Figure 1E . As in ( D ) , except that cells were treated with Cisplatin for 24 h ( mean ± SEM ; n = 3 independent experiments ) . F . U2OS WT , U2OS / SCAI KO and U2OS / SCAI KO / Strep - HA - SCAI cells were treated or not with MMC ( 9 nM ) for 48 h , fixed and co - stained with PCNA antibody and DAPI . Cell cycle distribution was analyzed by quantitative image - based cytometry ( QIBC ) ( ≥ 2000 cells analyzed per condition ) . Data from a representative experiment are shown . G . U2OS WT , U2OS / SCAI KO and U2OS / SCAI KO / Strep - HA - SCAI cells were treated or not with MMC ( 90 nM ) for 1 h , fixed 24 h later and co - stained with RPA2 antibody and DAPI . RPA2 foci were quantified by QIBC ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; ns , not significant , two - tailed paired t - test ) . H . As in ( G ) , except that cells were co - stained with γH2AX antibody and DAPI ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant , two - tailed paired t - test ) . I . Experimental workflow for metaphase chromosome morphology analysis ( top ) and representative images of metaphase spreads from indicated cell lines treated or not with MMC ( bottom ) . DNA was stained with DAPI . Scale bars , 10 µm . J . Quantification of radial chromosomes in ( I ) ( mean ± SD ; 180 cells analyzed per condition ; n = 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 , ns , not significant , two - tailed t - test ) . K . Quantification of chromosomal breaks / gaps in ( I ) ( mean ± SEM ; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 , ns , not significant , Mann - Whitney U test ) .

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Figure 1E. As in (D), except that cells were treated with Cisplatin for 24 h (mean±SEM; n=3 independent experiments). F. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (9 nM) for 48 h, fixed and co-stained with PCNA antibody and DAPI. Cell cycle distribution was analyzed by quantitative image-based cytometry (QIBC) (≥2000 cells analyzed per condition). Data from a representative experiment are shown.G. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). H. As in (G), except that cells were co-stained with γH2AX antibody and DAPI (≥3000 cells analyzed per condition; mean± SD; n=3 independent experiments; *, p<0.05; **, p<0.01; ns, not significant, two-tailed paired t-test).I. Experimental workflow for metaphase chromosome morphology analysis (top) and representative images of metaphase spreads from indicated cell lines treated or not with MMC (bottom). DNA was stained with DAPI. Scale bars, 10 µm. J. Quantification of radial chromosomes in (I) (mean±SD; 180 cells analyzed per condition; n=3 independent experiments; *, p<0.05; **, p<0.01, ns, not significant, two-tailed t-test). K. Quantification of chromosomal breaks/gaps in (I) (mean±SEM; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments; **, p<0.01; ****, p<0.0001, ns, not significant, Mann-Whitney U test).


Figure 2F . U2OS and U2OS / SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC ( 90 nM ) for 1 h , fixed 24 h later and co - stained with RPA2 antibody and DAPI . RPA2 foci were quantified by QIBC ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; ns , not significant , two - tailed paired t - test ) . G . As in ( F ) , except that cells were stained with γH2AX antibody ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * * , p < 0 . 01 ; ns , not significant , two - tailed paired t - test ) . I . Clonogenic survival of U2OS and U2OS / FANCA KO cells subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) . J . Clonogenic survival of U2OS , U2OS / SCAI KO , U2OS / FANCA KO , and U2OS / FANCA + SCAI DKO cells subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) . K . Immunoblot analysis of U2OS , U2OS / SCAI KO , U2OS / FANCA KO , and U2OS / FANCA SCAI DKO cell lines exposed or not to MMC as indicated .

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Figure 2F. U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). G. As in (F), except that cells were stained with γH2AX antibody (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; **, p<0.01; ns, not significant, two-tailed paired t-test).I. Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). J. Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI DKO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).K. Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.


Figure 3B . pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i ( 100 µM ) and associated proteins were analyzed by immunoblotting . F . pICLpt was replicated in mock - or SCAI - depleted egg extract in the presence of [ α - 32P ] dATP for the indicated times , and reactions were analyzed by native agarose gel electrophoresis . ∆ SCAI - C and ∆ SCAI - N denote SCAI immunodepletion with an antibody raised against the C - or N - terminus of SCAI , respectively ; RI , replication intermediates ; OC , open circular ; SC , supercoiled .

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Figure 3B. pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i (100 µM) and associated proteins were analyzed by immunoblotting.F. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. ∆SCAI-C and ∆SCAI-N denote SCAI immunodepletion with an antibody raised against the C- or N-terminus of SCAI, respectively; RI, replication intermediates; OC, open circular; SC, supercoiled.


Figure 4A . Analysis of SCAI - interacting proteins in Xenopus egg extracts . Volcano plot shows enrichment of proteins in both SCAI - C and SCAI - N immunoprecipitates ( IPs ) compared to an IgG control , plotted against the P - value ( n = 4 independent experiments ; permutation - based FDR < 0 . 01 , s0 = 2 ) . D . GFP IPs from U2OS or U2OS / GFP - SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies . Note that a small fraction of REV7 is still immunoprecipitated with GFP - SCAI in REV3 knockdown cells . This could be due to incomplete knockdown , and / or additional interactions of SCAI with the REV1 - Polζ complex that are independent of REV3 . J . pICLpt was replicated in egg extracts immunodepleted with indicated antibodies , isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting .

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Figure 4A. Analysis of SCAI-interacting proteins in Xenopus egg extracts. Volcano plot shows enrichment of proteins in both SCAI-C and SCAI-N immunoprecipitates (IPs) compared to an IgG control, plotted against the P-value (n=4 independent experiments; permutation-based FDR<0.01, s0=2).D. GFP IPs from U2OS or U2OS/GFP-SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies. Note that a small fraction of REV7 is still immunoprecipitated with GFP-SCAI in REV3 knockdown cells. This could be due to incomplete knockdown, and/or additional interactions of SCAI with the REV1-Polζ complex that are independent of REV3.J. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies, isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting.


Figure 5B . Immunoblot analysis of extracts immunodepleted with indicated antibodies . Asterisks denote antibody cross - reactivity . E . Representative images of metaphase spreads from U2OS / SCAI KO cells transfected with indicated siRNAs and processed as in Figure 1I . Scale bars , 10 µm . H . Clonogenic survival of U2OS and U2OS / SCAI KO cells transfected with non - targeting control ( CTRL ) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) .

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Figure 5B. Immunoblot analysis of extracts immunodepleted with indicated antibodies. Asterisks denote antibody cross-reactivity.E. Representative images of metaphase spreads from U2OS/SCAI KO cells transfected with indicated siRNAs and processed as in Figure 1I. Scale bars, 10 µm.H. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non-targeting control (CTRL) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).


Figure 1To verify the specificity of Cx43 - CreERT mice for astrocytes , mice were crossed to a tdTomato reporter line to induce Cre - dependent tdTomato expression after tamoxifen administration . TdTomato fluorescence was enhanced with an antibody against red fluorescent protein , and astrocytes were stained with antibodies against GFAP or S100β ( not shown ) . Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes ( arrowheads ) in 8 - month and 11 - month old animals . Expression was small in neurons ( stained with NeuN , not shown ) and negligible in NG2 cells and oligodendrocytes . Scale bars , 250 µm ( upper panel ) and 50 µm ( lower panel ) . The majority of peri - plaque astrocytes in APP / PS1 - Stat3WT were positive for activated / phosphorylated Stat3 ( pStat3 ) in the cortex ( Cx ) and hippocampus ( HC ) , whereas the fraction of pStat3 - positive astrocytes was significantly lower in APP / PS1 - Stat3KO mice in both regions ( n = 8 mice ( 3 females and 5 males ) per group ; age , 8 months ; Mann - Whitney test ) . The occurrence of pStat3 in WT - Stat3KO and WT - Stat3WT mice was negligible ( n = 8 mice ( 4 females and 4 males ) per group ; age , 8 months ; Mann - Whitney test ) . Examples of Stat3 activation in APP / PS1 - Stat3KO and APP / PS1 - Stat3WT . D , pStat3 ( arrowheads ) was abundantly present in reactive astrocytes ( identified by GFAP ) around plaques ( identified by IC16 ) . E , only few astrocytes were positive for pStat3 in APP / PS1 - Stat3WT mice . Scale bars , 50 µm . in postmortem brain sections from AD patients , we detected nuclear pStat3 ( arrows ) in the majority of peri - plaque reactive astrocytes ( identified by GFAP ) in the cortex and hippocampus ( plaques were stained with methoxy - XO4 , arrowheads ) . Scale bar , 50 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1To verify the specificity of Cx43-CreERT mice for astrocytes, mice were crossed to a tdTomato reporter line to induce Cre-dependent tdTomato expression after tamoxifen administration. TdTomato fluorescence was enhanced with an antibody against red fluorescent protein, and astrocytes were stained with antibodies against GFAP or S100β (not shown). Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes (arrowheads) in 8-month and 11-month old animals. Expression was small in neurons (stained with NeuN, not shown) and negligible in NG2 cells and oligodendrocytes. Scale bars, 250 µm (upper panel) and 50 µm (lower panel).The majority of peri-plaque astrocytes in APP/PS1-Stat3WT were positive for activated/phosphorylated Stat3 (pStat3) in the cortex (Cx) and hippocampus (HC), whereas the fraction of pStat3-positive astrocytes was significantly lower in APP/PS1-Stat3KO mice in both regions (n = 8 mice (3 females and 5 males) per group; age, 8 months; Mann-Whitney test). The occurrence of pStat3 in WT-Stat3KO and WT-Stat3WT mice was negligible (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test).Examples of Stat3 activation in APP/PS1-Stat3KO and APP/PS1-Stat3WT. D, pStat3 (arrowheads) was abundantly present in reactive astrocytes (identified by GFAP) around plaques (identified by IC16). E, only few astrocytes were positive for pStat3 in APP/PS1-Stat3WT mice. Scale bars, 50 µm.in postmortem brain sections from AD patients, we detected nuclear pStat3 (arrows) in the majority of peri-plaque reactive astrocytes (identified by GFAP) in the cortex and hippocampus (plaques were stained with methoxy-XO4, arrowheads). Scale bar, 50 µm.


