C . At day 21 , cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT - qPCR . Reduction in viral stimulation in Vs treated samples are represented as percentage values . ND - non determined . D . Aggregate plot for 3 patients from data ( C ) .

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C. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT-qPCR. Reduction in viral stimulation in Vs treated samples are represented as percentage values. ND - non determined. D. Aggregate plot for 3 patients from data (C).


A . The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells . The short black lines indicate individual expressions , the blue and yellow lines indicate the average expression level in each condition . The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4 .

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A. The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells. The short black lines indicate individual expressions, the blue and yellow lines indicate the average expression level in each condition. The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4.


B . The expression of EP subtypes in CD11b + cells isolated from tumor tissues and spleen of CT26 tumor - bearing mice by magnetic bead separation ( n = 3 ) .

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B. The expression of EP subtypes in CD11b+ cells isolated from tumor tissues and spleen of CT26 tumor-bearing mice by magnetic bead separation (n=3).


D . Representative flow cytometry plots revealing the effects of prostaglandin E2 ( PGE2 ) and EP1 - 4 antagonists on mouse bone marrow monocyte dendritic cell ( DC ) / macrophage differentiation ( n = 3 ) . E . The frequencies of F4 / 80 + CD11C − macrophages and F4 / 80 − CD11C + DCs under varying treatments as analyzed by flow cytometry analysis ( n = 3 ) .

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D. Representative flow cytometry plots revealing the effects of prostaglandin E2 (PGE2) and EP1-4 antagonists on mouse bone marrow monocyte dendritic cell (DC)/macrophage differentiation (n = 3). E. The frequencies of F4/80+CD11C− macrophages and F4/80−CD11C+ DCs under varying treatments as analyzed by flow cytometry analysis (n = 3).


G . Representative flow cytometry plots revealing the effects of PGE2 and EP1 - 4 antagonists on mouse bone marrow derived - MDSC differentiation . Upper subpanel : vehicle group and PGE2 group . Lower subpanel : ONO - 8711 ( EP1 antagonist ) group , PF - 04418948 ( EP2 antagonist ) group , L - 798106 ( EP3 antagonist ) group , and E7046 ( EP4 antagonist ) group . ( n = 3 ) . H . The frequencies of Ly6C + Ly6G − mMDSC and Ly6CmidLy6G + PMN - MDSCs under varied treatments as analyzed by flow cytometry analysis ( n = 3 ) .

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G. Representative flow cytometry plots revealing the effects of PGE2 and EP1-4 antagonists on mouse bone marrow derived-MDSC differentiation. Upper subpanel: vehicle group and PGE2 group. Lower subpanel: ONO-8711 (EP1 antagonist) group, PF-04418948 (EP2 antagonist) group, L-798106 (EP3 antagonist) group, and E7046 (EP4 antagonist) group. (n = 3). H. The frequencies of Ly6C+Ly6G− mMDSC and Ly6CmidLy6G+ PMN-MDSCs under varied treatments as analyzed by flow cytometry analysis (n = 3).


B . Dose - effect curve of TP - 16 in GloSensor ™ cAMP assay in EP4 - expressing 293 cells ( n = 3 ) .

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B. Dose-effect curve of TP-16 in GloSensor™ cAMP assay in EP4- expressing 293 cells (n=3).


C . Dose - effect curves of TP - 16 in PGE2 - induced calcium flux assay ( n = 3 ) .

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C. Dose-effect curves of TP-16 in PGE2-induced calcium flux assay (n=3).


D . The Schild plot of PGE2 in the presence of varying concentrations of TP - 16 . TP - 16 shifted the dose - response curve of PGE2 - induced intracellular cAMP levels in a dose - dependent manner ( n = 3 ) . E . The pA2 value and slope of the Schild plot .

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D. The Schild plot of PGE2 in the presence of varying concentrations of TP-16. TP-16 shifted the dose-response curve of PGE2-induced intracellular cAMP levels in a dose-dependent manner (n=3). E. The pA2 value and slope of the Schild plot.


B . The antitumor activities of E7046 ( 150 mg / kg ) and TP - 16 ( 37 . 5 , 75 , and150 mg / kg ) in CT26 tumor - bearing BALB / c mice ( n = 11 per group ) . Tumor growth curves in the CT26 model ( left panel ) . Bar graphs show the individual changes in tumor growth relative to after and before drug administration ( right panel ) . Tumor volumes reached 100 - 200 mm3 on day 7 .

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B. The antitumor activities of E7046 (150 mg/kg) and TP-16 (37.5, 75, and150 mg/kg) in CT26 tumor-bearing BALB/c mice (n = 11 per group). Tumor growth curves in the CT26 model (left panel). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.


C . Representative graph showing that the antitumor activity of TP - 16 is lost in CT26 tumors engrafted into BALB / c nude mice ( left panel ) ( n = 8 ) . Bar graphs show the individual changes in tumor growth relative to after and before drug administration ( right panel ) . Tumor volumes reached 100 - 200 mm3 on day 7 .

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C. Representative graph showing that the antitumor activity of TP-16 is lost in CT26 tumors engrafted into BALB/c nude mice (left panel) (n = 8). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.


D . Single - cell suspensions of tumor tissues from CT26 tumor - bearing BALB / c mice treated with vehicle or TP - 16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis . Representative flow cytometry and dot plots of intratumoral frequencies of CD8 + T cells ( n = 5 ) .

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D. Single-cell suspensions of tumor tissues from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).


E . Growth curves ( left panel ) and relative change after 18 days treatment ( right panel ) in colon cancer ( MC38 ) xenograft tumors in C57BL / 6 mice ( n = 7 ) . Tumor volumes reached 100 - 200 mm3 on day 7 .

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E. Growth curves (left panel) and relative change after 18 days treatment (right panel) in colon cancer (MC38) xenograft tumors in C57BL/6 mice (n = 7). Tumor volumes reached 100-200 mm3 on day 7.


F . Single - cell suspensions of tumor tissues from MC38 tumor - bearing C57BL / 6 mice treated with vehicle or TP - 16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis . Representative flow cytometry and dot plots of intratumoral frequencies of CD8 + T cells ( n = 5 ) .

