tomato (C, right top and bottom). Scale bars represent 200 µm, 10× magnification (left top and bottom), and 25 µm (right top and bottom), 20× magnification.


D , of number of neurons in CL region projecting to excitatory or inhibitory ACC neurons , as a fraction of total thalamic contribution ( top ) fraction of neurons co - expressing tdTomato c - Fos of total tdTomato + population in CL ( bottom ) . Foster mothers ( n = 3 ) , virgins with pups ( n = 4 ) , virgins with object ( n = 4 ) . Data were analyzed using student ' s t - are displayed as mean ± s . e . m ;

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

D, Quantification of the number of neurons in CL region projecting to excitatory or inhibitory ACC neurons, as a fraction of the total thalamic contribution (top) and fraction of neurons co-expressing tdTomato and c-Fos of the total tdTomato+ population in CL (bottom). Foster mothers (n=3), virgins with pups (n=4), virgins with object (n=4). Data were analyzed using student's t-test and are displayed as mean ± s.e.m;


, Representative in - situ hybridization of Gal in ILM taken from Allen Mouse Brain Atlas representative coronal showing expression of Gal neurons in Gal - Cre : : FLEX - YFP mice ( bottom right left ) .

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

, Representative in-situ hybridization images of Gal in the ILM taken from the Allen Mouse Brain Atlas representative coronal images showing the expression of Gal neurons in Gal-Cre::FLEX-YFP female mice (bottom right and left).


, Representative image of ILM immunolabeled for GFP c - Fos , following pup retrieval . Yellow arrows indicate c - Fos expressing Gal - positive Gal - negative neurons , respectively . Scale bars represent 100 µm , 20 × magnification .

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

, Representative image of the ILM immunolabeled for GFP and c-Fos, following pup retrieval test. Yellow and white arrows indicate c-Fos expressing Gal-positive and Gal-negative neurons, respectively. Scale bars represent 100 µm, 20× magnification.


G , Graph plots showing fraction of c - Fos expressing Gal - positive ( Galc - Fos + ) Gal - negative neurons ( DAPIc - Fos + ) in CL , PCN , CM after pup retrieval ( G , top ) or object exposure ( G , bottom ) . Data were analyzed using 2 - way ANOVA expressed as mean ± s . e . m . ; n = 3 ; * p < 0 . 05 .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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, Graph plots showing the fraction of c-Fos expressing Gal-positive (Galc-Fos+) and Gal-negative neurons (DAPIc-Fos+) in the CL, PCN, and CM after pup retrieval test (G, top) or object exposure (G, bottom). Data were analyzed using 2-way ANOVA and expressed as mean ± s.e.m.; n=3; *p < 0.05.


B , C , of pup retrieval latencies following ACC chemogenetic activation with hM3D ( n = 6 ) ( B ) or chemogenetic inhibition using hM4D ( n = 7 ) ( C ) compared to control groups in virgin ( n = 8 ) . Data were analyzed using 2 - way ANOVA with repeated measures are displayed as mean ± s . e . m * p < 0 . 05 , * * p < 0 . 01 . D , Same as ( C ) but for mothers ( n = 7 )

O O O O O B-ORGANISM O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C, Quantification of pup retrieval latencies following ACC chemogenetic activation with hM3D (n=6) (B) or chemogenetic inhibition using hM4D (n=7) (C) compared to control groups in virgin females (n=8). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as mean ± s.e.m* p< 0.05, ** p< 0.01. D, Same as (C) but for mothers (n=7)


E , Representative showing AAV - DIO - CAG - hM3D - 2A - tdTomato c - Fos expression in PFC of virgin , 2 hours following CNO / vehicle administration . Masks demonstrating distribution of DAPI + c - Fos ( green ) , DAPI + dTomato ( magenta ) their colocalization ( ) . Scale bars 100 µm , 20 × magnification .

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

E, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the PFC of virgin females, 2 hours following CNO/vehicle administration. Masks demonstrating distribution of DAPI + c-Fos (green), DAPI + dTomato (magenta) and their colocalization (white). Scale bars 100 µm, 20× magnification.


F , Fraction c - Fos + of total DAPI ( top ) or hM3D - 2A - dTomato ( bottom ) population in ACC , following CNO / vehicle administration . ( n = 3 ) . Data were analyzed using student ' s t - are displayed as mean ± s . e . m ; * p < 0 . 05 , * * p < 0 . 01 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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, Fraction c-Fos+ cells of the total DAPI (top) or hM3D-2A-dTomato (bottom) population in the ACC, following CNO/vehicle administration. (n=3). Data were analyzed using student's t-test and are displayed as mean ± s.e.m; * p< 0.05, ** p< 0.01.


G , Representative of CL , immunolabeled for c - Fos , following hM3D - mediated neuronal activation of ACC . Scale bars 200 µm , 20 × magnification .

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

G, Representative images of the CL, immunolabeled for c-Fos, following hM3D-mediated neuronal activation of the ACC. Scale bars 200 µm, 20× magnification.


H , Spearman correlation between hM3D - mediated activation of ACC ( as measured in ( F , top ) ) CL region .

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

H, Spearman correlation between the hM3D-mediated activation of the ACC (as measured in (F, top)) and the CL region.


B , Representative showing AAV - DIO - CAG - hM3D - 2A - tdTomato c - Fos expression in CL , 2 h after CNO administration . Scale bars 200 µm , 10 × magnification ( left ) 50 µm , 20 × magnification ( right ) .

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

B, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the CL, 2 h after CNO administration. Scale bars 200 µm, 10× magnification (left) and 50 µm, 20× magnification (right).


C , of pup retrieval latencies in virgin following CLGal + chemogenetic activation with hM3D compared to GFP - expressing control group ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures are presented as mean ± s . e . m ; * p < 0 . 05 .

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

C, Quantification of pup retrieval latencies in virgin females following CLGal+ chemogenetic activation with hM3D compared to GFP-expressing control group (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are presented as mean ± s.e.m; * p < 0.05.


HeLa were uninfected ( ' control ' ) or infected with B . abortus ( ' B . abortus ' ) . lysates were collected at indicated time points analyzed by Western blotting . ( A ) Representative immunoblots for pIRE1 , spliced - XBP1 , GAPDH . GAPDH was used for normalization . of bands was quantified using MultiGauge software . ( B , C ) Relative of pIRE1 ( B ) spliced - XBP1 ( C ) in uninfected control ( open circles ) Brucella - infected ( solid circles ) . at time 0 hr were assigned value 1 . Data are means ± SD from three independent experiments .

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

HeLa cells were uninfected ('control') or infected with B. abortus ('B. abortus'). Cell lysates were collected at the indicated time points and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and GAPDH. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software. (B, C) Relative protein levels of pIRE1 (B) and spliced-XBP1 (C) in uninfected control (open circles) and Brucella-infected (solid circles) cells. The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.


( D ) Representative immunoblots for pPERK , - ATF6 , GAPDH , relative of pPERK - ATF6 in control ( open bars ) Brucella - infected ( solid bars ) . GAPDH was used for normalization . of bands was quantified using MultiGauge software , results are shown in bar graphs . As a positive control for activation of PERK or ATF6 , HeLa were treated with 5 μg / ml of tunicamycin for 8hr ( ' Tm ' ) . at time 0 hr were assigned value 1 . Data are means ± SD from three independent experiments .

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

(D) Representative immunoblots for pPERK, cleaved-ATF6, and GAPDH, and relative protein levels of pPERK and cleaved-ATF6 in control (open bars) and Brucella-infected (solid bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. As a positive control for activation of PERK or ATF6, HeLa cells were treated with 5 μg/ml of tunicamycin for 8hr ('Tm'). The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.


( E ) Representative immunoblots for Sar1 , Sec23 , Sec24D , GAPDH , relative of Sar1 , Sec23 , Sec24D in control ( open bars ) Brucella - infected ( solid bars ) . GAPDH was used for normalization . of bands was quantified using MultiGauge software , results are shown in bar graphs . in control were assigned value 1 . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 .