Figure 2Overall astrocytic and microglial coverage , as assessed by GFAP and Iba1 stainings , remained unchanged in APP / PS1 - Stat3WT vs . APP / PS1 - Stat3KO mice ( n = 8 mice ( 4 females and 4 males ) per group ; age , 8 months ; Mann - Whitney test for each comparison ) . Scale bars , 500 µm . The volume of peri - plaque reactive astrocytes was increased by the Stat3 deletion ( Mann - Whitney test ) , and there was a nonsignificant trend towards more astrocytes branches and junctions and longer total process length ( Mann - Whitney test ; APP / PS1 - Stat3WT , n = 5 ( 2 female and 3 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 3 female and 4 male ) mice ; age , 8 - 10 months ) . Scale bar , 20 µm . Microglial volume was not affected by the Stat3 deletion ( Mann - Whitney test ) , but peri - plaque microglia had significantly more microglial branches and junctions per plaque , and the total process length of peri - plaque microglia was increased ( Mann - Whitney test ; same mice as in C - D ) . Scale bar , 20 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Overall astrocytic and microglial coverage, as assessed by GFAP and Iba1 stainings, remained unchanged in APP/PS1-Stat3WT vs. APP/PS1-Stat3KO mice (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test for each comparison). Scale bars, 500 µm.The volume of peri-plaque reactive astrocytes was increased by the Stat3 deletion (Mann-Whitney test), and there was a nonsignificant trend towards more astrocytes branches and junctions and longer total process length (Mann-Whitney test; APP/PS1-Stat3WT, n = 5 (2 female and 3 male) mice; APP/PS1-Stat3KO, n = 7 (3 female and 4 male) mice; age, 8-10 months). Scale bar, 20 µm.Microglial volume was not affected by the Stat3 deletion (Mann-Whitney test), but peri-plaque microglia had significantly more microglial branches and junctions per plaque, and the total process length of peri-plaque microglia was increased (Mann-Whitney test; same mice as in C-D). Scale bar, 20 µm.


Figure 3Aβ plaque burden , as assessed by plaque load and size using an anti - Aβ antibody , was strongly reduced in APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ( Mann - Whitney test ; scale bar , 300 µm ) . Plaque density , assessed by staining brain sections with thioflavin ( yellow ) , remained unchanged in APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ( Mann - Whitney test ; scale bar , 250 µm ) . Electrochemiluminescence ELISA after sequential extraction from whole - brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1 - 42 and Aβ1 - 40 were significantly reduced ( Mann - Whitney test ) , whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1 - 42 and Aβ1 - 40 ( Mann - Whitney test ) . Western Blot quantification of full - length amyloid precursor protein ( APP ) and its C - terminal fragments ( CTF ) showed no differences between both groups ( Mann - Whitney test ; for all datasets , n = 9 male mice for both groups ( age , 11 months ) ) .

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Figure 3Aβ plaque burden, as assessed by plaque load and size using an anti-Aβ antibody, was strongly reduced in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 300 µm).Plaque density, assessed by staining brain sections with thioflavin (yellow), remained unchanged in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 250 µm).Electrochemiluminescence ELISA after sequential extraction from whole-brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1-42 and Aβ1-40 were significantly reduced (Mann-Whitney test), whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1-42 and Aβ1-40 (Mann-Whitney test).Western Blot quantification of full-length amyloid precursor protein (APP) and its C-terminal fragments (CTF) showed no differences between both groups (Mann-Whitney test; for all datasets, n = 9 male mice for both groups (age, 11 months)).


Figure 4Internalization of Aβ ( stained with IC16 antibody or methoxy - XO4 ) was assessed using an engulfment assay , in which glial and Aβ structures were surface - rendered and Aβ volumes co - localized with glial volumes were quantified . Scale bars , 10 µm . Microglia ( left Y axes ) from APP / PS1 mice internalized significantly more Aβ positive for IC16 when Stat3 was deleted in astrocytes ( Mann - Whitney test ) , whereas no changes were seen in astrocytes ( right axes ; APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 11 ( 5 female and 6 male ) mice ; age , 11 months ; Mann - Whitney test ) . Microglia ( left Y axes ) from APP / PS1 mice internalized significantly more Aβ positive for methoxy - XO4 when Stat3 was deleted in astrocytes ( Mann - Whitney test ) , whereas no changes were seen in astrocytes ( right axes ; APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 11 ( 5 female and 6 male ) mice ; age , 11 months ; Mann - Whitney test ) . Western Blot quantification of protein levels of the Aβ - degrading enzymes neprilysin / CD10 and CD36 , as well as the Aβ - binding apoliprotein E ( apoE ) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE ( APP / PS1 - Stat3WT , n = 9 ( 5 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 9 ( 5 female and 4 male ) mice ; age , 11 months ; Mann - Whitney test for all comparisons ) . In contrast , TREM2 expression remained unchanged ( APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 4 female and 3 male ) mice ; age , 11 months ; Mann - Whitney test ) . Western Blot of protein levels of the Aβ - degrading enzymes neprilysin / CD10 and CD36 , as well as the Aβ - binding apoliprotein E ( apoE ) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE ( APP / PS1 - Stat3WT , n = 9 ( 5 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 9 ( 5 female and 4 male ) mice ; age , 11 months ; Mann - Whitney test for all comparisons ) . In contrast , TREM2 expression remained unchanged ( APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 4 female and 3 male ) mice ; age , 11 months ; Mann - Whitney test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Internalization of Aβ (stained with IC16 antibody or methoxy-XO4) was assessed using an engulfment assay, in which glial and Aβ structures were surface-rendered and Aβ volumes co-localized with glial volumes were quantified. Scale bars, 10 µm.Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for IC16 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for methoxy-XO4 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).Western Blot quantification of protein levels of the Aβ-degrading enzymes neprilysin/CD10 and CD36, as well as the Aβ-binding apoliprotein E (apoE) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE (APP/PS1-Stat3WT, n = 9 (5 female and 4 male) mice; APP/PS1-Stat3KO, n = 9 (5 female and 4 male) mice; age, 11 months; Mann-Whitney test for all comparisons). In contrast, TREM2 expression remained unchanged (APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 7 (4 female and 3 male) mice; age, 11 months; Mann-Whitney test).Western Blot of protein levels of the Aβ-degrading enzymes neprilysin/CD10 and CD36, as well as the Aβ-binding apoliprotein E (apoE) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE (APP/PS1-Stat3WT, n = 9 (5 female and 4 male) mice; APP/PS1-Stat3KO, n = 9 (5 female and 4 male) mice; age, 11 months; Mann-Whitney test for all comparisons). In contrast, TREM2 expression remained unchanged (APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 7 (4 female and 3 male) mice; age, 11 months; Mann-Whitney test).