F. Single-cell suspensions of tumor tissues from MC38 tumor-bearing C57BL/6 mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).


G . The representative bioluminescence images ( left panel ) and quantitative growth curves ( right panel ) of an orthotopic colorectal cancer mouse model , CT26 - Luc model ( n = 4 ) .

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G. The representative bioluminescence images (left panel) and quantitative growth curves (right panel) of an orthotopic colorectal cancer mouse model, CT26-Luc model (n = 4).


A , B . Tumors from CT26 tumor - bearing BALB / c mice treated with vehicle or TP - 16 for 2 weeks were harvested , and tumor single - cell suspensions were analyzed for tumor - associated myeloid cells by flow cytometry analysis . Representative graphs and quantification of F4 / 80 + MHC - II + immunosuppressive myeloid cells ( IMCs ) - M1 macrophages ( A ) and F4 / 80 + CD206 + IMCs - M2 macrophages ( B ) gated on CD45 + CD11b + myeloid cells ( n = 5 ) .

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A, B. Tumors from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were harvested, and tumor single-cell suspensions were analyzed for tumor-associated myeloid cells by flow cytometry analysis. Representative graphs and quantification of F4/80+MHC-II+ immunosuppressive myeloid cells (IMCs)-M1 macrophages (A) and F4/80+CD206+ IMCs-M2 macrophages (B) gated on CD45+CD11b+ myeloid cells (n = 5).


C . Representative flow cytometry analysis and quantification of Ly6C + Ly6G - IMCs - monocytic myeloid - derived suppressor cell ( mMDSC ) and Ly6CmidLy6G + IMCs - PMN - MDSC frequencies gated on CD45 + CD11b + myeloid cells ( n = 5 ) .

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C. Representative flow cytometry analysis and quantification of Ly6C+Ly6G- IMCs-monocytic myeloid-derived suppressor cell (mMDSC) and Ly6CmidLy6G+ IMCs-PMN-MDSC frequencies gated on CD45+CD11b+ myeloid cells (n = 5).


Relative expression of selected M1 , M2 markers on flow - sorted M1 macrophages ( D ) , M2 macrophages ( E ) from CT26 tumors . Data are normalized to β - actin expression levels ( n = 5 ) .

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Relative expression of selected M1, M2 markers on flow-sorted M1 macrophages (D), M2 macrophages (E) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5).


Relative expression of selected MDSC markers on flow - sorted mMDSCs ( F ) , and PMN - MDSCs ( G ) from CT26 tumors . Data are normalized to β - actin expression levels ( n = 5 ) .

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Relative expression of selected MDSC markers on flow-sorted mMDSCs (F), and PMN-MDSCs (G) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5).


H - K . Representative flow analysis and quantification of CD8 + T cell activation markers : Granzyme B ( H ) , IFN - γ ( I ) , TNF - α ( J ) , and PD - 1 ( K ) ( n = 5 ) .

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H-K. Representative flow analysis and quantification of CD8+ T cell activation markers: Granzyme B (H), IFN-γ (I), TNF-α (J), and PD-1 (K) (n = 5).


M - CSF induced mouse bone marrow derived macrophages ( BMMs ) were stimulated with 50 ng / mL mouse recombinant IFN - γ ( A ) in the absence or presence of PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected M1 markers ( Cxcl10 and Tnfa ) were measured by q - PCR . Data are normalized to β - actin expression levels ( n = 3 ) .

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M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 50 ng/mL mouse recombinant IFN-γ (A) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M1 markers (Cxcl10 and Tnfa) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).


B . M - CSF induced mouse bone marrow derived macrophages ( BMMs ) were stimulated with 20 ng / mL mouse recombinant IL - 4 ( B ) in the absence or presence of PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected M2 markers ( Arg - 1 and Ym - 1 ) were measured by q - PCR . Data are normalized to β - actin expression levels ( n = 3 ) .

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B. M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 20 ng/mL mouse recombinant IL-4 (B) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M2 markers (Arg-1 and Ym-1) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).


C . Bone marrow cells were cultured with 40 ng / mL GM - CSF / IL - 6 or co - stimulated with PGE2 ± varying concentrations of TP - 16 for 6 days . Representative quantification of Ly6C + Ly6G − monocytic myeloid - derived suppressor cells ( mMDSCs ) and Ly6CmidLy6G + PMN - MDSCs ( n = 3 ) .

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C. Bone marrow cells were cultured with 40 ng/mL GM-CSF/IL-6 or co-stimulated with PGE2 ± varying concentrations of TP-16 for 6 days. Representative quantification of Ly6C+Ly6G− monocytic myeloid-derived suppressor cells (mMDSCs) and Ly6CmidLy6G+ PMN-MDSCs (n = 3).


D . Bone marrow cells were treated with GM - CSF / IL - 6 for 6 days to induce MDSCs and thereafter , with PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected MDSC markers ( Arg - 1 , Ptgs2 , Il - 4 , and Il - 10 ) were measured by qPCR . Data are normalized to β - actin expression levels ( n = 3 ) .

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D. Bone marrow cells were treated with GM-CSF/IL-6 for 6 days to induce MDSCs and thereafter, with PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected MDSC markers (Arg-1, Ptgs2, Il-4, and Il-10) were measured by qPCR. Data are normalized to β-actin expression levels (n=3).


E , F . In vitro T cell suppression activity of CT26 - tumor infiltrating CD11b + myeloid cells collected at 2 weeks post - treatment with vehicle or 75 mg / kg TP - 16 . CD8 + T cells were labeled with CFSE and then stimulated with CD3 / CD28 antibodies for 3 days in the absence or presence of tumor - infiltrating CD11b + myeloid cells from vehicle - or 75 mg / kg TP - 16 - treated CT26 tumors . Representative flow cytometry histograms ( E ) and percentages ( F ) of proliferating CD8 + T cells when plated in a ratio of 2 : 1 , 1 : 1 , and 1 : 2 CD8 + T cells to CD11b + myeloid cells ( n = 3 ) .