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

(E) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (open bars) and Brucella-infected (solid bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. The protein levels in control cells were assigned the value 1. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.


( A ) Representative immunoblots showing co - immunoprecipitation with pIRE1 . After 5 hr of Tm treatment , immunoprecipitation was performed on HeLa lysates with an anti - pIRE1 antibody ( lanes labeled ' pIRE1 ' ) or control anti - rabbit IgG ( lanes labeled ' IgG ' ) , immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in ER - Golgi vesicular transport pathways or against ER - ( Sec61α , HSP47 , calnexin ) , ERGIC - ( ERGIC53 ) cis - Golgi - ( GM130 ) resident .

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

(A) Representative immunoblots showing co-immunoprecipitation with pIRE1. After 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-pIRE1 antibody (lanes labeled 'pIRE1') or control anti-rabbit IgG (lanes labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER-Golgi vesicular transport pathways or against ER- (Sec61α, HSP47, and calnexin), ERGIC- (ERGIC53) and cis-Golgi- (GM130) resident proteins.


( B ) Representative immunoblot showing co - immunoprecipitation of pIRE1 with Yip1A . Following 5 hr of Tm treatment , immunoprecipitation was performed on HeLa lysates with an anti - Yip1A antibody ( lane labeled ' Yip1A ' ) or control anti - rabbit IgG ( lane labeled ' IgG ' ) , immunoprecipitates were analyzed by Western blotting with an anti - pIRE1 antibody .

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

(B) Representative immunoblot showing the co-immunoprecipitation of pIRE1 with Yip1A. Following 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-Yip1A antibody (lane labeled 'Yip1A') or control anti-rabbit IgG (lane labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with an anti-pIRE1 antibody.


( C ) Representative confocal of HeLa double - stained for pIRE1 ( green ) Yip1A ( blue ) after 5 hr of Tm treatment . Insets are magnifications of boxed areas on main image . Co - localized were detected as large foci . Scale bars are 10 μm .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative confocal micrographs of HeLa cells double-stained for pIRE1 (green) and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were detected as large foci. Scale bars are 10 μm.


( D ) Representative confocal of HeLa triple - stained for pIRE1 ( green ) , Sec31 ( red ) , Yip1A ( blue ) after 5 hr of Tm treatment . Insets are magnifications of boxed areas on main image . Co - localized were identified as large , bright foci ( arrows ) . Scale bars are 10 μm .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Representative confocal micrographs of HeLa cells triple-stained for pIRE1 (green), Sec31 (red), and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were identified as large, bright foci (arrows). Scale bars are 10 μm.


HeLa were transfected with each siRNA for 24 hr , then treated with Tm to induce UPR . lysates were prepared at indicated time points analyzed by Western blotting . ( A ) Representative immunoblots for pIRE1 , spliced - XBP1 , β - tubulin . β - tubulin was used for normalization . of bands was quantified using MultiGauge software .

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

HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm to induce the UPR. Cell lysates were prepared at the indicated time points and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and β-tubulin. β-tubulin was used for normalization. The intensity of the bands was quantified using the MultiGauge software.


( B - D ) Relative of pIRE1 ( B ) , relative mRNA of spliced - XBP1 ( C ) , relative of spliced - XBP1 ( C ) in control ( solid circles ) Yip1A - knockdown ( open circles ) . or mRNA in control at beginning of Tm treatment were assigned value 1 . Data are means ± SD from three independent experiments .

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

(B-D) Relative protein levels of pIRE1 (B), relative mRNA levels of spliced-XBP1 (C), and relative protein levels of spliced-XBP1 (C) in control (solid circles) and Yip1A-knockdown (open circles) cells. The protein or mRNA levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments.


( E ) Representative immunoblots for Sar1 , Sec23 , Sec24D , GAPDH , relative of Sar1 , Sec23 , Sec24D in control ( solid circles ) , Yip1A - knockdown ( open circles ) , IRE1 - knockdown ( solid gray circles ) . GAPDH was used for normalization . of bands was quantified using MultiGauge software , results are shown in graphs . in control at beginning of Tm treatment were assigned value 1 . Data are means ± SD from three independent experiments .

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

(E) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid circles), Yip1A-knockdown (open circles), and IRE1-knockdown (solid gray circles) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the line graphs. The protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments.


( F ) Representative confocal of control ( upper panels ) or Yip1A - knockdown ( lower panels ) during Tm treatment . Fixed at indicated time points were stained for pIRE1 . are outlined with dashed lines . Scale bars are 10 μm . numbers of pIRE1 foci per were counted , shown in graph . Data are means ± SD ( N = 30 ) .

O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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 confocal micrographs of control (upper panels) or Yip1A-knockdown (lower panels) cells during the Tm treatment. Fixed cells at the indicated time points were stained for pIRE1. Cells are outlined with white dashed lines. Scale bars are 10 μm. The numbers of pIRE1 foci per cell were counted, and shown in the line graph. Data are means ± SD (N = 30).


( G ) Representative immunoblot for pIRE1 after native PAGE , showing two high - order complexes of pIRE1 ( pIRE1 - I pIRE1 - II ) . Lane ' S ' represents lysate from HeLa transfected with control scramble siRNA , lane ' Y ' represents lysate from HeLa transfected with Yip1A siRNA . Numbers on left - side correspond to standard molecular . of bands was quantified by using MultiGauge software , results are shown in bar graphs . in control at beginning of Tm treatment were assigned value 1 . Data are means ± SD from three independent experiments . * * : p < 0 . 01 .

O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O 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 immunoblot for pIRE1 after native PAGE, showing two high-order complexes of pIRE1 (pIRE1-I and pIRE1-II). Lane 'S' represents lysate from HeLa cells transfected with control scramble siRNA, and lane 'Y' represents lysate from HeLa cells transfected with Yip1A siRNA. Numbers on the left-hand side correspond to the standard molecular weight. The intensity of the bands was quantified by using the MultiGauge software, and the results are shown in the bar graphs. Protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments. **: p<0.01.


HeLa were transfected with each siRNA for 24 hr , then treated with Tm for 5 hr to induce UPR . ( A , B ) Representative confocal of control ( left - panels ) , Yip1A - knockdown ( middle panels ) , IRE1 - knockdown ( right - had panels ) after 0 hr or 5 hr of Tm treatment ( A ) . ER structure was visualized with an anti - calnexin antibody . Large vacuoles were observed in control ( arrowheads ) , but not in Yip1A - or IRE1 - knockdown . Scale bars are 10 μm . of with vacuoles was counted , is shown in bar graph ( B ) . Data are means ± SD from three independent experiments ( N = 100 ) . * * : p < 0 . 01 .

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

HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm for 5 hr to induce the UPR. (A, B) Representative confocal micrographs of control (left-hand panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (right-had panels) cells after 0 hr or 5 hr of Tm treatment (A). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control cells (arrowheads), but not in Yip1A- or IRE1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (B). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.


( C , D ) Representative confocal of control , Atg9 - knockdown , WIPI1 - knockdown , DFCP1 - knockdown after 0 hr or 5 hr of Tm treatment ( C ) . ER structure was visualized with an anti - calnexin antibody . Large vacuoles were observed in control DFCP1 - knockdown ( arrowheads ) , but rarely seen in Atg9 - or WIPI1 - knockdown . Scale bars are 10 μm . of with vacuoles was counted , is shown in bar graph ( D ) . Data are means ± SD from three independent experiments ( N = 100 ) . * * : p < 0 . 01 .

O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Representative confocal micrographs of control, Atg9-knockdown, WIPI1-knockdown, and DFCP1-knockdown cells after 0 hr or 5 hr of Tm treatment (C). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control and DFCP1-knockdown cells (arrowheads), but rarely seen in Atg9- or WIPI1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (D). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.