Figure 5Quantitative PCR from cortex of APP / PS1 - Stat3KO compared to APP / PS1 - Stat3WT mice revealed lower expression of ' A1 ' markers Amigo2 and C3 , whereas Ggta1 remained unchanged . In turn , the ' A2 ' marker Tm4sf1 was upregulated and there was a nonsignificant trend for a higher expression of B3gnt5 ( n = 6 mice ( 3 females and 3 males ) per group ; age , 8 months ; Mann - Whitney test ) . Confirming lower expression of the ' A1 ' marker C3d , western blot analysis indicated lower protein levels of C3d in APP / PS1 - Stat3KO . APP / PS1 - Stat3WT , n = 6 ( 2 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 3 female and 3 male ) mice ; age , 8 months ; Mann - Whitney test ) . Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri - plaque reactive astrocytes ( arrows ; plaques were visualized with methoxy - XO4 , arrowheads ; scale bars , 50 µm ; APP / PS1 - Stat3WT , n = 6 ( 2 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 3 female and 3 male ) mice ; age , 8 months ; Mann - Whitney test ) . Whole - brain levels of the pro - inflammatory cytokines IL - 1β and TNF - α were significantly reduced in APP / PS1 - Stat3KO mice ( Mann - Whitney test ) , whereas no changes were seen for IL - 10 ( APP / PS1 - Stat3WT , n = 13 ( 6 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 13 ( 8 female and 5 male ) mice ; age , 11 months ; Mann - Whitney test ) . These changes were paralleled by a decrease in the area covered by dystrophic neurites in APP / PS1 - Stat3KO compared to APP / PS1 - Stat3WT mice , as assessed by LAMP1 staining ( Mann - Whitney test ; n = 10 male mice for both groups ; age , 8 months ; scale bars , 300 µm ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Quantitative PCR from cortex of APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice revealed lower expression of 'A1' markers Amigo2 and C3, whereas Ggta1 remained unchanged. In turn, the 'A2' marker Tm4sf1 was upregulated and there was a nonsignificant trend for a higher expression of B3gnt5 (n = 6 mice (3 females and 3 males) per group; age, 8 months; Mann-Whitney test).Confirming lower expression of the 'A1' marker C3d, western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri-plaque reactive astrocytes (arrows; plaques were visualized with methoxy-XO4, arrowheads; scale bars, 50 µm; APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).Whole-brain levels of the pro-inflammatory cytokines IL-1β and TNF-α were significantly reduced in APP/PS1-Stat3KO mice (Mann-Whitney test), whereas no changes were seen for IL-10 (APP/PS1-Stat3WT, n = 13 (6 female and 7 male) mice; APP/PS1-Stat3KO, n = 13 (8 female and 5 male) mice; age, 11 months; Mann-Whitney test).These changes were paralleled by a decrease in the area covered by dystrophic neurites in APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice, as assessed by LAMP1 staining (Mann-Whitney test; n = 10 male mice for both groups; age, 8 months; scale bars, 300 µm).


