( G ) Maximal projection of a confocal z - stack of MZsinhcaf - / - or WT 4 - stage injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . arrowheads point to cleavage furrows of 4 - .

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(G) Maximal intensity projection of a confocal z-stack of MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. White arrowheads point to cleavage furrows of 4-cell embryos.


( H ) Relative GFP - Buc density at cleavage furrow of MZsinhcaf - / - ( n = 12 ) or WT ( n = 13 ) 4 - stage injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . Data shown are mean ± ( n = number of analyzed ) .

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(H) Relative GFP-Buc density at the cleavage furrow of MZsinhcaf -/- (n = 12) or WT (n = 13) 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. Data shown are mean ± SEM (n = the number of embryos analyzed).


( I ) Statistical of relative of GFP - Buc / a - Tubulin . Data shown are mean ± ( n = 3 , biological replicates ) . ( J ) Western blots for GFP - Buc expression in MZsinhcaf - / - or WT 4 - stage injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical was performed using one - way ANOVA followed by Tukey ' s post - hoc . Scale bars , 100 μm .

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(I) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (J) Western blots for GFP-Buc expression in MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


A . Graphs showing relative abundance of undigested as a function of time during digestion with micrococcal nuclease ( MNase ) for three types of wrapped around CENP - A nucleosome ( red ) H3 nucleosome ( green ) . For α - satellite with initial of 171 bp , ranges ( 141 - 171 ) corresponding to lengths above NCP ( nucleosome particle ) is presented . Data are presented as mean ( SD ) for each time point on three independent experiments .

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

A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments.


B . Native PAGE gel stained with Commassie blue showing complexes formed between CENP - A or H3 nucleosome CENP - CCR . Lane 1 : CENP - A nucleosome , Lanes 2 - 4 : Increasing amounts of CENP - CCR are added to CENP - A nucleosome . Generation of a sharp band with slower mobility indicates formation of a specific CENP - A / CENP - CCR . Lane 5 : H3 nucleosome . Lanes 6 - 8 : Increasing amounts of CENP - CCR are added to H3 nucleosome . Smear on gel indicates formation of non - specific CENP - A / CENP - CCR complexes . C . Same experiment as in ( B ) using CENP - Cmotif . Lane 1 . CENP - A nucleosome . Lanes 2 - 4 . Increasing amounts of CENP - Cmotif are added to CENP - A nucleosome . Upon binding CENP - Cmotif , CENP - A nucleosome migrates slower through gel . Note only modest change in mobility due to small of CENP - Cmotif , comparing to CENP - CCR in ( B ) . Lane 5 . H3 nucleosome . Lanes 6 - 8 . Increasing amounts of CENP - Cmotif are added to H3 nucleosome . Smear on gel indicates formation of non - specific CENP - A / CENP - Cmotif complexes .

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

B. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes. C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes.


D . Native gel showing CENP - CCR competing out CENP - Cmotif bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome , Lane 2 : CENP - A / CENP - Cmotif . Lane 3 - 6 : Increasing amounts of CENP - CCR are added to pre - formed CENP - A / CENP - Cmotif . Formation of slower migrating bands indicates that longer CENP - CCR is replacing shorter CENP - Cmotif bound to CENP - A nucleosome .

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

D. Native gel showing CENP-CCR competing out CENP-Cmotif bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome, Lane 2: CENP-A/CENP-Cmotif complex. Lane 3-6: Increasing amounts of CENP-CCR are added to the pre-formed CENP-A/CENP-Cmotif complex. Formation of slower migrating bands indicates that longer CENP-CCR is replacing shorter CENP-Cmotif bound to the CENP-A nucleosome.