HeLa were infected with B . abortus , then transfected with each siRNA at 1 hr p . i . lysates were collected at indicated time points , analyzed by Western blotting . ( A ) Representative immunoblots for pIRE1 , spliced - XBP1 , GAPDH . GAPDH was used for normalization . of bands was quantified using MultiGauge

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HeLa cells were infected with B. abortus, and then transfected with each siRNA at 1 hr p.i. Cell lysates were collected at the indicated time points, and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and GAPDH. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software


( B - D ) Relative of pIRE1 ( B ) spliced - XBP1 ( C ) , relative mRNA of spliced - XBP1 ( D ) in control ( solid circles ) Yip1A - knockdown ( open circles ) . at time 0 hr were assigned value 1 . Data are means ± SD from three independent experiments .

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(B-D) Relative protein levels of pIRE1 (B) and spliced-XBP1 (C), and relative mRNA levels of spliced-XBP1 (D) in control (solid circles) and Yip1A-knockdown (open circles) cells. The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.


( E ) Representative immunoblots for pPERK , - ATF6 , GAPDH , relative of pPERK - ATF6 in control ( solid bars ) Yip1A - knockdown ( open bars ) . GAPDH was used for normalization . of bands was quantified using MultiGauge software , results are shown in bar graphs . As a positive control for activation of PERK or ATF6 , HeLa were treated with 5 μg / ml tunicamycin for 8 hr ( ' Tm ' ) . at time 0 hr were assigned value 1 . Data are means ± SD from three independent experiments .

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(E) Representative immunoblots for pPERK, cleaved-ATF6, and GAPDH, and relative protein levels of pPERK and cleaved-ATF6 in control (solid bars) and Yip1A-knockdown (open bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. As a positive control for activation of PERK or ATF6, HeLa cells were treated with 5 μg/ml tunicamycin for 8 hr ('Tm'). The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.


. ( F ) Representative immunoblots for Sar1 , Sec23 , Sec24D , GAPDH , relative of Sar1 , Sec23 , Sec24D in control ( solid bars ) Yip1A - knockdown ( open bars ) at 24 hr p . i . GAPDH was used for normalization . of bands was quantified using MultiGauge software , results are shown in bar graphs . in control were assigned value 1 . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 .

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. (F) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid bars) and Yip1A-knockdown (open bars) cells at 24 hr p.i. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. The protein levels in control cells were assigned the value 1. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.


( G ) Representative immunoblots for Sar1 , Sec23 , Sec24D , GAPDH , relative of Sar1 , Sec23 , Sec24D in control ( solid bars ) IRE1 - knockdown ( open bars ) at 24 hr p . i . GAPDH was used for normalization . of bands was quantified using MultiGauge software , results are shown in bar graphs . in control were assigned value 1 . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 .

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(G) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid bars) and IRE1-knockdown (open bars) cells at 24 hr p.i. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. The protein levels in control cells were assigned the value 1. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.


HeLa were infected with B . abortus , then transfected with each siRNA at 1 hr p . i . ( A ) Intracellular growth of B . abortus within control ( solid bars ) , Yip1A - knockdown ( open bars ) , IRE1 - knockdown ( solid gray bars ) . CFUs were enumerated at indicated time points . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 .

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HeLa cells were infected with B. abortus, and then transfected with each siRNA at 1 hr p.i. (A) Intracellular growth of B. abortus within control (solid bars), Yip1A-knockdown (open bars), and IRE1-knockdown (solid gray bars) cells. CFUs were enumerated at the indicated time points. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.


( B , C ) Representative confocal of control ( left - panel ) Yip1A - knockdown ( right - panel ) at 24 hr p . i . ( B ) . Fixed were double - stained for Yip1A ( green ) B . abortus ( red ) . infected are outlined with dashed lines . Scale bars are 10 μm . knockdown efficiency of Yip1A in infected was evaluated by quantifying of immunofluorescence for Yip1A , result is shown in bar graph ( C ) .

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(B, C) Representative confocal micrographs of control (left-hand panel) and Yip1A-knockdown (right-hand panel) cells at 24 hr p.i. (B). Fixed cells were double-stained for Yip1A (green) and B. abortus (red). The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The knockdown efficiency of Yip1A in infected cells was evaluated by quantifying the intensity of immunofluorescence staining for Yip1A, and the result is shown in the bar graph (C).


( D , E ) Representative confocal of control ( left - panel ) IRE1 - knockdown ( right - panel ) at 24 hr p . i . ( D ) . Fixed were double - stained for IRE1 ( green ) B . abortus ( red ) . infected are outlined with dashed lines . Scale bars are 10 μm . knockdown efficiency of IRE1 in infected was evaluated by quantifying of immunofluorescence for IRE1 , result is shown in bar graph ( E ) .

(D, E) Representative confocal micrographs of control (left-hand panel) and IRE1-knockdown (right-hand panel) cells at 24 hr p.i. (D). Fixed cells were double-stained for IRE1 (green) and B. abortus (red). The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The knockdown efficiency of IRE1 in infected cells was evaluated by quantifying the intensity of immunofluorescence staining for IRE1, and the result is shown in the bar graph (E).


( F , G ) Representative confocal of control ( left - panels ) , Yip1A - knockdown ( middle panels ) , IRE1 - knockdown ( right - panels ) at 8 hr , 16 hr , 24 hr p . i . ( F ) . Fixed were stained for B . abortus . infected are outlined with dashed lines . Scale bars are 10 μm . To assess replication efficiency , of infected with fewer than ten B . abortus was determined at 24hr p . i . , result is shown in bar graph ( G ) .

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(F, G) Representative confocal micrographs of control (left-hand panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (right-hand panels) cells at 8 hr, 16 hr, and 24 hr p.i. (F). Fixed cells were stained for B. abortus. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. To assess the replication efficiency, the percentage of infected cells with fewer than ten B. abortus was determined at 24hr p.i., and the result is shown in the bar graph (G).


( A ) Representative electron of Brucella - infected control ( left - panel ) Yip1A - knockdown ( right - panel ) at 24 hr p . i . Insets are magnifications of boxed areas on main image , showing typical forms of BCVs . In control , BCVs can be seen in form of ER - derived - bound compartments ( inset in left - panel , defined as ' I ' ) . Note presence of ribosomes on ( arrowheads ) . In Yip1A - knockdown , were not sequestered into such compartments ( inset in right - panel , defined as ' II ' ) . Scale bars are 2 μm .

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

(A) Representative electron micrographs of Brucella-infected control (left-hand panel) and Yip1A-knockdown (right-hand panel) cells at 24 hr p.i. Insets are magnifications of the boxed areas on the main image, showing the typical forms of BCVs. In control cells, BCVs can be seen in the form of ER-derived membrane-bound compartments (inset in left-hand panel, defined as 'I'). Note the presence of ribosomes on the membrane (arrowheads). In Yip1A-knockdown cells, the bacteria were not sequestered into such compartments (inset in right-hand panel, defined as 'II'). Scale bars are 2 μm.


( B ) Representative electron micrograph of Brucella - infected IRE1 - knockdown at 24 hr p . i . Inset is a magnification of boxed in main image , showing that were not sequestered into ER - derived . Scale bar is 2 μm .

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(B) Representative electron micrograph of Brucella-infected IRE1-knockdown cells at 24 hr p.i. Inset is a magnification of the boxed area in the main image, showing that the bacteria were not sequestered into ER-derived membrane. Scale bar is 2 μm.


) percentages of two forms of BCVs ( I II ) present in control Yip1A - knockdown at 24 hr p . i . total numbers of BCVs analyzed were 67 for control 37 for Yip1A - knockdown .

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) The percentages of the two forms of BCVs (I and II) present in control and Yip1A-knockdown cells at 24 hr p.i. The total numbers of BCVs analyzed were 67 for control cells and 37 for Yip1A-knockdown cells.