Figure 6For in vivo two - photon imaging , astrocytes ( arrows ) and neurons ( arrowheads ) were labeled with the calcium indicator OGB - 1 , and astrocytes were co - labeled with sulforhodamine 101 ( SR101 ; arrows ) . Aβ plaques were labeled with the intravital dye methoxy - XO4 ( open arrowheads ) . Scale bar , 50 µm . Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP / PS1 - Stat3KO mice was reduced to levels comparable to WT - Stat3WT mice , but significantly increased in APP / PS1 - Stat3WT mice ( One - Way ANOVA followed by Bonferroni ' s multiple comparison test ; n = 6 mice ( 3 female and 3 male ) for each group ; age , 8 months ) . Similarly , neuronal activity was also reduced to levels comparable to WT - Stat3WT mice in APP / PS1 - Stat3KO mice , but significantly increased in APP / PS1 - Stat3WT mice ( One - Way ANOVA followed by Bonferroni ' s multiple comparison test ; same mice as in B ) . The cumulative distributions of neuronal calcium transients in APP / PS1 - Stat3KO mice were not different from those of WT - Stat3WT mice ( p = 0 . 31 , Kolmogorov - Smirnov test ) , but significantly different from those of APP / PS1 - Stat3WT mice ( p = 0 . 001 , Kolmogorov - Smirnov test ) . Two - photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) . Two - photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) . MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) . This network normalization induced a reduction of activated Stat3 ( pStat3 ) in astrocytes assessed in fixed brain sections of the same mice ( Mann - Whitney test ) . Scale bars , 50 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6For in vivo two-photon imaging, astrocytes (arrows) and neurons (arrowheads) were labeled with the calcium indicator OGB-1, and astrocytes were co-labeled with sulforhodamine 101 (SR101; arrows). Aβ plaques were labeled with the intravital dye methoxy-XO4 (open arrowheads). Scale bar, 50 µm.Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP/PS1-Stat3KO mice was reduced to levels comparable to WT-Stat3WT mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; n = 6 mice (3 female and 3 male) for each group; age, 8 months).Similarly, neuronal activity was also reduced to levels comparable to WT-Stat3WT mice in APP/PS1-Stat3KO mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; same mice as in B). The cumulative distributions of neuronal calcium transients in APP/PS1-Stat3KO mice were not different from those of WT-Stat3WT mice (p = 0.31, Kolmogorov-Smirnov test), but significantly different from those of APP/PS1-Stat3WT mice (p = 0.001, Kolmogorov-Smirnov test).Two-photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).Two-photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). This network normalization induced a reduction of activated Stat3 (pStat3) in astrocytes assessed in fixed brain sections of the same mice (Mann-Whitney test). Scale bars, 50 µm.


Figure 7Spatial learning and memory were assessed in the Morris Water Maze paradigm . APP / PS1 - Stat3KO mice showed faster latencies to reach the hidden platform compared with APP / PS1 - Stat3WT on days 4 and 5 , but were similar to WT - Stat3WT and WT - Stat3KO mice ( Two - way repeated - measures ANOVA followed by Bonferroni post - hoc test ; P values are for APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ) . The area under the curve ( AUC ) for the latency to reach the hidden platform was similar in APP / PS1 - Stat3WT compared to WT - Stat3WT and WT - Stat3KO mice , but significantly higher in APP / PS1 - Stat3KO mice ( Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ) . The swimming velocity was similar in all groups ( Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ) . In the probe trial 24 h after the last training day , the time mice spent in the target quadrant ( TQ ) was different from chance in all groups except for APP / PS1 - Stat3WT mice ( p < 0 . 05 , one - tailed one - sample t test ) . APP / PS1 - Stat3KO , WT - Stat3WT and WT - Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants ( AO ) , whereas APP / PS1 - Stat3WT spent equal times in the target and all other quadrants ( Wilcoxon matched - pairs signed rank test for each comparison ; APP / PS1 - Stat3WT , n = 15 ( 8 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 15 ( 9 female and 6 male ) mice ; age , 8 - 9 months ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Spatial learning and memory were assessed in the Morris Water Maze paradigm. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on days 4 and 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test; P values are for APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice).The area under the curve (AUC) for the latency to reach the hidden platform was similar in APP/PS1-Stat3WT compared to WT-Stat3WT and WT-Stat3KO mice, but significantly higher in APP/PS1-Stat3KO mice (Kruskal-Wallis test followed by Dunn's multiple comparisons test).The swimming velocity was similar in all groups (Kruskal-Wallis test followed by Dunn's multiple comparisons test).In the probe trial 24 h after the last training day, the time mice spent in the target quadrant (TQ) was different from chance in all groups except for APP/PS1-Stat3WT mice (p < 0.05, one-tailed one-sample t test). APP/PS1-Stat3KO, WT-Stat3WT and WT-Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants (AO), whereas APP/PS1-Stat3WT spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for each comparison; APP/PS1-Stat3WT, n = 15 (8 female and 7 male) mice; APP/PS1-Stat3KO, n = 15 (9 female and 6 male) mice; age, 8-9 months).


Figure 8APP / PS1 mice were systemically treated with SH - 4 - 54 for 6 weeks and compared to age - matched APP / PS1 mice treated with vehicle . In the Morris Water Maze test , treatment with SH - 4 - 54 led to significantly faster latencies to reach the hidden platform on the last training day ( Two - way repeated - measures ANOVA followed by Bonferroni post - hoc test ) . The area under the curve ( AUC ) for the latency to reach the hidden platform was also smaller in APP / PS1 mice treated with the Stat3 inhibitor ( Mann - Whitney test ) . The swimming velocity was not affected by the treatment ( Mann - Whitney test ) . In the probe trial test , APP / PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant ( TQ ) compared to the mean of all other quadrants ( AO ) , whereas APP / PS1 mice treated with vehicle spent equal times in the target and all other quadrants ( Wilcoxon matched - pairs signed rank test for all comparisons ; APP / PS1 - Stat3WT , n = 12 ( 5 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 12 ( 4 female and 8 male ) mice ; age , 8 months ) . ( E ) To verify target engagement in the brain , the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two - photon microscopy of calcium activity . Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons ( Mann - Whitney test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8APP/PS1 mice were systemically treated with SH-4-54 for 6 weeks and compared to age-matched APP/PS1 mice treated with vehicle. In the Morris Water Maze test, treatment with SH-4-54 led to significantly faster latencies to reach the hidden platform on the last training day (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test).The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (Mann-Whitney test).The swimming velocity was not affected by the treatment (Mann-Whitney test).In the probe trial test, APP/PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant (TQ) compared to the mean of all other quadrants (AO), whereas APP/PS1 mice treated with vehicle spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for all comparisons; APP/PS1-Stat3WT, n = 12 (5 female and 7 male) mice; APP/PS1-Stat3KO, n = 12 (4 female and 8 male) mice; age, 8 months).(E) To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (Mann-Whitney test).