E . Native gel showing inability of CENP - Cmotif to compete out CENP - CCR bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome . Lane 2 6 : CENP - CCR / CENP - A nucleosome . Lanes 3 - 5 : Increasing amounts of CENP - Cmotif are added to pre - formed CENP - A / CENP - CCR . Formation of smear at high amounts of CENP - Cmotif added , indicates that CENP - Cmotif , at high concentrations , non - specifically binds CENP - A / CENP - CCR rather than replacing bound CENP - CCR . Lane 7 - 9 : Increasing amounts of CENP - CCR - short are added to pre - formed CENP - A / CENP - CCR . Formation of bands with higher mobility indicates that smaller CENP - CCR - short is effectively replacing bigger CENP - CCR bound to CENP - A nucleosome .

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

E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.


G . Native gels showing impaired ability of mutated CENP - CCR to form complexes with CENP - A nucleosome . molar of CENP - CCR / CENP - A nucleosomes is shown above each lane .

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G. Native gels showing the impaired ability of mutated CENP-CCR to form complexes with the CENP-A nucleosome. The molar ratio of CENP-CCR/CENP-A nucleosomes is shown above each lane.


A . Graph showing relative abundance of undigested ( 145 bp ) as a function of time during digestion with micrococcal nuclease ( MN - ase ) for CENP - A nucleosome ( red ) , CENP - A / CENP - CCR ( blue ) CENP - A / CENP - Cmotif ( black ) .

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A. Graph showing the relative abundance of undigested DNA (145 bp) as a function of time during digestion with micrococcal nuclease (MN-ase) for the CENP-A nucleosome (red), the CENP-A/CENP-CCR complex (blue) and the CENP-A/CENP-Cmotif complex (black).


D . Same type of data as in ( A ) for CENP - A nucleosome assembled with tailless H2A ( H2A , 1 - 109 ) alone ( pink ) in with CENP - CCR ( blue ) .

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D. Same type of data as in (A) for the CENP-A nucleosome assembled with tailless H2A (H2A, 1-109) alone (pink) and in complex with CENP-CCR (light blue).


( A ) Representative single immunofluorescent for dsDNA at 6 h , 24 h , 3 d of reperfusion after MCAO in penumbra as well as in sham brain . Scale bars , 5 μm .

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(A) Representative single immunofluorescent staining for dsDNA at 6 h, 24 h, and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain. Scale bars, 5 μm.


( B ) Representative of double - immunofluorescent stained for 53BP - 1 dsDNA ( upper panel ) , Iba - 1 dsDNA ( middle panel ) , for GFAP dsDNA ( lower panel ) . Scale bars , 5 μm .

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(B) Representative images of double-immunofluorescent stained for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel). Scale bars, 5 μm.


( C ) Western blot of cGAS STING using lysates prepared from indicated tissues of mice brain following MCAO . ( D ) Quantitative for Western blot .

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(C) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO. (D) Quantitative analysis for Western blot analysis.


( E ) Immunohistochemical for cGAS at 3 d of reperfusion after tMCAO in peri - ischemic as well as in corresponding regions of sham control brains . Scale bars , 50 μm , 20 μm in insets . ( F ) Quantitative of cGAS immunohistochemical . n = 6 mice per group . * * P < 0 . 01 , two - unpaired Student ' s t .

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(E) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri-ischemic area as well as in the corresponding regions of sham control brains. Scale bars, 50 μm, 20 μm in insets. (F) Quantitative analysis of cGAS immunohistochemical staining. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test.


( G ) Representative double immunofluorescent stainings for cGAS ( red ) 53BP1 ( green ) . n = 6 mice per group .

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(G) Representative double immunofluorescent stainings for cGAS (red) and 53BP1 (green). n=6 mice per group.


Mice received daily intraperitoneal ( IP ) injections of A151 ( 300 μg ) or an equal volume of vehicle for 3 consecutive days after MCAO induction . ( A ) mRNA expression of cGAS STING after MCAO were determined using qRT - PCR . n = 6 mice per group . * *

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Mice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for 3 consecutive days after MCAO induction. (A) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT-PCR. n = 6 mice per group. **


( B ) expression of cGAS , STING , NF - κB in brains of indicated groups were measured using Western blot . ( C ) Bar graph shows relative expression of cGAS , STING , NF - κB in brains of indicated groups . n = 6 mice per group .