( D , E ) Representative confocal of control ( upper panels ) , Yip1A - knockdown ( middle panels ) , IRE1 - knockdown ( lower panels ) double - stained for Lamp2 ( a marker for late endosomes / lysosomes ; green ) B . abortus ( red ) at 24 hr p . i . BCVs co - localized with Lamp2 are indicated by arrowheads . infected are outlined with dashed lines . Scale bars are 10 μm . of Lamp2 - positive BCVs was determined , is shown in graph ( E ) .

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

(D, E) Representative confocal micrographs of control (upper panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (lower panels) cells double-stained for Lamp2 (a marker for late endosomes/lysosomes; green) and B. abortus (red) at 24 hr p.i. BCVs co-localized with Lamp2 are indicated by arrowheads. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The percentage of Lamp2-positive BCVs was determined, and is shown in the line graph (E).


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


. ( F ) Magnification of electron micrograph of Brucella - infected control in ( A ) , showing large vacuoles studded locally with ribosomes ( arrows ) .

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. (F) Magnification of the electron micrograph of the Brucella-infected control cell in (A), showing large vacuoles studded locally with ribosomes (arrows).


( G - I ) Representative confocal of Brucella - infected control at 24 hr p . i . triple - stained for HA - Sec61α ( a marker for rough ER ; green ) , B . abortus ( red ) , Lamp2 ( a marker for endosomes / lysosomes ; blue ) ( G ) . An expression construct for HA - Sec61α was co - transfected with scramble siRNA . infected is outlined with dashed lines . Scale bars are 10 μm . Magnifications of boxed areas ' a ' ' b ' are shown in ( H ) ( I ) , respectively . Large vacuoles adjacent to replicating were stained for both Sec61α Lamp2 ( H ) . B . abortus was co - stained with Sec61α but not with Lamp2 ( I ) .

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

(G-I) Representative confocal micrographs of Brucella-infected control cells at 24 hr p.i. triple-stained for HA-Sec61α (a marker for rough ER; green), B. abortus (red), and Lamp2 (a marker for endosomes/lysosomes; blue) (G). An expression construct for HA-Sec61α was co-transfected with scramble siRNA. The infected cell is outlined with white dashed lines. Scale bars are 10 μm. Magnifications of the boxed areas 'a' and 'b' are shown in (H) and (I), respectively. Large vacuoles adjacent to replicating bacteria were stained for both Sec61α and Lamp2 (H). B. abortus was co-stained with Sec61α but not with Lamp2 (I).


HeLa were infected with B . abortus , then transfected with each siRNA at 1 hr p . i . ( A ) Intracellular growth of B . abortus within control ( blue bars ) , Atg9 - knockdown ( red bars ) , WIPI1 - knockdown ( green bars ) , DFCP1 - knockdown ( purple bars ) . CFUs were counted at indicated time points after infection . Data are means ± SD from three independent experiments . * * : p < 0 . 01 .

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HeLa cells were infected with B. abortus, and then transfected with each siRNA at 1 hr p.i. (A) Intracellular growth of B. abortus within control (blue bars), Atg9-knockdown (red bars), WIPI1-knockdown (green bars), and DFCP1-knockdown (purple bars) cells. CFUs were counted at the indicated time points after infection. Data are means ± SD from three independent experiments. **: p<0.01.


( B , C ) Representative confocal of control ( top row ) , Atg9 - knockdown ( second row ) , WIPI1 - knockdown ( third row ) , DFCP1 - knockdown ( bottom row ) double - stained for Lamp2 ( green ) B . abortus ( red ) at 24 hr p . i . BCVs co - localized with Lamp2 are indicated by arrowheads . infected are outlined with dashed lines . Scale bars are 10 μm . of Lamp2 - positive BCVs was determined , is shown in graph ( C ) .

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(B, C) Representative confocal micrographs of control (top row), Atg9-knockdown (second row), WIPI1-knockdown (third row), and DFCP1-knockdown (bottom row) cells double-stained for Lamp2 (green) and B. abortus (red) at 24 hr p.i. BCVs co-localized with Lamp2 are indicated by arrowheads. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The percentage of Lamp2-positive BCVs was determined, and is shown in the line graph (C).


Kaplan - Meier of correlation between expression level of LOC113230 overall survival of colon adenocarcinoma ( COAD ) from TCGA database . P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank .

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Kaplan-Meier analysis of the correlation between expression level of LOC113230 and overall survival of colon adenocarcinoma (COAD) patients from TCGA database. P<0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


qRT - PCR of LOC113230 expression level in 65 paired CRC samples .

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qRT-PCR analysis of LOC113230 expression level in 65 paired CRC samples.


Representative - Scope immunostaining of LOC113230 in CRC tissue corresponding normal tissue from TMA chips . Scale bar represents 100 μM .

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Representative RNA-Scope immunostaining images of LOC113230 levels in CRC tissue and corresponding normal tissue from TMA chips. Scale bar represents 100 μM.


Kaplan - Meier for overall survival of CRC with high low expression of LOC113230 from TMA chips . * P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank .

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Kaplan-Meier analysis for overall survival of CRC patients with high and low expression of LOC113230 from TMA chips. *P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


Overexpression of LOC113230 in SW - 1116 inhibited lung metastasis . Left : Exponentially growing SW - 1116 - LOC113230 - Luc SW - 1116 - Vector - Luc were injected into tail vein of 8 - week - old nude mice intensities of lung metastatic at 8th week were analyzed by in vivo photon flux imaging equipment . red circles in are to help to see tumors . Right : Box plot ( central band : median ; box limits : first third quartile ; whiskers : minimum maximum ) of lung photon flux at 8th .

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Overexpression of LOC113230 in SW-1116 cells inhibited lung metastasis. Left: Exponentially growing SW-1116 - LOC113230-Luc and SW-1116-Vector-Luc cells were injected into the tail vein of 8-week-old nude mice and intensities of lung metastatic tumor cells at the 8th week were analyzed by in vivo photon flux imaging equipment. The red circles in images are to help to see the tumors. Right: Box plot (central band: median; box limits: first and third quartile; whiskers: minimum and maximum) of lung photon flux at the 8th weeks.


5 ng / ml TGF - β DMSO were applied to stimulate SW - 1116 - Vector or SW - 1116 - LOC113230 for 24 48 hrs , respectively . migration was detected by transwell assays . Scale bar represents 100 μM .

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5 ng/ml TGF-β and DMSO were applied to stimulate SW-1116 -Vector or SW-1116 -LOC113230 cells for 24 and 48 hrs, respectively. Cell migration was detected by transwell assays. Scale bar represents 100 μM.


I ) Stable expression of LOC113230 300 - 800 inhibited migration , while LOC113230 antisense showed no apparent effect on migration . Scale bar represents 100 μM .

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I)Stable expression of LOC113230 300-800 inhibited cell migration, while LOC113230 antisense showed no apparent effect on cell migration. Scale bar represents 100 μM.


( A ) Top : taken by confocal microscopy showed localization of LOC113230 detected by scope assays in SW - 1116 . Scale bar represents 50 μM . Bottom : qRT - PCR assays showed LOC113230 expression level in fractions extracted from SW - 1116 .

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(A)Top: Images taken by confocal microscopy showed the localization of LOC113230 detected by RNA scope assays in SW-1116. Scale bar represents 50 μM. Bottom: qRT-PCR assays showed LOC113230 expression level in the nuclear and cytoplasmic fractions extracted from SW-1116 cells.


SW - 1116 bearing stable expression or knockdown of LOC113230 were treated with MG132 ( 5 μM ) for 12 hrs . lysates were immunoprecipitated ( IP ) assays were performed with α - Flag antibody . co - eluted were detected by western blot assays with ubiquitin ( Ub ) , ASS1 α - Flag antibodies , respectively .

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SW-1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub), ASS1 and α-Flag antibodies, respectively.


SW - 1116 stably expressing ASS1WT , or ASS1 K234R LOC113230 or not were treated with MG132 ( 5 μM ) for 12 hrs . lysates were immunoprecipitated ( IP ) assays were performed with α - Flag antibody . co - eluted were detected by western blot assays with ubiquitin ( Ub ) α - Flag antibodies , respectively .