Figure 9SH - 4 - 54 significantly decreased plaque growth , as assessed by IC16 immunohistochemistry , while plaque load remained unchanged . SH - 4 - 54 significantly There was also no significant change in dystrophic neurite area during the treatment time ( Mann - Whitney test for all comparisons ; scale bars , 500 µm ) . The fraction of pStat3 - positive astrocytes in the peri - plaque region was strongly reduced by the treatment with the Stat3 inhibitor ( arrowheads indicate pStat3 signals ; scale bars , 100 µm ; Mann - Whitney test ) . The fraction of pStat3 - positive astrocytes in the peri - plaque region was strongly reduced by the treatment with the Stat3 inhibitor ( arrowheads indicate pStat3 signals ; scale bars , 100 µm ; Mann - Whitney test ) . While no changes were seen in morphological parameters of peri - plaque astrocytes , there was a significant increase in the process length of near - plaque microglia , indicating higher microglial complexity ( Mann - Whitney test for all comparisons ; APP / PS1 - Stat3WT , n = 12 ( 6 female and 6 male ) mice ; APP / PS1 - Stat3KO , n = 12 ( 7 female and 5 male ) mice ; age , 8 months ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9SH-4-54 significantly decreased plaque growth, as assessed by IC16 immunohistochemistry, while plaque load remained unchanged.SH-4-54 significantly There was also no significant change in dystrophic neurite area during the treatment time (Mann-Whitney test for all comparisons; scale bars, 500 µm).The fraction of pStat3-positive astrocytes in the peri-plaque region was strongly reduced by the treatment with the Stat3 inhibitor (arrowheads indicate pStat3 signals; scale bars, 100 µm; Mann-Whitney test).The fraction of pStat3-positive astrocytes in the peri-plaque region was strongly reduced by the treatment with the Stat3 inhibitor (arrowheads indicate pStat3 signals; scale bars, 100 µm; Mann-Whitney test).While no changes were seen in morphological parameters of peri-plaque astrocytes, there was a significant increase in the process length of near-plaque microglia, indicating higher microglial complexity (Mann-Whitney test for all comparisons; APP/PS1-Stat3WT, n = 12 (6 female and 6 male) mice; APP/PS1-Stat3KO, n = 12 (7 female and 5 male) mice; age, 8 months).


Figure 1Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin . Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin . Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPO , erythropoietin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPO, erythropoietin.


Figure 3Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.


Figure 4Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting B , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting E ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290APP / PS1 - 21 mice ( 5 m ) treated with rhEPO ( 10000 IU / kg / d ) or ARA290 ( 0 . 03 mg / kg / d ) ( i . p . , once a day , for 14 days ) ( n = 10 ) , and Aβ plaques in cortex and hippocampus ( n = 5 ) were detected by IHC ( I ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290 ; IHC , Immunohistochemical staining . Proteins were extracted from spleens of 5 - or 11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , and soluble Aβ levels were measured by ELISA ( n = 5 ) ( J Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ELISA , 11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , Aβ plaques in the cortex and hippocampus of mice of 11 - month - old ( M ) ( n = 5 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining . R Aβ plaques in the cortex and hippocampus ( n = 4 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting B, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting E). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290APP/PS1-21 mice (5 m) treated with rhEPO (10000 IU/kg/d) or ARA290 (0.03 mg/kg/d) (i.p., once a day, for 14 days) (n=10), and Aβ plaques in cortex and hippocampus (n = 5) were detected by IHC (I). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290; IHC, Immunohistochemical staining.Proteins were extracted from spleens of 5- or 11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), and soluble Aβ levels were measured by ELISA (n = 5) (J Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ELISA, 11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), Aβ plaques in the cortex and hippocampus of mice of 11-month-old (M) (n = 5) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.R Aβ plaques in the cortex and hippocampus (n = 4) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.


Figure 5A , Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . B Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Both non - cognitive and cognitive behavioral impairments of APP / PS1 - 21 mice ( 11 m ) were analyzed by nest construction assay , social interaction assay , NORT , FCT ( n = 10 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . NORT , novel object recognition test ; FCT , fear conditioning test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.B Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Both non-cognitive and cognitive behavioral impairments of APP/PS1-21 mice (11 m) were analyzed by nest construction assay, social interaction assay, NORT, FCT (n = 10). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. NORT, novel object recognition test; FCT, fear conditioning test.