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(B) The expression levels of cGAS, STING, and NF-κB proteins in the brains of indicated groups were measured using Western blot analysis. (C) Bar graph shows the relative protein expression levels of cGAS, STING, and NF-κB in the brains of indicated groups. n = 6 mice per group.


( D ) mRNA expression of AIM2 inflammasome components ( AIM2 , ASC , caspase - 1 ) , effector of pyroptosis GSDMD , IL - 1β , IL - 18 after MCAO were determined using qRT - PCR . n = 6 mice per group .

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(D) The mRNA expression levels of the AIM2 inflammasome components (AIM2, ASC, and caspase-1), effector of pyroptosis GSDMD, IL-1β, and IL-18 after MCAO were determined using qRT-PCR. n = 6 mice per group.


( E ) expression of AIM2 inflammasome components , GSDMD , IL - 1β in brains of indicated groups was measured using Western blot . ( F ) Bar graph shows relative expression of AIM2 inflammasome components , GSDMD , IL - 1β in brains of indicated groups . n = 6 mice per group .

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(E) The expression of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups was measured using Western blot analysis. (F) Bar graph shows the relative protein expression levels of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups. n = 6 mice per group.


( A ) Representative of immunohistochemical of IL - 1β , caspase - 1 , GSDMD in brains of indicated group . Scale bars , 50 μm . ( B ) Quantitative for immunohistochemical . n = 6 mice per group .

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(A) Representative images of immunohistochemical staining of IL-1β, caspase-1, and GSDMD in the brains of indicated group. Scale bars, 50 μm. (B) Quantitative analysis for immunohistochemical staining. n = 6 mice per group.


( C ) Representative of double - immunofluorescent stained for GFAP GSDMD , for Iba - 1 GSDMD , for NeuN GSDMD in post - ischemic brains after tMCAO .

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(C) Representative images of double-immunofluorescent stained for GFAP and GSDMD, for Iba-1 and GSDMD, and for NeuN and GSDMD in post-ischemic brains after tMCAO.


( D ) Representative of double - immunofluorescent stained for Iba - 1 GSDMD , for Iba - 1 caspase - 1 , for Iba - 1 IL - 1β of sham + vehicle , MCAO + vehicle , MCAO + A151 groups . Scale bars , 50 μm . ( E ) Bar graph shows relative immunofluorescence of GSDMD , caspase - 1 , IL - 1β in brains of indicated groups . n = 6 mice per group . DAPI : nuclei .

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

(D) Representative images of double-immunofluorescent stained for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. (E) Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. DAPI staining: nuclei.


( A ) Expression of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 ( FL ) , GSDMD in microglia lysates was detected using western blot . ( B ) Quantitative for Western blot of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 GSDMD .

(A) Expression of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 (FL), and GSDMD in primary microglia lysates was detected using western blot. (B) Quantitative analysis for Western blot analysis of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 and GSDMD.


( C ) of caspase - 1 p20 IL - 1β ( p17 ) in supernatant of cultured microglia were determined by western blot . ( D ) Quantitative for Western blot of caspapse - 1 IL - 1β . * P < 0 . 05 , * * P < 0 . 01 compared with untreated group , # P < 0 . 05 , # # P < 0 . 01 compared with LPS + poly ( dA : dT ) group .

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(C) The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by western blot. (D) Quantitative analysis for Western blot analysis of caspapse-1 and IL-1β. *P < 0.05, **P<0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group.


( E - F ) Immunofluorescence was conducted to detect expression of cGAS , NF - κB , pyroptosis associated molecules ( GSDMD , caspase - 1 , IL - 1β ) in BV2 . n = 3 in each group . Scale bars , 50 μm .

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(E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm.