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E, F. In vitro T cell suppression activity of CT26-tumor infiltrating CD11b+ myeloid cells collected at 2 weeks post-treatment with vehicle or 75 mg/kg TP-16. CD8+ T cells were labeled with CFSE and then stimulated with CD3/CD28 antibodies for 3 days in the absence or presence of tumor-infiltrating CD11b+ myeloid cells from vehicle- or 75 mg/kg TP-16-treated CT26 tumors. Representative flow cytometry histograms (E) and percentages (F) of proliferating CD8+ T cells when plated in a ratio of 2:1, 1:1, and 1:2 CD8+ T cells to CD11b+ myeloid cells (n = 3).


A . The growth of CT26 tumor volume when treated with vehicle , TP - 16 ( 75 mg / kg , p . o . , daily ) , anti - PD - 1 antibody ( 50 μg , i . p . , twice weekly ) or their combination ( left ) and percent change in tumor volumes between days 0 and 20 ( right ) ( n = 12 ) .

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A. The growth of CT26 tumor volume when treated with vehicle, TP-16 (75 mg/kg, p.o., daily), anti-PD-1 antibody (50 μg, i.p., twice weekly) or their combination (left) and percent change in tumor volumes between days 0 and 20 (right) (n=12).


B . BALB / c mice with subcutaneous CT26 tumors were treated with vehicle , TP - 16 ( 75 mg / kg , p . o . , daily ) , PD - 1 antibody ( 50 μg , i . p . , twice weekly ) or combined therapies for 2 weeks . Animal survival ( time to tumor burden reaching 2000 mm3 ) was analyzed by the Kaplan - Meier method using GraphPad Prism ( n = 10 ) . P values were calculated using the Log - rank test .

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B. BALB/c mice with subcutaneous CT26 tumors were treated with vehicle, TP-16 (75 mg/kg, p.o., daily), PD-1 antibody (50 μg, i.p., twice weekly) or combined therapies for 2 weeks. Animal survival (time to tumor burden reaching 2000 mm3) was analyzed by the Kaplan-Meier method using GraphPad Prism (n=10). P values were calculated using the Log-rank test.


C . Representative immunofluorescence staining images of tumor sections from CT26 tumor - bearing mice stained for CD8 and DAPI . Scale bars , 50 μm . D . Representative immunostaining images of tumor sections from CT26 tumor - bearing mice stained for p - STAT3 , p - AKT and PD - L1 . Scale bars , 100 μm .

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C. Representative immunofluorescence staining images of tumor sections from CT26 tumor-bearing mice stained for CD8 and DAPI. Scale bars, 50 μm. D. Representative immunostaining images of tumor sections from CT26 tumor-bearing mice stained for p-STAT3, p-AKT and PD-L1. Scale bars, 100 μm.


F . The heatmap of T cell activation biomarkers for RNA - seq analysis . The unit of heatmap scale is expression value after centering and scaling by genes .

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F. The heatmap of T cell activation biomarkers for RNA-seq analysis. The unit of heatmap scale is expression value after centering and scaling by genes.


Representative resected colons ( B ) after 5 weeks of indicated treatment ( n = 7 ) .

Representative resected colons (B) after 5 weeks of indicated treatment (n=7).


total number of colon tumors per mouse ( C , left ) and tumor size distribution ( C , right ) , and colon length ( D ) after 5 weeks of indicated treatment ( n = 7 ) .

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total number of colon tumors per mouse (C, left) and tumor size distribution (C, right), and colon length (D) after 5 weeks of indicated treatment (n=7).


E . Representative hematoxylin and eosin ( H & E ) staining and immunostaining for CD8 , p - STAT3 , p - AKT , and ARG - 1 of tumors treated with vehicle , TP - 16 , anti - PD - 1 antibody , or TP - 16 and anti - PD - 1 combined . Scale bars , 500 μm ( left images ) , 50 μm ( right images ) .

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E. Representative hematoxylin and eosin (H&E) staining and immunostaining for CD8, p-STAT3, p-AKT, and ARG-1 of tumors treated with vehicle, TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined. Scale bars, 500 μm (left images), 50 μm (right images).


F . Quantitative polymerase chain reaction ( q - PCR ) analysis of the mRNA level of genes associated with immunosuppression ( n = 6 ) .

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F. Quantitative polymerase chain reaction (q-PCR) analysis of the mRNA level of genes associated with immunosuppression (n = 6).


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 .

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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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(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 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(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 .

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

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

(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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

(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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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.


) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) 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 the electron micrograph of the Brucella - infected control cell 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

(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).


a , Ultrasonic vocalizations ( USV ) in isolated miR379 - 410 ko mouse pups emitted on postnatal day P3 , P6 , P9 and P12 . Left panel : total number of ultrasonic vocalizations ( USV ) in isolated miR379 - 410 wt and ko mouse pups emitted on average ( P 3 , 6 , 9 and 12 ) when isolated from the mother , wt n = 13 ( male n = 9 , female n = 4 ) , ko n = 20 ( male n = 11 , female n = 9 ) , t31 = 2 . 274 , * p = 0 . 0301 , unpaired Student ' s t - test . Right panel : developmental course for total number of USV , wt n = 13 ( male n = 9 , female n = 4 ) , ko n = 20 ( male n = 11 , female n = 9 ) , ( development : F3 , 87 = 25 . 463 , p < 0 . 001 ; genotype : F1 , 29 = 4 . 979 , p = 0 . 03 ; development x genotype : F3 , 87 = 1 . 403 , p = 0 . 22 , rm - ANOVA ) ; P 9 : t31 = 2 . 096 , * p = 0 . 044 ; unpaired Student ` s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Ultrasonic vocalizations (USV) in isolated miR379-410 ko mouse pups emitted on postnatal day P3, P6, P9 and P12. Left panel: total number of ultrasonic vocalizations (USV) in isolated miR379-410 wt and ko mouse pups emitted on average (P 3, 6, 9 and 12) when isolated from the mother, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), t31=2.274, *p=0.0301, unpaired Student's t-test. Right panel: developmental course for total number of USV, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), (development: F3,87=25.463, p<0.001; genotype: F1,29=4.979, p=0.03; development x genotype: F3,87=1.403, p= 0.22, rm-ANOVA); P 9: t31=2.096, *p=0.044; unpaired Student`s t-test.


b , Reciprocal social interaction in juvenile miR379 - 410 wt - and ko mice pairs ( P23 ) . Aberrant time spending in social interaction activity in miR379 - 410 ko - pairs compared to their control wt littermate pairs . Left panel : wt - pairs male n = 13 , ko - pairs male n = 10 , t21 = 2 . 433 , * p = 0 . 0240 , unpaired Student ' s t - test . Middle panel : wt - pairs female n = 15 , ko - pairs female n = 13 , t26 = 1 . 873 , ns p = 0 . 0723 , unpaired Student ' s t - test . Right panel : pooled data of wt - pairs n = 28 ( male n = 13 , female n = 15 ) , ko - pairs n = 23 ( male n = 10 , female n = 13 ) , t49 = 2 . 969 , * * p = 0 . 005 ; unpaired Student ` s t - test .