Figure 6Splenic macrophages from APP / PS1 - 21 ( 5 - or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Splenic macrophages from APP / PS1 - 21 11 - month - old ) or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) B ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies ( n = 3 ) ( G ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( I ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin . PMs were treated with Aβ42 ( 0 , 5 , 7 . 5 or 10 μM ) for 12 hr , and cell lysates were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( K ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . PMs were treated with Aβ42 ( 0 , 5 , 10 μM ) for 12 hr , and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( M ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Splenic macrophages from APP/PS1-21 (5- or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Splenic macrophages from APP/PS1-21 11-month-old) or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) B). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies (n = 3) (G). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (I). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.PMs were treated with Aβ42 (0, 5, 7.5 or 10 μM) for 12 hr, and cell lysates were detected by immunoblotting with the indicated antibodies (n = 3) (K). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.PMs were treated with Aβ42 (0, 5, 10 μM) for 12 hr, and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (M). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.


Figure 7E Aβ plaques in the cortex and hippocampus ( n = 5 ) were detected by IHC . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . G Expression of MAP - 2 or the apoptotic neurons of hippocampus were detected by IF staining and then statistically quantified ( n = 5 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IF , Immunofluorescent ; MAP - 2 , microtubule - associated protein 2 ; NeuN , neuronal nuclei ; TUNEL , Terminal dUTP Nick End Labeling .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7E Aβ plaques in the cortex and hippocampus (n = 5) were detected by IHC. Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.G Expression of MAP-2 or the apoptotic neurons of hippocampus were detected by IF staining and then statistically quantified (n = 5). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IF, Immunofluorescent; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclei; TUNEL, Terminal dUTP Nick End Labeling.


Figure 2A - H ) slices through electron cryotomograms of H . gracilis cells ( left panels ) , central slices through subtomogram averages ( STA , middle panels ) and schematic representations ( right panels ) representing the various assembly stages observed . White arrow in ( A ) points to the MS - complex ; green arrows point to the novel 53 - nm - wide cytoplasmic ring . Scale bars in ( H ) are 50 nm for the cryotomogram slice and 20 nm for the STA and apply to all panels . Empty panels represent stages where not enough particles were found to produce a STA . OM = outer membrane , IM = inner membrane .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-H) slices through electron cryotomograms of H. gracilis cells (left panels), central slices through subtomogram averages (STA, middle panels) and schematic representations (right panels) representing the various assembly stages observed. White arrow in (A) points to the MS-complex; green arrows point to the novel 53-nm-wide cytoplasmic ring. Scale bars in (H) are 50 nm for the cryotomogram slice and 20 nm for the STA and apply to all panels. Empty panels represent stages where not enough particles were found to produce a STA. OM=outer membrane, IM=inner membrane.


Figure 3A - D ) slices through electron cryotomograms of H . pylori ( left panels ) indicating the presence of various flagellar intermediates . Middle panels show central slices through STAs . Right panels show schematic representations . Question marks in the schematic in ( C ) indicate that it is unclear whether the densities surrounding the upper part of the C - ring are the same as the novel cytoplasmic rings seen in the MS - complex . Empty panels indicate that there were not enough examples to produce a STA . E & F ) cryotomogram slices , STAs , and schematics of H . pylori fliP * cells showing the MS - complex ( E ) and the C - complex ( F ) . The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel . White arrows in ( A - F ) point to the particles . G & H ) central slices through the STAs of the MS - complexes of the motile H . pylori ( G ) and H . pylori FliP * mutant ( H ) with the diameters of their various constituent rings indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-D) slices through electron cryotomograms of H. pylori (left panels) indicating the presence of various flagellar intermediates. Middle panels show central slices through STAs. Right panels show schematic representations. Question marks in the schematic in (C) indicate that it is unclear whether the densities surrounding the upper part of the C-ring are the same as the novel cytoplasmic rings seen in the MS-complex. Empty panels indicate that there were not enough examples to produce a STA.E & F) cryotomogram slices, STAs, and schematics of H. pylori fliP* cells showing the MS-complex (E) and the C-complex (F). The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel. White arrows in (A-F) point to the particles.G & H) central slices through the STAs of the MS-complexes of the motile H. pylori (G)and H. pylori FliP* mutant (H) with the diameters of their various constituent rings indicated.


Figure 5A - G ) Slices through electron cryotomograms ( left panels ) , central slices of subtomogram averages ( middle panels ) , and schematic representations ( right panels ) of the MS - and C - complexes ( white arrows on the slices ) of different mutants in H . pylori fliP * . Light and dark green arrows in ( A and D ) point to the ~ 53 - nm and ~ 67 - nm cytoplasmic rings . Scale bars for cryotomogram slices are 50 nm , and 20 nm for STAs . Empty panels indicate that there were not enough examples to produce a STA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-G) Slices through electron cryotomograms (left panels), central slices of subtomogram averages (middle panels), and schematic representations (right panels) of the MS- and C-complexes (white arrows on the slices) of different mutants in H. pylori fliP*. Light and dark green arrows in (A and D) point to the ~53-nm and ~67-nm cytoplasmic rings. Scale bars for cryotomogram slices are 50 nm, and 20 nm for STAs. Empty panels indicate that there were not enough examples to produce a STA.