( A ) Gating strategy of brain - infiltrating immune including CD4 + T ( CD3 + CD4 + ) , CD8 + T ( CD3 + CD8 + ) , B ( CD3 - CD19 + ) , NK ( CD3 - NK1 . 1 + ) , monocyte / macrophages ( CD11b + CD45highF4 / 80 + ) , neutrophils ( CD11b + CD45highLy6G + ) , microglia ( CD11b + CD45int ) their expression of IL ( interleukin ) - 6 , TNF - α ( necrosis factor - α ) , TGF - β ( transforming growth factor - β ) , IL - 4 . FMO , minus one . ( B ) Quantitative of of brain - infiltrating leukocytes in brains of MCAO mice receiving indicated treatment . ( C ) Bar graph shows of leukocytes subtype in spleens of MCAO mice receiving indicated treatment . n = 6 mice per group . ( D ) Quantitative of microglia in brains of MCAO mice receiving indicated treatment . n = 6 mice per group . *

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(A) Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), NK cells (CD3-NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. (B) Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. (C) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. (D) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *


( E ) Bar graph shows percentages of microglia expressing IL - 6 , TNF - α , TGF - β , IL - 10 in brains of MCAO mice receiving indicated treatment . n = 6 mice per group .

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(E) Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group.


( F ) Representative of immunofluorescent for ly6G in peri - infarct areas of MCAO mice receiving indicated treatment . ( G ) Bar graph shows number of ly6G + neutrophils . n = 6 mice per group .

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(F) Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. (G) Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group.


( H ) Representative of immunofluorescent for Iba - 1 in peri - infarct areas of MCAO mice receiving indicated treatment . ( I ) Bar graph shows number of Iba - 1 + microglia . n = 6 mice per group .

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(H) Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. (I) Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group.


( B ) Neurological were performed to evaluate motor , sensory , balance functions in mice receiving A151 , or vehicle at days 1 , 3 , 7 , 14 after MCAO . n = 10 mice per group .

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(B) Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving A151, or vehicle at days 1, 3, 7, and 14 after MCAO. n = 10 mice per group.


( C ) Representative TTC of brain sections from MCAO mice on day 3 after MCAO , infarct is shown in .

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(C) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO, the infarct area is shown in white.


( D ) Bar graph shows percentages of infarct volume of indicated groups . n = 10 mice per group . *

(D) Bar graph shows percentages of infarct volume of indicated groups. n = 10 mice per group. *


( E ) Flow cytometry plots ( F ) summarized results show percentages of Annexin V + PI + in brains of MCAO mice receiving A151 or vehicle . n = 6 mice per group .

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(E) Flow cytometry plots and (F) summarized results show percentages of Annexin V+ PI+ cells in the brains of MCAO mice receiving A151 or vehicle. n = 6 mice per group.


( G ) Representative histology for TUNEL . ( H ) Bar gragh shows percentages of TUNEL - positive . n = 6 mice per group .

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(G) Representative histology images for TUNEL staining. (H) Bar gragh shows percentages of TUNEL-positive cells. n = 6 mice per group.


( A ) Representative double immunofluorescent stainings for Iba - 1 cGAS , for GFAP cGAS . Scale bars , 10 μm .

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(A) Representative double immunofluorescent stainings for Iba-1 and cGAS, and for GFAP and cGAS. Scale bars, 10 μm.


( D ) Representative genotyping for WT , cGAS - KD , cGAS - KO mice .

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(D) Representative genotyping analysis for WT, cGAS-KD, and cGAS-KO mice.


( F ) Representative of TTC of brain sections from vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , A151 - treated cGAS - KO mice on day 3 after tMCAO . Three representative rostrocaudal of TTC are shown . ( G ) Bar graph shows percentages of infarct volume of indicated group . n = 6 mice per group . ( H ) Bar graph shows modified Neurological Severity Score ( mNSS ) in vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , A151 - treated cGAS - KO mice n = 10 mice per group .

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(F) Representative images of TTC staining of brain sections from vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice on day 3 after tMCAO. Three representative rostrocaudal levels of TTC staining are shown. (G) Bar graph shows percentages of infarct volume of indicated group. n = 6 mice per group. (H) Bar graph shows modified Neurological Severity Score (mNSS) in vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice n = 10 mice per group.