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

b, Reciprocal social interaction in juvenile miR379-410 wt- and ko mice pairs (P23). Aberrant time spending in social interaction activity in miR379-410 ko-pairs compared to their control wt littermate pairs. Left panel: wt-pairs male n=13, ko-pairs male n=10, t21=2.433, *p=0.0240, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=1.873, ns p=0.0723, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=2.969, **p=0.005; unpaired Student`s t-test.


c , Emitted USVs during social interaction activity in genotype - matched pairs in juvenile mice ( P23 ) . Left panel : wt - pairs male n = 13 , ko - pairs male n = 15 , t21 = 2 . 704 , * p = 0 . 0133 , unpaired Student ' s t - test . Middle panel : wt - pairs female n = 15 , ko - pairs female n = 13 , t26 = 2 . 869 , * * p = 0 . 0081 , unpaired Student ' s t - test . Right panel : pooled data of wt - pairs n = 28 ( male n = 13 , female n = 15 ) , ko - pairs n = 23 ( male n = 10 , female n = 13 ) , t49 = 3 . 992 , * * * p < 0 . 001 ; unpaired Student ` s t - test .

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

c, Emitted USVs during social interaction activity in genotype-matched pairs in juvenile mice (P23). Left panel: wt-pairs male n=13, ko-pairs male n=15, t21=2.704, *p=0.0133, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=2.869, **p=0.0081, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=3.992, ***p<0.001; unpaired Student`s t-test.


d , Social preference index in adolescent mice - defined as the ratio of time sniffing a stranger mouse vs . an object - is shown . Left panel : wt male n = 26 , ko male n = 26 , t50 = 1 . 075 , ns p = 0 . 2876 , unpaired Student ' s t - test . Middle panel : wt female n = 27 , ko female n = 30 , t55 = 2 . 017 , * p = 0 . 0486 , unpaired Student ' s t - test . Right panel : pooled data of wt n = 53 ( male n = 26 , female n = 27 ) , ko n = 56 ( male n = 26 , female n = 30 ) , t107 = 2 . 283 , * p = 0 . 0244 ; unpaired Student ` s t - test .

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

d, Social preference index in adolescent mice - defined as the ratio of time sniffing a stranger mouse vs. an object - is shown. Left panel: wt male n=26, ko male n=26, t50=1.075, ns p=0.2876, unpaired Student's t-test. Middle panel: wt female n=27, ko female n=30, t55=2.017, *p=0.0486, unpaired Student's t-test. Right panel: pooled data of wt n=53 (male n=26, female n=27), ko n=56 (male n=26, female n=30), t107=2.283, *p=0.0244; unpaired Student`s t-test.


A , B Single - molecule FISH ( smFISH ) on mouse hippocampal neurons . ( A ) Representative pictures of smFISH on BDNF - treated WT ( top ) and KO ( bottom ) mouse hippocampal neurons ( DIV6 ) using a probe directed against the Mirg transcript ( green ) . ( Middle ) Hoechst ( blue ) . ( Right ) MAP2 immunostaining ( red ) . Pictures show a zoom - in on the cell body , since the signal detected in WT neurons was restricted to the nuclei ( scale bar : 5μm ) . ( B ) Representative pictures of smFISH on mouse wild type hippocampal neuron cultures ( DIV6 ; basal ) using a probe specific for the Mirg transcript ( green ) . ( Middle ) Hoechst ( blue ) . ( Right ) MAP2 immunostaining ( red ) . Top : overview , scale bar : 10μm ; bottom : zoom - in , scale bar : 5μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Single-molecule FISH (smFISH) on mouse hippocampal neurons. (A) Representative pictures of smFISH on BDNF-treated WT (top) and KO (bottom) mouse hippocampal neurons (DIV6) using a probe directed against the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Pictures show a zoom-in on the cell body, since the signal detected in WT neurons was restricted to the nuclei (scale bar: 5μm). (B) Representative pictures of smFISH on mouse wild type hippocampal neuron cultures (DIV6; basal) using a probe specific for the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Top: overview, scale bar: 10μm; bottom: zoom-in, scale bar: 5μm.


C , D , Patch - clamp electrophysiological recordings in cultured mouse hippocampal neurons ( DIV 8 - 10 ) isolated from wt or miR379 - 410 ko animals ( n = 3 per group ) . ( C ) Cumulative distribution ( left panel , D = 0 . 25 , p < 0 . 0001 , KS - test ) and mean ( right panel , t16 = 2 . 736 , * p = 0 . 0146 , unpaired Student ` s t - test ) of mEPSC inter - event intervals ; wt n = 9 , ko n = 9 cells analyzed . ( D ) Cumulative distribution ( left panel , D = 0 . 34741 , p < 0 . 0001 , KS - test ) and mean ( right panel , t18 = 2 . 357 , * p = 0 . 0299 , unpaired Student ` s t - test ) of mEPSC amplitudes ; wt n = 10 , ko n = 10 cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Patch-clamp electrophysiological recordings in cultured mouse hippocampal neurons (DIV 8-10) isolated from wt or miR379-410 ko animals (n=3 per group). (C) Cumulative distribution (left panel, D=0.25, p<0.0001, KS-test) and mean (right panel, t16=2.736, *p=0.0146, unpaired Student`s t-test) of mEPSC inter-event intervals; wt n=9, ko n=9 cells analyzed. (D) Cumulative distribution (left panel, D=0.34741, p<0.0001, KS-test) and mean (right panel, t18=2.357, *p=0.0299, unpaired Student`s t-test) of mEPSC amplitudes; wt n=10, ko n=10 cells analyzed.


e , Representative confocal images of CA1 pyramidal neuron apical dendrites from adult Thy1 - GFP / miR379 - 410 wt and Thy1 - GFP / miR379 - 410 ko mouse tissue . Scale bar : 10 µm . f , Spine density in CA1 pyramidal neurons of adult male Thy1 - GFP / miR379 - 410 wt and ko mice ; wt n = 5 , ko n = 6 animals . Mean of basal and apical neurons per mouse are shown , t9 = 3 . 662 , * * p = 0 . 0052 , unpaired Student ` s t - test . g , Spine volume in CA1 pyramidal neurons of adult Thy1 - GFP / miR379 - 410 wt and ko mice ; wt n = 5 , ko n = 6 animals . Mean of basal and apical neurons per mouse are shown , t9 = 2 . 265 , * p = 0 . 0497 , unpaired Student ` s t - test .