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

SW-1116 cells stably expressing ASS1WT, or ASS1 K234R and LOC113230 or not were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub) and α-Flag antibodies, respectively.


Arginine production from each group was determined by a commercially available Arginine kit . were stimulated with 5 ng / ml TGF - β or DMSO for 24 hrs . Urea production for each group was determined by a commercially available urea kit . were stimulated with 5 ng / ml TGF - β or DMSO for 24 hrs .

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Arginine production from each group was determined by a commercially available Arginine assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs. Urea production for each group was determined by a commercially available urea assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs.


Depletion of ASS1 decreased migration in SW - 1116 . Scale bar represents 100 μM .

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Depletion of ASS1 decreased cell migration in SW-1116 cells. Scale bar represents 100 μM.


Transwell assays showed effect of ASS1WT ASS1K234R on Caco - 2 - Vector or Caco - 2 - LOC113323 migration . Scale bar represents 100 μM . .

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Transwell assays showed the effect of ASS1WT and ASS1K234R on Caco-2-Vector or Caco-2-LOC113323 cell migration. Scale bar represents 100 μM..


( N ) Kaplan - Meier for overall survival of with high or low expression of ASS1 . P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank .

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(N) Kaplan-Meier analysis for overall survival of patients with high or low expression of ASS1. P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


interaction of endogenous TRAF2 , ASS1 LRPPRC was detected in SW - 1116 by co - IP assays . GST - TRAF2 , HA - LRPPRC , His - ASS1 Biotin - LOC113230 were expressed in E . coli BL21 , were used for in vitro binding assays .

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The interaction of endogenous TRAF2, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays. GST-TRAF2, HA-LRPPRC, His-ASS1 and Biotin-LOC113230 were expressed in E. coli BL21 cells, and were used for in vitro binding assays.


SW - 1116 - LOC113230 transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control ( SiRNA - NC ) were treated with MG132 ( 5 µM ) for 12 hrs . lysates were immunoprecipitated with α - Flag antibody . precipitate input were analyzed by immunoblotting . NC : Negative control .

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

SW-1116-LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control (SiRNA-NC) were treated with MG132 (5 µM) for 12 hrs. Cell lysates were immunoprecipitated with α-Flag antibody. The precipitate proteins and input proteins were analyzed by immunoblotting. NC: Negative control.


B : brain MRI of II - 1 ( FLAIR ) . A : sagittal interemispheric showing profound cerebellar atrophy ( arrow ) ; B : transverse showing involvement of dentate nuclei of cerebellum ; C : coronal revealing cortical atrophy , a wide third ventricle , high signal changes in thalami , particularly on left ( arrow ) , D : thalamic lesion is indicated by an arrow .

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

B: brain MRI images of II-1 (FLAIR). A: sagittal interemispheric sequence showing profound cerebellar atrophy (arrow); B: transverse sequence showing involvement of the dentate nuclei of cerebellum; C: coronal sequence revealing cortical atrophy, a wide third ventricle, and high signal changes in the thalami, particularly on the left (arrow), D: the thalamic lesion is indicated by an arrow.


C : histochemical of skeletal muscle from II - 2 . Combined COX / SDH shows scattered COX negative ( blue ) fibres ( arrows ) . black bar corresponds to 100 µm .

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C: histochemical staining of skeletal muscle from II-2. Combined COX/SDH staining shows scattered COX negative (blue) fibres (arrows). The black bar corresponds to 100 µm.


D : respiratory chain activities in skeletal muscle . Specific activities ( nmol min - 1 mg - 1 ) of I , II IV are reduced in II - 2 ( red ) compared to controls mean ( blue ) ; CS activity is also low ( < 50 % of controls ) suggesting a reduction of mitochondrial mass ( see main text for further details ) . Each activity was measured in triplicate .

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D: respiratory chain complex activities in skeletal muscle. Specific activities (nmol min-1 mg-1) of complex I, II and IV are reduced in II-2 (red) compared to the controls mean (blue); CS activity is also low (<50% of controls) suggesting a reduction of mitochondrial mass (see main text for further details). Each activity was measured in triplicate.


E : Western blot of PITRM1 in fibroblasts ( left ) skeletal muscle ( right ) of controls ( CTR ) subject II - 2 . Densitometric using Genetools software is shown below blots . In blue is PITRM1WT in red PITRM1R183Q .

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E: Western blot analysis of PITRM1 in primary fibroblasts (left) and skeletal muscle (right) of controls (CTR) and subject II-2. Densitometric quantification using the Genetools software is shown below the blots. In blue is PITRM1WT and in red PITRM1R183Q.


F : proteolytic activity on two oligopeptides ( AnaSpec ) , reported at bottom of each histogram , by 6 - His tagged PITRM1WT PITRM1R183Q expressed in E . coli affinity - purified by Ni - agarose chromatography . Values are expressed as arbitrary units per sec per µg of ( a . u . s - 1µg - 1 ) . Experiments were performed in duplicate .

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F: proteolytic activity on two fluorescent oligopeptides (AnaSpec), reported at the bottom of each histogram, by 6-His tagged PITRM1WT and PITRM1R183Q proteins expressed in E. coli and affinity-purified by Ni-agarose chromatography. Values are expressed as arbitrary units per sec per µg of protein (a.u. s-1µg-1). Experiments were performed in duplicate.


G : degradation rate of Aβ1 - 42 by purified 6 - His tagged PITRM1WT PITRM1R183Q . of these experiments is displayed below blots . Experiments were performed in duplicate .

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

G: degradation rate of Aβ1-42 by purified 6-His tagged PITRM1WT and PITRM1R183Q proteins. Quantification of these experiments is displayed below the blots. Experiments were performed in duplicate.


A : growth in glucose ( GLU ) galactose ( GAL ) . C29V , C47V , TpLV : immortalized fibroblast lines from control individuals ; PITRM1V : immortalized fibroblasts from subject II - 2 , carrying PITRM1R183Q mutation . pLKO . 1 : empty vector ; sh38 : shRNA38 ; sh41 : shRNA41 . Each was measured six times . Error bars indicate ± SD . Statistic was by two - way ANOVA post - hoc Bonferroni * * * p < 0 . 001 .

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A: cell growth in glucose (GLU) and galactose (GAL). C29V, C47V, TpLV: immortalized fibroblast cell lines from control individuals; PITRM1V: immortalized fibroblasts from subject II-2, carrying the PITRM1R183Q mutation. pLKO.1: empty vector; sh38: shRNA38; sh41: shRNA41. Each cell line was measured six times. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA post-hoc Bonferroni test ***p<0.001.


B : Western - blot of PITRM1 in C29V transduced with empty vector plKO . 1 two shRNA , sh38 sh41 . SDHB b - Actin are used as loading controls .

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

B: Western-blot analysis of PITRM1 in C29V cells transduced with the empty vector plKO.1 and the two shRNA, sh38 and sh41. SDHB and b-Actin are used as loading controls.


C : co - localization of PITRM1 ( red ) with TOM20 ( T20 , green ) in human fibroblast from a control ( CTR ) subject II - 2 . Note that of PITRM1 immunofluorescence is much lower in II - 2 than in CTR ( see main text for further details ) . Nuclei are stained in blue by DAPI . A bar corresponds to 10 µM .

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

C: co-localization of PITRM1 (red) with TOM20 (T20, green) in human fibroblast cells from a control (CTR) and subject II-2. Note that the intensity of PITRM1 immunofluorescence is much lower in II-2 cells than in CTR cells (see main text for further details). Nuclei are stained in blue by DAPI. A white bar corresponds to 10 µM.