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

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


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

(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 of AEP positive C / EBPβ positive , respectively . density of both AEP positive C / EBPβ positive were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± . * P = 0 . 0390 ( * P < 0 . 05 ) , * * * P = 0 . 0002 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( C ) Quantitative of AT8 positive ( left ) TauN368 positive ( right ) , respectively . density of both AT8 positive TauN368 positive were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . n = 5 in each group , data are shown as mean ± . * P = 0 . 0194 ( * P < 0 . 05 ) , * * * P = 0 . 0001 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( D ) Quantitative of Aβ positive ( left ) APPC586 positive ( right ) , respectively . of Aβ positive density APPC586 positive were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± . * 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 of AT8 positive ( upper ) T22 positive ( lower ) , respectively . of both AT8 positive T22 positive were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± . * * 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 .

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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


( A ) Immunofluorescent of AEP 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 . arrows indicate that AEP p - C / EBP β signals ( green ) were colocalized with ATTO 550 signals ( red ) , respectively .

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


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

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


( C ) Immunofluorescent of APPC586 ( red ) Aβ ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( D ) Quantitative of APP C586 positive Aβ positive , respectively . density of both APP C586 positive Aβ positive were significantly increased by colonic - injection of AD brain extracts decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± . * * * * P < 0 . 0001 , one - way ANOVA .

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(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 of T22 ( red ) HT7 ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( F ) Quantitative of T22 positive . density of T22 positive was significantly increased by colonic - injection of AD brain extracts decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± . * * * P = 0 . 0007 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA .

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(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 ) , virgin ( 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 ) ignoring ( D ) behaviors of virgin , of total session duration in each of three pup exposure days ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures are displayed as measurements with connecting 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 day 3 . mPFC region is indicated by dashed lines , which is also shown in 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 Z - score for difference in fraction of c - Fos expressing , between day 1 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 mPFC immunostained for c - Fos with anatomical borders of ACC indicated by dashed lines , in foster mothers ( FM ) virgin exposed to pups ( V pups ) or objects ( V object ) on day 1 day 3 of behavioral testing . Scale bar 100 µm , 20 × magnification .

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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 mouse brain following anterograde labeling with AAV - GFP in ACC ( I ) ; representative coronal showing site of injection axonal distribution pattern in dorsal striatum ( II ) central lateral nucleus of thalamus ( III , IV ) . in aI - aIV were taken from 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 site of injection . arrows in magnified image ( C , right ) point to double - labeled putative starter 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 thalamus ( I ) monosynaptic input to ACC from claustrum ( II ) ventral CA1 ( III ) . Scale bars 500 µm , 10 × magnification ( left ) 100 µm , 20 × magnification ( right ) .

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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 dorsal thalamus , following c - Fos immunolabeling on day 1 ( left ) day 3 ( right ) , with 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 + in different ILM subnuclei , virgin exposed to pups ( left ) , in foster mothers ( middle ) , virgin exposed to object ( right ) , on day 1 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 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 PFC , following labeling schemes described in ( A ) , for mapping inputs onto ACC excitatory ( B , top ) inhibitory ( B , bottom ) neurons . High magnification ( B , right top bottom ) show double - labeled , putative starter for transsynaptic retrograde labeling . Scale bars represent 200 µm , 10 × magnification ( left top bottom ) , 50 µm ( right top 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 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 .

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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 , 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 ;

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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 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 ) .

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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 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 .

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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 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 .

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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 , 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 )

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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 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 .

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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 + 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 .

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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 of CL , immunolabeled for c - Fos , following hM3D - mediated neuronal activation of 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 hM3D - mediated activation of ACC ( as measured in ( F , top ) ) 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 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 ) .

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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 .

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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 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 .

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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 , - 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 .

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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 , 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 .

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(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 .

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

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

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

(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 .

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

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

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

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

(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 O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

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

(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

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

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 .

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

(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 .

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

(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 .

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

. (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 .

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

(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 .

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

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 ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR 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 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 ) .

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

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

(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 .

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

(B) 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 .

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

(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).


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

O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 .

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

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 ) .

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

(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 .

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

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.