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

e, Representative confocal images of CA1 pyramidal neuron apical dendrites from adult Thy1-GFP/miR379-410 wt and Thy1-GFP/miR379-410 ko mouse tissue. Scale bar: 10 µm. f, Spine density in CA1 pyramidal neurons of adult male Thy1-GFP/miR379-410 wt and ko mice; wt n=5, ko n=6 animals. Mean of basal and apical neurons per mouse are shown, t9=3.662, **p=0.0052, unpaired Student`s t-test. g, Spine volume in CA1 pyramidal neurons of adult Thy1-GFP/miR379-410 wt and ko mice; wt n=5, ko n=6 animals. Mean of basal and apical neurons per mouse are shown, t9=2.265, *p=0.0497, unpaired Student`s t-test.


b , qPCR validation of predicted direct miR379 - 410 targets using RNA from hippocampi of juvenile ( P 22 - 24 ) mice , except of Shank 3 where adult miR - 379 - 410 wt and ko animals were measured . If not indicated , both , male and female samples were used . Cnih2 ( wt n = 12 , ko n = 9 , t19 = 2 . 344 , * p = 0 . 0301 ) , Dlgap3 ( wt n = 12 , ko n = 9 , t19 = 2 . 128 , * p = 0 . 0467 ) , Prr7 ( male wt n = 7 , ko n = 4 , t9 = 2 . 719 , * p = 0 . 0236 ) , Src ( wt n = 12 , ko n = 9 , t19 = 2 . 410 , * p = 0 . 0263 ) , Lzts2 ( male wt n = 7 , ko n = 4 , t9 = 2 . 554 , * p = 0 . 0310 ) , Mpp2 ( male wt n = 7 , ko n = 4 , t9 = 2 . 369 , * p = 0 . 0420 ) , Prr12 ( wt n = 6 , ko n = 4 , t8 = 2 . 375 , * p = 0 . 0449 ) , Shank1 ( wt n = 11 , ko n = 9 , t18 = 2 . 310 , * p = 0 . 0329 ) , Shb ( male wt n = 6 , ko n = 4 , t8 = 3 . 451 , * * p = 0 . 0087 ) and Shank3 ( adult male wt n = 3 , ko n = 5 , t6 = 2 . 538 , * p = 0 . 0442 ) ; unpaired Student ` s t - test . Data are presented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, qPCR validation of predicted direct miR379-410 targets using RNA from hippocampi of juvenile (P 22-24) mice, except of Shank 3 where adult miR-379-410 wt and ko animals were measured. If not indicated, both, male and female samples were used. Cnih2 (wt n=12, ko n=9, t19=2.344, *p=0.0301), Dlgap3 (wt n=12, ko n=9, t19=2.128, *p=0.0467), Prr7 (male wt n=7, ko n=4, t9=2.719, *p=0.0236), Src (wt n=12, ko n=9, t19=2.410, *p=0.0263), Lzts2 (male wt n=7, ko n=4, t9=2.554, *p=0.0310), Mpp2 (male wt n=7, ko n=4, t9=2.369, *p=0.0420), Prr12 (wt n=6, ko n=4, t8=2.375, *p=0.0449), Shank1 (wt n=11, ko n=9, t18=2.310, *p=0.0329), Shb (male wt n=6, ko n=4, t8=3.451, **p=0.0087) and Shank3 (adult male wt n=3, ko n=5, t6=2.538, *p=0.0442); unpaired Student`s t-test. Data are presented as mean ± s.e.m.