D : mitochondrial potential ( ΔP ) . In healthy with high mitochondrial ΔP , JC - 1 forms complexes known as J - that show an intense red . On contrary in unhealthy with low ΔP , JC - 1 remains in monomeric form , showing only green . Values are referred to as % of those of control means , taken as 100 % . results are mean of 3 independent experiments . Error bars indicate ± SD . Statistic was by two - way ANOVA * * p < 0 . 01 ; * * * * p < 0 . 0001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: mitochondrial membrane potential (ΔP). In healthy cells with high mitochondrial ΔP, JC-1 forms complexes known as J-aggregates that show an intense red fluorescence. On the contrary in unhealthy cells with low ΔP, JC-1 remains in the monomeric form, showing only green fluorescence. Values are referred to as % of those of the control means, taken as 100%. The results are the mean of 3 independent experiments. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA **p <0.01; ****p<0.0001.


A : oxidative growth . W303 - 1Bcym1Δ strains harbouring wild - type CYM1 allele ( CYM1wt ) , cym1R163Q mutant allele or empty vector were serially diluted from 107 to 104 / ml . Five microliters of each dilution were spotted on SC agar plates without uracil , supplemented with 2 % glucose , 2 % glycerol or 2 % ethanol . Plates were incubated at 37 ° C for 3 - 7 days .

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A: oxidative growth. W303-1Bcym1Δ strains harbouring the wild-type CYM1 allele (CYM1wt), the cym1R163Q mutant allele or the empty vector were serially diluted from 107 to 104 cells/ml. Five microliters of each dilution were spotted on SC agar plates without uracil, supplemented with 2% glucose, 2% glycerol or 2% ethanol. Plates were incubated at 37°C for 3-7 days.


B : oxygen consumption rate ( OCR ) . grown at 37 ° C SC medium without uracil were supplemented with 0 . 5 % glucose . Values were normalized to OCR of CYM1wt ( 49 nmol O2 min − 1 mg − 1 ) represented as mean of at least three values ± SD . Statistical was by unpaired , two - tail Student ' s t : * * * p < 0 . 001 .

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B: oxygen consumption rate (OCR). Cells grown at 37°C SC medium without uracil were supplemented with 0.5% glucose. Values were normalized to the OCR of the CYM1wt strain (49 nmol O2 min−1 mg−1) and represented as the mean of at least three values ± SD. Statistical analysis was by unpaired, two-tail Student's t test: ***p<0.001.


C : reduced versus oxidized cytochrome spectra . Peaks at 550 , 560 602 nm correspond to cytochromes c , b aa3 , respectively . height of each peak relative to baseline is an index of cytochrome content .

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C: reduced versus oxidized cytochrome spectra. Peaks at 550, 560 and 602 nm correspond to cytochromes c, b and aa3, respectively. The height of each peak relative to the baseline is an index of cytochrome content.


D : respiratory chain activities . Biochemical activities of Succinate Quinone DCPIP Reductase , SQDR ( CII ) , NADH - cytochrome c oxidoreductase activity NCCR ( CIII ) , cytochrome c oxidase ( CIV ) were measured on a mitochondrial - enriched fraction from grown at 37 ° C as in panel B . Values were normalized to that of CYM1wt represented as mean of three independent experiments ± SD . Statistical was by unpaired , two - tail Student ' s t : * * p < 0 . 01 ; * * * p < 0 . 001 .

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

D: respiratory chain complex activities. Biochemical activities of Succinate Quinone DCPIP Reductase, SQDR (CII), NADH-cytochrome c oxidoreductase activity NCCR (CIII), and cytochrome c oxidase (CIV) were measured on a mitochondrial-enriched fraction from cells grown at 37°C as in panel B. Values were normalized to that of CYM1wt strain and represented as the mean of three independent experiments ± SD. Statistical analysis was by unpaired, two-tail Student's t test: **p<0.01; ***p<0.001.


E : Western - blot using an anti - HA monoclonal antibody visualizing CYM1wt cym1R163Q recombinant both fused in frame with HA epitope on C - terminus . Western - blot on total extract . Total extracts were obtained by strains expressing HA - tagged CYM1wt cym1R163Q . PGK was used as a loading control signals were normalized to wt . was performed on 5 independent blots . F : prolonged exposure of a Western - blot containing CYM1wt cym1R163Q recombinant reveals presence of a band corresponding to a degradation product in cym1R163Q lanes ( arrow ) .

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

E: Western-blot using an anti-HA monoclonal antibody visualizing the CYM1wt and cym1R163Q recombinant proteins both fused in frame with the HA epitope on the C-terminus. Western-blot analysis on total protein extract. Total protein extracts were obtained by strains expressing HA-tagged CYM1wt and cym1R163Q. PGK was used as a loading control and signals were normalized to the wt. The quantification was performed on 5 independent blots.F: prolonged exposure of a Western-blot containing the CYM1wt and cym1R163Q recombinant proteins reveals the presence of a band corresponding to a degradation product in the cym1R163Q lanes (arrow).


G : Western blot of Aβ1 - 42myc monomer dimer incubated with purified mitochondrial extracts from grown at 37 ° C in SC medium supplemented with 0 . 15 % glucose 2 % galactose . VDAC is used as a loading control . Each experiment was performed in triplicate .

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

G: Western blot of Aβ1-42myc monomer and dimer incubated with purified mitochondrial extracts from cells grown at 37°C in SC medium supplemented with 0.15% glucose and 2% galactose. VDAC is used as a loading control. Each experiment was performed in triplicate.


A : Western - blot of Pitrm1 in brain , muscle liver of two 4 - mo Pitrm1 + / - mice two Pitrm1 + / + . Densitometric is reported in histograms below blots . Pitrm1 + / + is in blue , Pitrm1 + / - in red . Error bars indicate ±

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A: Western-blot analysis of Pitrm1 protein in brain, muscle and liver of two 4-mo male Pitrm1+/- mice and two Pitrm1+/+ littermates. Densitometric analysis is reported in the histograms below the blots. Pitrm1+/+ is in blue, Pitrm1+/- in red. Error bars indicate ±SD


B : representative hindlimb - clasping phenomenon is shown in a 4 mo Pitrm1 + / - mouse , consisting in strong adduction of hindlimbs when is suspended by tail ; a littermate Pitrm1 + / + control displays normal reflex , consisting in wide abduction of limbs . examined Pitrm1 + / - displayed this abnormal reflex from 2 months of .

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

B: representative hindlimb-clasping phenomenon is shown in a 4 mo Pitrm1+/- male mouse, consisting in strong adduction of the hindlimbs when the animal is suspended by the tail; a littermate Pitrm1+/+ control displays the normal reflex, consisting in wide abduction of the limbs. All examined Pitrm1+/- animals displayed this abnormal reflex from 2 months of age.


C : rotarod . Blue red lines refer to Pitrm1 + / + ( n = 9 ) Pitrm1 + / - ( n = 7 ) 4 mo , respectively . Error bars indicate ± SD . Statistical was by unpaired , two - tail Student ' s t . * p < 0 . 05 ; * * p < 0 . 01 .

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C: rotarod test. Blue and red lines refer to Pitrm1+/+ (n=9) and Pitrm1+/- (n=7) 4 mo animals, respectively. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05; ** p<0.01.


D : negative geotaxis . experiment was carried out on same group of as in panel B . Error bars indicate ± SD . Statistical was by unpaired , two - tail Student ' s t . * p < 0 . 05 .

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

D: negative geotaxis test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. * p<0.05.


E : cylinder . experiment was carried out on same group of as in panel b . Error bars indicate ± SD . Statistical was by unpaired , two - tail Student ' s t . * * * p < 0 . 001 .

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E: cylinder test. The experiment was carried out on the same group of animals as in panel b. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *** p<0.001.


F : pole . experiment was carried out on same group of as in panel B . Error bars indicate ± SD . Statistical was by unpaired , two - tail Student ' s t . * * * p < 0 . 001 .

F: pole test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. ***p<0.001.


A : Western - blot of amyloid - precursor ( APP ) in 6 mo mice . GAPDH was used as a loading control . Densitometric on a total of 5 independent samples is shown in histogram on right .

