( 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 .

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

(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 .

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

(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 B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD 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 O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD 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 B-GENEPROD I-GENEPROD I-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 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 .

O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE 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 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

(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 .

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

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

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O B-TISSUE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE 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 O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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.


( C ) Immunofluorescent staining of cleaved APPC586 ( red ) and Aβ ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( D ) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells , respectively . The density of both cleaved APP C586 positive cells and Aβ 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 . 0001 , one - way ANOVA .

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

(C) Immunofluorescent staining of cleaved APPC586 (red) and Aβ (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (D) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells, respectively. The density of both cleaved APP C586 positive cells and Aβ 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<0.0001, one-way ANOVA.


( E ) Immunofluorescent staining of T22 ( red ) and HT7 ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( F ) Quantitative analysis of T22 positive cells . The density of T22 positive cells was 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 . 0007 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA .

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

(E) Immunofluorescent staining of T22 (red) and HT7 (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (F) Quantitative analysis of T22 positive cells. The density of T22 positive cells was 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=0.0007 (***P < 0.001), ****P<0.0001, one-way ANOVA.


B , Total pup retrieval time in biological mothers ( n = 6 ) , foster mothers ( n = 7 ) , and virgin females ( n = 10 ) on 3 consecutive days .

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B, Total pup retrieval time in biological mothers (n=6), foster mothers (n=7), and virgin females (n=10) on 3 consecutive days.


C , D Fraction of parental ( C ) and ignoring ( D ) behaviors of virgin females , of the total session duration in each of the three pup exposure days ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are displayed as individual measurements with the line connecting the mean value ; * p < 0 . 05 .

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C,D Fraction of parental (C) and ignoring (D) behaviors of virgin females, of the total session duration in each of the three pup exposure days (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as individual measurements with the line connecting the mean value; *p < 0.05.


F , A representative image of a coronal brain section immunostained for c - Fos after pup retrieval on day 1 and day 3 . The mPFC region is indicated by dashed lines , which is also shown in the insets in higher magnification . Scale bar indicates 500 µm , 10 × magnification .

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F, A representative image of a coronal brain section immunostained for c-Fos after pup retrieval on day 1 and day 3. The mPFC region is indicated by dashed lines, which is also shown in the insets in higher magnification. Scale bar indicates 500 µm, 10× magnification.


G , Heat - map of the Z - score for the difference in the fraction of c - Fos expressing cells , between day 1 and day 3 of behavioral testing . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) .

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G, Heat-map of the Z-score for the difference in the fraction of c-Fos expressing cells, between day 1 and day 3 of behavioral testing. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7).


H , Representative coronal sections of the mPFC immunostained for c - Fos with the anatomical borders of the ACC indicated by dashed lines , in foster mothers ( FM ) and virgin females exposed to pups ( V pups ) or objects ( V object ) on day 1 and day 3 of behavioral testing . Scale bar 100 µm , 20 × magnification .

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

H, Representative coronal sections of the mPFC immunostained for c-Fos with the anatomical borders of the ACC indicated by dashed lines, in foster mothers (FM) and virgin females exposed to pups (V pups) or objects (V object) on day 1 and day 3 of behavioral testing. Scale bar 100 µm, 20× magnification.


A , Reconstruction of the mouse brain following anterograde labeling with AAV - GFP in the ACC ( I ) ; representative coronal images showing site of injection and axonal distribution pattern in the dorsal striatum ( II ) and central lateral nucleus of the thalamus ( III , IV ) . Images in aI - aIV were taken from the Allen Mouse Brain Connectivity

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A, Reconstruction of the mouse brain following anterograde labeling with AAV-GFP in the ACC (I); representative coronal images showing site of injection and axonal distribution pattern in the dorsal striatum (II) and central lateral nucleus of the thalamus (III, IV). Images in aI-aIV were taken from the Allen Mouse Brain Connectivity Atlas


C , A representative coronal section showing labeling pattern at the site of injection . White arrows in the magnified image ( C , right ) point to double - labeled putative starter cells for transsynaptic retrograde labeling .

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C, A representative coronal section showing labeling pattern at the site of injection. White arrows in the magnified image (C, right) point to double-labeled putative starter cells for transsynaptic retrograde labeling.