c , d , Cnih2 , Prr7 and Src are direct miR - 379 - 410 target mRNAs . ( C ) 3 ' - UTR luciferase reporter gene assays in cultured rat cortical neurons using the indicated 3 ' UTR reporter genes ( wt or seed mutant ) together with miRNA mimics . Cnih2 ( n = 3 per group ; wt : * * p = 0 . 0020 , mut : p = 0 . 9267 ; mimic : F1 , 8 = 19 . 92 , p = 0 . 0021 ; utr - construct : F1 , 8 = 4 . 760 , p = 0 . 0607 ; mimic x utr - construct : F1 , 8 = 12 . 99 , p = 0 . 0069 ) , Prr7 ( n = 3 per group ; wt : * * * * p < 0 . 0001 , mut : * p = 0 . 0103 ; wt vs . mut mimic miR329 : * * p = 0 . 0014 ; mimic : F1 , 8 = 153 . 1 , p < 0 . 0001 ; utr - construct : F1 , 8 = 5 . 631 , p = 0 . 0450 ; mimic x utr - construct : F1 , 8 = 38 . 54 , p = 0 . 0003 ) ; Src ( n = 3 per group ; wt : * p = 0 . 0110 , mut : p = 0 . 4724 ; mimic : F1 , 8 = 16 . 99 , p = 0 . 0033 ; utr - construct : F1 , 8 = 0 . 6809 , p = 0 . 4332 ; mimic vs . utr - construct : F1 , 8 = 3 . 920 , p = 0 . 0831 ) ; two - way ANOVA . ( D ) Same as in ( C ) , but in the presence of antimiRs ( pLNAs ) in hippocampal neurons after 48h of PTX stimulation . Cnih2 ( n = 4 per group ; wt : p = 0 . 2688 ; mut : p = 0 . 8668 ; wt ctr vs . mut ctr : p = 0 . 6463 ; anti - miRNA : F1 , 12 = 3 . 629 , p = 0 . 0810 ; utr - construct : F1 , 12 = 0 . 7407 , p = 0 . 4063 ; anti - miRNA x utr - construct : F1 , 12 = 0 . 6679 , p = 0 . 4297 ) , Prr7 ( n = 4 per group ; wt : * p = 0 . 0123 ; mut : p = 0 . 9917 ; wt ctr vs . mut ctr : * p = 0 . 0152 ; anti - miRNA : F1 , 12 = 6 . 082 , p = 0 . 0297 ; utr - construct : F1 , 12 = 5 . 271 ; p = 0 . 0405 ; anti - miRNA x utr - construct : F1 , 12 = 8 . 212 , p = 0 . 0142 ) ; Src ( n = 3 per group ; wt : * p = 0 . 0370 ; mut : p = 0 . 3540 ; wt ctr vs . mut ctr : p = 0 . 9906 ; anti - miRNA : F1 , 12 = 13 . 43 , p = 0 . 0064 ; utr - construct : F1 , 12 = 0 . 5672 , p = 0 . 4730 ; anti - miRNA x utr - construct : F1 , 12 = 1 . 362 , p = 0 . 2768 ) ; two - way ANOVA . Data are presented as mean ± s . d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Cnih2, Prr7 and Src are direct miR-379-410 target mRNAs. (C) 3'-UTR luciferase reporter gene assays in cultured rat cortical neurons using the indicated 3'UTR reporter genes (wt or seed mutant) together with miRNA mimics. Cnih2 (n=3 per group; wt: **p=0.0020, mut: p=0.9267; mimic: F1,8=19.92, p=0.0021; utr-construct: F1,8=4.760, p=0.0607; mimic x utr-construct: F1,8=12.99, p=0.0069), Prr7 (n=3 per group; wt: ****p<0.0001, mut: *p=0.0103; wt vs. mut mimic miR329: **p=0.0014; mimic: F1,8=153.1, p<0.0001; utr-construct: F1,8=5.631, p=0.0450; mimic x utr-construct: F1,8=38.54, p=0.0003); Src (n=3 per group; wt: *p=0.0110, mut: p=0.4724; mimic: F1,8=16.99, p=0.0033; utr-construct: F1,8=0.6809, p=0.4332; mimic vs. utr-construct: F1,8=3.920, p=0.0831); two-way ANOVA. (D) Same as in (C), but in the presence of antimiRs (pLNAs) in hippocampal neurons after 48h of PTX stimulation. Cnih2 (n=4 per group; wt: p=0.2688; mut: p=0.8668; wt ctr vs. mut ctr: p=0.6463; anti-miRNA: F1,12=3.629, p=0.0810; utr-construct: F1,12=0.7407, p=0.4063; anti-miRNA x utr-construct: F1,12=0.6679, p=0.4297), Prr7 (n=4 per group; wt: *p=0.0123; mut: p=0.9917; wt ctr vs. mut ctr: *p=0.0152; anti-miRNA: F1,12=6.082, p=0.0297; utr-construct: F1,12=5.271; p=0.0405; anti-miRNA x utr-construct: F1,12=8.212, p=0.0142); Src (n=3 per group; wt: *p=0.0370; mut: p=0.3540; wt ctr vs. mut ctr: p=0.9906; anti-miRNA: F1,12=13.43, p=0.0064; utr-construct: F1,12=0.5672, p=0.4730; anti-miRNA x utr-construct: F1,12=1.362, p=0.2768); two-way ANOVA. Data are presented as mean ± s.d.


( a ) Hippocampal tissues obtained from Nrf2 ( − / − ) ( 6 months old , one male and two female ) or wild - type ( WT ) ( 11 months old , two male and one female ) mice were immunoblotted with a Nrf2 antibody , 12E8 , PHF1 , a monoclonal to total tau ( Tau5 ) or a polyclonal to total tau ( Tau ) antibody . The asterisk to the left of the Nrf2 blot designates the position at which a nonspecific band migrated . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( b ) Bar graphs represent the relative optical density of phosphorylated or total tau in the hippocampal tissues . n = 6 ( These data are based on the mouse hippocampi immunoblotted in Supplementary Fig . 1 ) . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * * P0 . 01 ; * * * P0 . 001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hippocampal tissues obtained from Nrf2 (−/−) (6 months old, one male and two female) or wild-type (WT) (11 months old, two male and one female) mice were immunoblotted with a Nrf2 antibody, 12E8, PHF1, a monoclonal to total tau (Tau5) or a polyclonal to total tau (Tau) antibody. The asterisk to the left of the Nrf2 blot designates the position at which a nonspecific band migrated. The relative molecular masses (kDa) are indicated to the left of each blot. (b) Bar graphs represent the relative optical density of phosphorylated or total tau in the hippocampal tissues. n=6 (These data are based on the mouse hippocampi immunoblotted in Supplementary Fig. 1). Data shown are mean±s.e. and were analysed using Student's t-test. (**P0.01; ***P0.001)


( c , d ) Left panels show brain sections stained for tau phosphorylated at Ser262 / Ser356 ( 12E8 ) ( c ) and Ser396 / Ser404 ( PHF1 ) ( d ) . The area of delineated by dotted lines is enlarged and shown in the right corner of the image . Right panels shows the same slice stained with DAPI 4 ' , 6 - diamidino - 2 - phenylindole . Scale bar , 50 μm . DG , dentate gyrus ; Hi , dentate hilar area .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panels show brain sections stained for tau phosphorylated at Ser262/Ser356 (12E8) (c) and Ser396/Ser404 (PHF1) (d). The area of delineated by dotted lines is enlarged and shown in the right corner of the image. Right panels shows the same slice stained with DAPI 4',6-diamidino-2-phenylindole. Scale bar, 50 μm. DG, dentate gyrus; Hi, dentate hilar area.


Brain cortical tissue obtained from Nrf2 ( − / − ) ( 6 months old , one male and two female ) or wild - type ( 11 months old , two male and one female ) mice were separated into sarkosyl - soluble ( SS ) and insoluble ( SI ) fractions as described in the Methods . The level of total tau was analysed by immunoblotting using a monoclonal antibody to total tau ( Tau5 ) . The relative molecular masses ( kDa ) are indicated to the left of each blot . Bar graph represents the relative optical density of sarkosyl - insoluble tau normalized with that of sarkosyl - soluble tau . n = 3 . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * P0 . 05 ) .