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

A: Western-blot analysis of amyloid-precursor protein (APP) in 6 mo male mice. GAPDH was used as a loading control. Densitometric analysis on a total of 5 independent samples is shown in the histogram on the right.


B : morphological of an AD subject of Pitrm1 + / - Pitrm1 + / + mouse brains . TF : Thioflavin T ( thalamus ) ; CR : Congo - red ( brain cortex ) ; PL : polarised ( same sections as those stained with CR ) ; Aβ1 - 42 immunostaining ( pons ) ; Ub : ubiquitin immunohistochemistry shown as brownish ( brain cortex ) . bar indicates 20 µm .

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

B: morphological analysis of an AD subject and of Pitrm1+/- and Pitrm1+/+ mouse brains. TF: Thioflavin T (thalamus); CR: Congo-red (brain cortex); PL: polarised light (same sections as those stained with CR); Aβ1-42 immunostaining (pons); Ub: ubiquitin immunohistochemistry shown as brownish staining (brain cortex). The white bar indicates 20 µm.


A : upper panel : import of Aβ1 - 42 into mitochondria ; lower panel : relative from two independent experiments . Values of Aβ1 - 42 signal were normalized to HSP60 signal resulting value at 5 min without trypsin was arbitrarily chosen as 1 . Error bars indicate ± SD . Statistical was by unpaired , two - tail Student ' s t . * p < 0 . 05 .

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A: upper panel: import of Aβ1-42 into mitochondria; lower panel: relative quantification from two independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the resulting value at 5 min without trypsin was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05.


B : upper panel : chase experiment to assess clearance of Aβ1 - 42 ; lower panel : from three independent experiments . Values of Aβ1 - 42 signal were normalized to HSP60 signal value was arbitrarily chosen as 1 . Error bars indicate ± SD . Statistical was by unpaired , two - tail Student ' s t . * p < 0 . 05 , * * p < 0 . 01 .

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B: upper panel: pulse and chase experiment to assess clearance of Aβ1-42; lower panel: quantification from three independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the pulse value was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05, **p<0.01.


A : MEFs from Pitrm1 + / + , Pitrm1 + / - , Mfn1 - / - , Mfn2 - / - , as well as h - PITRM1 R183Q h - PITRM1 WT were exposed to Aβ1 - 40 peptide for 18h . Note that Aβ1 - 40 signal was still evident in Pitrm1 + / - h - PITRM1 R183Q . B : of three independent experiments similar to those shown in panel A . Statistical was by unpaired , two - tail Student ' s t . * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A: MEFs from Pitrm1+/+, Pitrm1+/-, Mfn1-/-, Mfn2-/-, as well as h-PITRM1 R183Q and h- PITRM1 WT were exposed to Aβ1-40 peptide for 18h. Note that Aβ1-40 signal was still evident in Pitrm1+/- and h-PITRM1 R183Q cells.B: quantification of three independent experiments similar to those shown in panel A. Statistical analysis was by unpaired, two-tail Student's t test. **p<0.01, ***p<0.001.


FACS of relative TCRβ + T content in blood ( left panel ) tumors ( right panel ) of mice at end of treatments as indicated in A . αCD4 / αCD8 : n = 10 ; IgG : n = 6 .

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FACS analysis of relative TCRβ+ T cell content in blood (left panel) and tumors (right panel) of mice at the end of treatments as indicated in A. αCD4/αCD8: n=10; IgG: n=6.


burden ( left panel ) , number ( middle panel ) average ( right panel ) per mouse assessed during colon resection at end of treatments . αCD4 / αCD8 : n = 10 ; IgG : n = 9 .

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Tumor burden (left panel), tumor number (middle panel) and average tumor size (right panel) per mouse assessed during colon resection at the end of treatments. αCD4/αCD8: n=10; IgG: n=9.


Immune contexture in healthy colon colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Relative TCRβ + T content in colon ( n = 10 ) ( n = 22 ) . relative content of types was determined in tissue - derived single suspension using type specific antibodies FACS .

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Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative TCRβ+ T cell content in colon (n=10) and tumor (n=22). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Immune contexture in healthy colon colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Anti - CD3 immunostaining on normal colon ( left panel ) a representative colon section derived from an Apcfl / fl - Cdx2CreERT2 mouse ( right panel : higher magnification of indicated in middle panel ) .

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

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Anti-CD3 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).


Immune contexture in healthy colon colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Relative CD11b + myeloid content in colon ( n = 16 ) tumors ( n = 16 ) . relative content of types was determined in tissue - derived single suspension using type specific antibodies FACS .

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

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative CD11b+ myeloid cell content in colon (n=16) and tumors (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Immune contexture in healthy colon colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Anti - Gr1 immunostaining on normal colon ( left panel ) a representative colon section derived from an Apcfl / fl - Cdx2CreERT2 mouse ( right panel : higher magnification of indicated in middle panel ) .

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Anti-Gr1 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).


Immune contexture in healthy colon colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Relative CD11b + MHCII - Gr1hi neutrophil ( E ) CD11b + MHCII - Gr1lo monocyte ( F ) content in colon ( n = 13 ) tumors ( n = 16 ) . relative content of types was determined in tissue - derived single suspension using type specific antibodies FACS .

O O O O B-TISSUE B-DISEASE I-DISEASE O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD B-CELL_TYPE O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative CD11b+ MHCII- Gr1hi neutrophil (E) and CD11b+ MHCII- Gr1lo monocyte (F) content in colon (n=13) and tumors (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Immune contexture in healthy colon colon tumors of Apcfl / fl - Cdx2CreERT2 mice . , Correlation of T numbers to sample - matched - derived neutrophil ( left panel ) monocyte ( right panel ) numbers ( n = 16 ) . relative content of types was determined in tissue - derived single suspension using type specific antibodies FACS .

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

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. , Correlation of tumor T cell numbers to sample-matched tumor-derived neutrophil (left panel) and monocyte (right panel) numbers (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


of T subsets among total CD45 + hematopoietic in colon ( n = 10 ) ( n = 11 ) . H , CD4 + TCRβ + T . I , CD8 + TCRβ + T . J , Foxp3 + CD4 + TCRβ + regulatory T ( Tregs ) . relative content of types was determined in tissue - derived single suspension using type specific antibodies FACS .

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

Percentage of T cell subsets among total CD45+ hematopoietic cells in colon (n=10) and tumor (n=11). H, CD4+ TCRβ+ T cells. I, CD8+ TCRβ+ T cells. J, Foxp3+ CD4+ TCRβ+ regulatory T cells (Tregs). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


of Tregs among total T ( CD45 + TCRβ + ) in normal colon ( n = 10 ) or colon tumors ( n = 11 ) . relative content of types was determined in tissue - derived single suspension using type specific antibodies FACS .

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Percentage of Tregs among total T cells (CD45+ TCRβ+) in normal colon (n=10) or colon tumors (n=11). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


In vitro co - culture of activated T with increasing ratios of neutrophils , monocytes or macrophages . T proliferation index is numbers of proliferated T after three days of indicated co - culture condition relative to number of proliferated T when cultured alone . CD4 + CD8 + T were derived from lymph nodes of wild - type mice . Neutrophils , monocytes macrophages were derived from colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Each dot represents an neutrophil ( n = 4 ) , monocyte ( n = 3 ) or macrophage ( n = 4 ) sample . Bars represent mean + / - SD .

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

In vitro co-culture of activated T cells with increasing ratios of neutrophils, monocytes or macrophages. T cell proliferation index is numbers of proliferated T cells after three days of indicated co-culture condition relative to the number of proliferated T cells when cultured alone. CD4+ and CD8+ T cells were derived from lymph nodes of wild-type mice. Neutrophils, monocytes and macrophages were derived from colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Each dot represents an individual neutrophil (n=4), monocyte (n=3) or macrophage (n=4) sample. Bars represent mean +/- SD.


neutrophil ( left panel ) monocyte ( right panel ) content after αGr1 + CXCR2i ( n = 7 ) or IgG + DMSO ( n = 10 ) treatments .