D , Representative coronal sections showing bidirectional connectivity patterns with the thalamus ( I ) and monosynaptic input to the ACC from the claustrum ( II ) and ventral CA1 ( III ) . Scale bars 500 µm , 10 × magnification ( left images ) and 100 µm , 20 × magnification ( right images ) .

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D, Representative coronal sections showing bidirectional connectivity patterns with the thalamus (I) and monosynaptic input to the ACC from the claustrum (II) and ventral CA1 (III). Scale bars 500 µm, 10× magnification (left images) and 100 µm, 20× magnification (right images).


G , Representative coronal sections of the dorsal thalamus , following c - Fos immunolabeling on day 1 ( left ) and day 3 ( right ) , with the ILM subnuclei indicated ; CM central medial nucleus , CL central lateral nucleus , PCN paracentral nucleus , RH rhomboid nucleus . Scale bar 200 µm , 10 × magnification . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) .

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G, Representative coronal sections of the dorsal thalamus, following c-Fos immunolabeling on day 1 (left) and day 3 (right), with the ILM subnuclei indicated; CM central medial nucleus, CL central lateral nucleus, PCN paracentral nucleus, RH rhomboid nucleus. Scale bar 200 µm, 10× magnification. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7).


H , Fraction of c - Fos + cells in different ILM subnuclei , virgin exposed to pups ( left ) , in foster mothers ( middle ) , and virgin exposed to object ( right ) , on day 1 and day 3 of behavioral testing . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are expressed as mean ± s . e . m ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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H, Fraction of c-Fos+ cells in different ILM subnuclei, virgin exposed to pups (left), in foster mothers (middle), and virgin exposed to object (right), on day 1 and day 3 of behavioral testing. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are expressed as mean ± s.e.m; *p < 0.05, **p < 0.01, ***p < 0.001.


B , Representative coronal sections of the PFC , following the labeling schemes described in ( A ) , for mapping inputs onto ACC excitatory ( B , top ) and inhibitory ( B , bottom ) neurons . High magnification images ( B , right top and bottom ) show the double - labeled , putative starter cells for transsynaptic retrograde labeling . Scale bars represent 200 µm , 10 × magnification ( left top and bottom ) , and 50 µm ( right top and bottom ) , 20 × magnification .

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B, Representative coronal sections of the PFC, following the labeling schemes described in (A), for mapping inputs onto ACC excitatory (B, top) and inhibitory (B, bottom) neurons. High magnification images (B, right top and bottom) show the double-labeled, putative starter cells for transsynaptic retrograde labeling. Scale bars represent 200 µm, 10× magnification (left top and bottom), and 50 µm (right top and bottom), 20× magnification.


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 .

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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 , 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 ;

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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 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 ) .

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, 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 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 .

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, 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 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 .

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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 , 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 )

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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 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 .

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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 + 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 .

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

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

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


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 ) .

O O O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O 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 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 , 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 .

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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 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 .

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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 , 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 .

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(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 , 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 .

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

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

O O O O B-EXP_ASSAY O 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 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 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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O 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 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 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 .

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

(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 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-CELL_LINE 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O 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 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 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 .

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

(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 , 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 .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD 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 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 B-SMALL_MOLECULE 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 (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 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 ) .

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

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 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 O O B-CELL_LINE 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 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

(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 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 .

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

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

(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 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

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

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 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 .

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

(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 , 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 .

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

(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 , 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 .

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

. (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 , 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 .

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

(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 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-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

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

(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 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 ) .

(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 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 ) .

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

(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 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 .

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

(A) 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 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 .

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

(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.


) 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 .

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

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


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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 ) .

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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 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 ) .

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(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 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 .

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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 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 ) .

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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 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 .

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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 analysis 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 RNA - Scope immunostaining images of LOC113230 levels in CRC tissue and 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 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 .

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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 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 .

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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 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 .

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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 cell migration , while LOC113230 antisense showed no apparent effect on cell 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 : 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 .

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

(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 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 .

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

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 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 .

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

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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 cell migration in SW - 1116 cells . 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 the effect of ASS1WT and ASS1K234R on Caco - 2 - Vector or Caco - 2 - LOC113323 cell 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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and PITRM1R183Q proteins . Quantification of these experiments is displayed below the blots . Experiments were performed in duplicate .

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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 : 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 .

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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 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 .

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

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 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 O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O 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 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 .

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