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

Brain cortical tissue obtained from Nrf2 (−/−) (6 months old, one male and two female) or wild-type (11 months old, two male and one female) mice were separated into sarkosyl-soluble (SS) and insoluble (SI) fractions as described in the Methods. The level of total tau was analysed by immunoblotting using a monoclonal antibody to total tau (Tau5). The relative molecular masses (kDa) are indicated to the left of each blot. Bar graph represents the relative optical density of sarkosyl-insoluble tau normalized with that of sarkosyl-soluble tau. n=3. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05).


( a ) Primary cortical neurons ( DIV 5 ) were treated with SFN ( 10 μM , SFN ) for 12 or 24 h . ( c , d ) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ) .

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

(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM, SFN) for 12 or 24 h. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01).


( b ) Stably transfected CN1 . 4 cortical neurons were maintained in the presence of doxycycline ( 1 μg ml − 1 ) to induce the expression of tau for 24 h , and subsequently treated with SFN for 12 or 24 h . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies , respectively . Tau was detected with a polyclonal phospho - independent tau antibody ( Tau ) . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( c , d ) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ) .

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

(b) Stably transfected CN1.4 cortical neurons were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN for 12 or 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau was detected with a polyclonal phospho-independent tau antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01).


Mouse brains were obtained from wild - type ( 11 months old , one male ; 5 months old , one female ; 12 months old , two female ) and Nrf2 ( − / − ) ( 10 months old , two male and two female ) mice . ( a ) Mouse brain tissues were homogenized in the lysis buffer , and 10 μg of lysates was incubated with 2 μg of GST - tau protein at 37 ° C for 30 min in the presence or absence of 1 mM ATP . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies , respectively . GST - tau was detected with a monoclonal GST antibody ( GST - tau ; Supplementary Fig . 4 ) . Bar graph of the relative optical density of phosphorylated tau normalized to actin . Data shown are mean ± s . e . of duplicated independent experiments and were analysed using Student ' s t - test .

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

Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (a)Mouse brain tissues were homogenized in the lysis buffer, and 10 μg of lysates was incubated with 2 μg of GST-tau protein at 37°C for 30 min in the presence or absence of 1 mM ATP. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies, respectively. GST-tau was detected with a monoclonal GST antibody (GST-tau; Supplementary Fig. 4). Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of duplicated independent experiments and were analysed using Student's t-test.


Mouse brains were obtained from wild - type ( 11 months old , one male ; 5 months old , one female ; 12 months old , two female ) and Nrf2 ( − / − ) ( 10 months old , two male and two female ) mice . ( b ) Mouse brain tissues were homogenized in the phosphatase storage buffer , and phosphatase activity in the lysates was quantified using the serine / threonine phosphatase assay system ( Promega ) by measuring the dephosphorylation of a phosphopeptide , RRA ( pT ) VA in the presence or absence of okadaic acid ( OA , 20 nM ) . PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture . n = 4 . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (b) Mouse brain tissues were homogenized in the phosphatase storage buffer, and phosphatase activity in the lysates was quantified using the serine/threonine phosphatase assay system (Promega) by measuring the dephosphorylation of a phosphopeptide, RRA(pT)VA in the presence or absence of okadaic acid (OA, 20 nM). PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture. n=4. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test.


CN1 . 4mousecortical cells stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . On the next day ( a ) , the cells were washed twice with PBS and the media were replaced with Dox - deficient media with either vehicle , epoxomicin ( 12 . 5 nM , Epox ) , MG132 ( 7 μM ) or 3 - methyladenine ( 10 mM , 3 - MA ) for 24 h . Samples in first lane were from cells maintained in the presence of Dox without medium exchange .

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

CN1.4mousecortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. On the next day (a), the cells were washed twice with PBS and the media were replaced with Dox-deficient media with either vehicle, epoxomicin (12.5 nM, Epox), MG132 (7 μM) or 3-methyladenine (10 mM, 3-MA) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.


CN1 . 4 mouse cortical cells stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . ( b ) Cells were treated with either chloroquine ( 50 μM , CQ ) or bafilomycin A1 ( 10 nM , Baf A1 ) for 24 h . Samples in first lane were from cells maintained in the presence of Dox without medium exchange .

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

CN1.4 mouse cortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. (b) Cells were treated with either chloroquine (50 μM, CQ) or bafilomycin A1 (10 nM, Baf A1) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.


( c ) HEK293 - TN cells were co - transfected with the plasmid expressing tau together either with siRNA of beclin - 1 or scramble RNA as a control , which was kept for 48 h .

O O O O O O O O O O O O O O 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) HEK293-TN cells were co-transfected with the plasmid expressing tau together either with siRNA of beclin-1 or scramble RNA as a control, which was kept for 48 h.


( d ) Stably transfected CN1 . 4 cortical cells were maintained in the presence of Dox ( 1 μg ml − 1 ) to induce the expression of tau for 24 h , and subsequently treated with SFN ( 10 μM ) for 12 h . The culture medium was exchanged for fresh media containing Dox ( 1 μg ml − 1 ) but no SFN after washing with PBS . The cells were then incubated for an additional 18 h in the absence or presence of CQ ( 50 μM ) . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies , respectively . Tau , beclin - 1 and LC3 were detected with the appropriate polyclonal antibodies . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( e ) Bar graphs represent the relative optical density of phosphorylated tau , tau or LC3 - II normalized with that of actin . n = 4 . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ) .

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

(d) Stably transfected CN1.4 cortical cells were maintained in the presence of Dox (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN (10 μM) for 12 h. The culture medium was exchanged for fresh media containing Dox (1 μg ml−1) but no SFN after washing with PBS. The cells were then incubated for an additional 18 h in the absence or presence of CQ (50 μM). The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau, beclin-1 and LC3 were detected with the appropriate polyclonal antibodies. The relative molecular masses (kDa) are indicated to the left of each blot. (e) Bar graphs represent the relative optical density of phosphorylated tau, tau or LC3-II normalized with that of actin. n=4. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01).