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Tumor neutrophil (left panel) and monocyte (right panel) content after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=10) treatments.


Average volume per mouse at end of αGr1 + CXCR2i ( n = 9 ) or IgG + DMSO ( n = 12 ) treatments as indicated in B .

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Average tumor volume per mouse at the end of αGr1 + CXCR2i (n=9) or IgG + DMSO (n=12) treatments as indicated in B.


Anti - CD3 immunostaining on representative sections derived from mice after indicated treatments .

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Anti-CD3 immunostaining on representative tumor sections derived from mice after indicated treatments.


FACS of CD45 + TCRβ + , CD45 + TCRβ + CD8 + CD45 + TCRβ + CD4 + Foxp3 + in extracted tumors after αGr1 + CXCR2i ( n = 8 ) or IgG + DMSO ( n = 7 ) treatments .

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FACS analysis of CD45+ TCRβ+ cells, CD45+ TCRβ+ CD8+ cells and CD45+ TCRβ+ CD4+ Foxp3+ cells in extracted tumors after αGr1 + CXCR2i (n=8) or IgG + DMSO (n=7) treatments.


FACS of IFNγ ( left panel ) IL17A ( right panel ) expression in extracted T after αGr1 + CXCR2i ( n = 7 ) or IgG + DMSO ( n = 7 ) treatments .

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FACS analysis of IFNγ (left panel) and IL17A (right panel) expression in tumor extracted T cells after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=7) treatments.


Measurement of cytokines present in culture supernatants of T co - cultured with either - derived neutrophils ( T + Ne ; n = 3 ) or with - derived monocytes ( T + Mo ; n = 3 ) . Cytokines with a log2 fold change > 0 . 6 or < - 0 . 4 were considered as differentially present in supernatants . Shown is a of scaled cytokines ( z - score ) differentially present in T + Ne compared to T + Mo . Adjusted p - values were calculated by moderated t - ( limma ) Benjamini post were < 0 . 05 only for MMP9 RELMβ . Supernatants of T alone ( T only ; n = 2 ) served as internal control . T cultured with monocytes did not have differential presence of cytokines compared to T alone .

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

Measurement of cytokines present in cell culture supernatants of T cells co-cultured with either tumor-derived neutrophils (T + Ne; n=3) or with tumor-derived monocytes (T + Mo; n=3). Cytokines with a log2 fold change >0.6 or <-0.4 were considered as differentially present in supernatants. Shown is a heatmap of scaled cytokines levels (z-score) differentially present in T + Ne compared to T + Mo. Adjusted p-values were calculated by moderated t-test (limma) and Benjamini post test and were <0.05 only for MMP9 and RELMβ. Supernatants of T cells alone (T only; n=2) served as internal control. T cells cultured with monocytes did not have differential presence of cytokines compared to T cells alone.


Co - immunostaining for MMP9 ( green ) S100A9 ( red ) on a representative section derived from an Apcfl / fl - Cdx2CreERT2 mouse . Right panel shows a higher magnification of indicated of left panel .

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Co-immunostaining for MMP9 (green) and S100A9 (red) on a representative tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse. Right panel shows a higher magnification of indicated area of the left panel.


mRNA expression of Mmp9 ( C ) or Tgfb1 , Tgfb2 Tgfb3 ( D ) in indicated FACS sorted types isolated from mouse colon tumors ( n = 4 ) . Data are derived from - sequencing are displayed as reads per kilo base per million mapped reads ( rpkm ) .

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mRNA expression levels of Mmp9 (C) or Tgfb1, Tgfb2 and Tgfb3 (D) in indicated FACS sorted cell types isolated from mouse colon tumors (n=4). Data are derived from RNA-sequencing and are displayed as reads per kilo base per million mapped reads (rpkm).


Neutrophil - T co - cultures treated with vehicle ( T + Ne ) , 10µm Galunisertib ( T + Ne + TGFBRi ) , T proliferation after three days of indicated co - culture conditions . T proliferation index is numbers of proliferated T under indicated condition relative to number of proliferated T when cultured alone .

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Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.


Neutrophil - T co - cultures treated with vehicle ( T + Ne ) , 10µm of MMP inhibitor GM6001 ( T + Ne + MMPi ) of free active TGFβ1 in culture supernatants of indicated conditions determined by ELISA .

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Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) Protein levels of free active TGFβ1 in cell culture supernatants of indicated conditions determined by ELISA.


Neutrophil - T co - cultures treated with vehicle ( T + Ne ) , 10µm of MMP inhibitor GM6001 ( T + Ne + MMPi ) T proliferation after three days of indicated co - culture conditions . T proliferation index is numbers of proliferated T under indicated condition relative to number of proliferated T when cultured alone .

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Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.


Neutrophil - T co - cultures treated with vehicle ( T + Ne ) , 10µm Galunisertib ( T + Ne + TGFBRi ) , 10µm of MMP inhibitor GM6001 ( T + Ne + MMPi ) or with 10µm Galunisertib plus 10µm GM6001 ( T + Ne + TGFBRi + MMPi ) . T proliferation after three days of indicated co - culture conditions . T proliferation index is numbers of proliferated T under indicated condition relative to number of proliferated T when cultured alone .

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

Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) or with 10µm Galunisertib plus 10µm GM6001 (T + Ne + TGFBRi + MMPi). T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.


Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg / kg of MMP2 / 9 - inhibitor SB3 - CT ( MMPi ) . , Co - immunostaining for pSMAD3 ( green ) IGFBP7 ( magenta ) on representative - containing colon sections derived from Apcfl / fl - Cdx2CreERT2 mice treated with vehicle ( A ) MMPi ( B ) .

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Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). , Co-immunostaining for pSMAD3 (green) and IGFBP7 (magenta) on representative tumor-containing colon sections derived from Apcfl/fl-Cdx2CreERT2 mice treated with vehicle (A) MMPi (B).


Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg / kg of MMP2 / 9 - inhibitor SB3 - CT ( MMPi ) . of pSMAD3 + ( left panel ) pSMAD3 per pSMAD3 + ( right panel ) on at least five arbitrarily selected areas per section of MMPi ( n = 6 ) or vehicle ( n = 6 ) treated mice .

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Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). Quantification of pSMAD3+ cells (left panel) and pSMAD3 staining intensity per pSMAD3+ cell (right panel) on at least five arbitrarily selected tumor areas per section of MMPi (n=6) or vehicle (n=6) treated mice.


effect of in vivo TGFBRi MMPi treatment of mice for two on colon formation . Apcfl / fl - Cdx2CreERT2 mice were treated with Tamoxifen , one day post treatment , injected with either MMPi ( n = 10 ) or TGFBRi SB431542 ( n = 10 ) at 40mg / kg for two with five injections per week . Vehicle treated mice : n = 11 . Average per mouse was assessed during colon resection one week after end of treatments ( E ) .

O O O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-DISEASE O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O B-TISSUE O O O O O O O O O O O

effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation. Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, one day post treatment, injected with either MMPi (n=10) or the TGFBRi SB431542 (n=10) at 40mg/kg for two weeks with five injections per week. Vehicle treated mice: n=11. Average tumor size per mouse was assessed during colon resection one week after the end of treatments (E).


evaluation of S100A9 + neutrophil CD8 + T infiltration in histologic sections of human CRC tumors . , Anti - S100A9 immunostaining on a representative human CRC section . Arrowheads indicate neutrophil infiltration at boarder . Co - immunostaining for CD8 ( brown ) S100A9 ( gray - blue ) in same CRC sample as shown in A .

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

evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. , Anti-S100A9 immunostaining on a representative human CRC section. Arrowheads indicate neutrophil infiltration at the tumor boarder. Co-immunostaining for CD8 (brown) and S100A9 (gray-blue) in the same CRC sample as shown in A.