( a ) Primary cortical neurons ( DIV 5 ) were treated with SFN ( 10 μM ) for 12 h , and the relative mRNA level of each gene was compared with that of cells not treated with SFN using qRT - PCR .

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 B-CONTROLLED_VAR O O O O O

(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM) for 12 h, and the relative mRNA level of each gene was compared with that of cells not treated with SFN using qRT-PCR.


( b ) In hippocampal tissues from Nrf2 ( − / − ) ( 5 months old , one male ; 11 months old , one female ; 5 months old , one female ; 12 months old , three female ) or wild - type ( 10 months old , four male and two female ) mice , the relative mRNA level of each gene compared with that of wild - type mice . n = 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In hippocampal tissues from Nrf2 (−/−) (5 months old, one male; 11 months old, one female; 5 months old, one female; 12 months old, three female) or wild-type (10 months old, four male and two female) mice, the relative mRNA level of each gene compared with that of wild-type mice. n=6.


( c ) The levels of p62 / SQSTM1 , Keap1 , mouse NDP52 ( mNDP52 ) and Hsp70 in Nrf2 ( − / − ) or wild - type mice were examined by immunoblotting . The asterisk on the Nrf2 panel indicates a nonspecific band .

O O O O O O O O O O O O O O O O O O O O O O O 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) The levels of p62/SQSTM1, Keap1, mouse NDP52 (mNDP52) and Hsp70 in Nrf2 (−/−) or wild-type mice were examined by immunoblotting. The asterisk on the Nrf2 panel indicates a nonspecific band.


( d , e : upper panel ) Primary cortical neurons ( DIV 5 ) ( d ) and SH - SY5Y cells ( e ) were treated with SFN for 24 h , and the expression levels of rat and human NDP52 ( rNDP52 and hNDP52 ) examined by immunoblotting using anti - NDP52 antibody . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( d , e : low panel ) Primary cortical neurons ( DIV 5 ) ( d ) and SH - SY5Y cells ( e ) transiently transfected with the pGL4 . 14 / human NDP52 promoter ( 2 , 173 bp ) luciferase reporter plasmid were treated with SFN for 24 h , and assayed for the luciferase activity .

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

(d,e: upper panel) Primary cortical neurons (DIV 5) (d) and SH-SY5Y cells (e) were treated with SFN for 24 h, and the expression levels of rat and human NDP52 (rNDP52 and hNDP52) examined by immunoblotting using anti-NDP52 antibody. The relative molecular masses (kDa) are indicated to the left of each blot. (d,e: low panel) Primary cortical neurons (DIV 5) (d) and SH-SY5Y cells (e) transiently transfected with the pGL4.14/human NDP52 promoter (2,173 bp) luciferase reporter plasmid were treated with SFN for 24 h, and assayed for the luciferase activity.


( f ) SH - SY5Y cells were treated with SFN for 12 h , and cell lysates were used for the ChIP assay using anti - Nrf2 antibody as described in the Methods . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ) .

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

(f) SH-SY5Y cells were treated with SFN for 12 h, and cell lysates were used for the ChIP assay using anti-Nrf2 antibody as described in the Methods. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01; ***P0.001).


( b ) CN1 . 4 cortical cells and SH - SY5Y cells were transiently transfected with the reporter plasmids with the human NDP52 promoter ( 2 , 317 bp or deleted mutants ) , and luciferase activity was assayed 24 h after transfection .

(b) CN1.4 cortical cells and SH-SY5Y cells were transiently transfected with the reporter plasmids with the human NDP52 promoter (2,317 bp or deleted mutants), and luciferase activity was assayed 24 h after transfection.


( c ) SH - SY5Y cells were co - transfected with the reporter plasmids with human NDP52 promoter ( wild or deleted mutants ) along with either the mock plasmid ( pcDNA3 . 1 ) or the plasmid expressing Nrf2 , and the luciferase activity was assayed 24 h after transfection . ( d ) SH - SY5Y cells were transiently co - transfected with the reporter plasmids with human NDP52 promoter or its mutant plasmid in which the putative ARE sequence was site - directly mutated ( X ) , together with the mock plasmid ( pcDNA3 . 1 ) or the plasmid expressing human Nrf2 . The luciferase activity was assayed after 24 h . The induced luciferase activity with co - expression with Nrf2 was not observed in a mutant NDP52 promoter in which all three ARE sites ( − 2 , 057 , − 1 , 023 and − 94 ) were site - directly mutated , indicating these three sites of five ARE cis - acting elements are functional . Data shown are mean ± s . d . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SH-SY5Y cells were co-transfected with the reporter plasmids with human NDP52 promoter (wild or deleted mutants) along with either the mock plasmid (pcDNA3.1) or the plasmid expressing Nrf2, and the luciferase activity was assayed 24 h after transfection. (d) SH-SY5Y cells were transiently co-transfected with the reporter plasmids with human NDP52 promoter or its mutant plasmid in which the putative ARE sequence was site-directly mutated (X), together with the mock plasmid (pcDNA3.1) or the plasmid expressing human Nrf2. The luciferase activity was assayed after 24 h. The induced luciferase activity with co-expression with Nrf2 was not observed in a mutant NDP52 promoter in which all three ARE sites (−2,057, −1,023 and −94) were site-directly mutated, indicating these three sites of five ARE cis-acting elements are functional. Data shown are mean±s.d. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01; ***P0.001).


( a ) Primary cortical neurons were transduced with lentivirus expressing sh - RNA for Nrf2 or rat NDP52 ( rNDP52 ) , or a scrambled sh - RNA at DIV 1 , and were maintained until DIV 6 . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using 12E8 - and PHF1 - specific antibodies , respectively . Total tau was detected with a polyclonal tau - specific antibody ( Tau ) . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( b ) Bar graph of the relative optical density of phosphorylated tau normalized to actin . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * * P0 . 001 )

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

(a) Primary cortical neurons were transduced with lentivirus expressing sh-RNA for Nrf2 or rat NDP52 (rNDP52), or a scrambled sh-RNA at DIV 1, and were maintained until DIV 6. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8- and PHF1-specific antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot. (b) Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; ***P0.001)