Figure 1MITOL - KO MEFs was vulnerable to ER stress . Control MEFs ( MITOLF / F ) and MITOL - KO MEFs ( MITOL - / - ) were treated with DMSO as control , 0 . 8 µM thapsigargin ( Tg ) , 0 . 7 µg / mL tunicamycin ( Tu ) or 1 . 2 µg / mL brefeldin A ( Br ) for 24 hours . All experiments using Tu below were performed at concentration of 0 . 7 µg / mL . The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers . Viable cells were detected by cell viability assay using cell counting kit - 8 . MITOL KO increased ER stress - induced apoptosis . MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V - FITC for the detection of apoptosis using flow cytometry . MITOL re - expression in MITOL - KO MEFs rescued ER stress vulnerability . MEFs were transfected with MITOL - coding vector ( WT ) , MITOL C65 / 68S - coding vector ( CS ) , or empty vector ( Vec ) 24 hours prior to Tu treatment for 18 hours , followed by immunoblotting with indicated antibodies . Cleaved caspase - 3 ( cC3 ) and cleaved PARP ( cPARP ) were used as apoptosis markers . MITOL KO enhanced ER stress - induced Bax translocation and cytochrome c release . Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours , followed by immunoblotting with indicated antibodies . MITOL KO induced excessive mitochondrial depolarization by ER stress . MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM , followed by flow cytometric analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1MITOL-KO MEFs was vulnerable to ER stress. Control MEFs (MITOLF/F) and MITOL-KO MEFs (MITOL-/-) were treated with DMSO as control, 0.8 µM thapsigargin (Tg), 0.7 µg/mL tunicamycin (Tu) or 1.2 µg/mL brefeldin A (Br) for 24 hours. All experiments using Tu below were performed at concentration of 0.7 µg/mL. The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers. Viable cells were detected by cell viability assay using cell counting kit-8.MITOL KO increased ER stress-induced apoptosis. MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V-FITC for the detection of apoptosis using flow cytometry.MITOL re-expression in MITOL-KO MEFs rescued ER stress vulnerability. MEFs were transfected with MITOL-coding vector (WT), MITOL C65/68S-coding vector (CS), or empty vector (Vec) 24 hours prior to Tu treatment for 18 hours, followed by immunoblotting with indicated antibodies. Cleaved caspase-3 (cC3) and cleaved PARP (cPARP) were used as apoptosis markers.MITOL KO enhanced ER stress-induced Bax translocation and cytochrome c release. Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours, followed by immunoblotting with indicated antibodies.MITOL KO induced excessive mitochondrial depolarization by ER stress. MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM, followed by flow cytometric analysis.


Figure 2IRE1α inhibition compensated MITOL - KO MEFs for vulnerability to ER stress . MEFs were transfected with scramble or siIRE1α ( # 1 , # 2 ) . After 24 hours , these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) . MEFs were treated with 4µ8C ( 25 µM ) for 2 hours prior to Tu treatment for 24 hours ( B ) , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) . MITOL KO did not affect the autophosphorylation of IRE1α . MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos - tag SDS - PAGE . MITOL KO enhanced RIDD and xbp1 splicing . MEFs were incubated with Tu for 4 hours . Expression levels of various UPR associated genes were determined by qRT - PCRMITOL KO enhanced RIDD and xbp1 splicing . MEFs were incubated with Tu for 4 hours . MEFs were transfected with either miR - 17 or miR - 34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours , followed by luciferase reporter assay ( F ) . Error bars represent SD ( n = 3 ) . MEFs were incubated with Tu for 4 hours . Expression levels of various UPR associated genes were determined by qRT - PCRIRE1α - mediated JNK phosphorylation was prolonged by MITOL KO . MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) . JNK inhibition reduced ER stress - induced apoptosis of MITOL - KO MEFs . MEFs were treated with 20 µM JNK inhibitor - II ( JNK inh ) 2 hours prior to Tu treatment for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2IRE1α inhibition compensated MITOL-KO MEFs for vulnerability to ER stress. MEFs were transfected with scramble or siIRE1α (#1, #2). After 24 hours, these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).MEFs were treated with 4µ8C (25 µM) for 2 hours prior to Tu treatment for 24 hours (B), followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE.MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCRMITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. MEFs were transfected with either miR-17 or miR-34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours, followed by luciferase reporter assay (F). Error bars represent SD (n=3).MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCRIRE1α-mediated JNK phosphorylation was prolonged by MITOL KO. MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies. Error bars represent SD (n=3).JNK inhibition reduced ER stress-induced apoptosis of MITOL-KO MEFs. MEFs were treated with 20 µM JNK inhibitor-II (JNK inh) 2 hours prior to Tu treatment for 24 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


Figure 3MITOL KO facilitated IRE1α oligomerization . MEFs were transfected with IRE1α - GFP expression vector for 24 hours prior to Tu treatment for indicated periods ( A ) . The right panels show 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection in each independent experiment ( A ) . To evaluate the oligomerization level of endogenous IRE1α , cells without any transfection were solubilized and separated by sucrose density - gradient centrifugation , followed by immunoblotting with anti - IRE1α antibody ( B ) . IRE1α oligomers were stabilized by MITOL KO . MEFs transfected with IRE1α - GFP were treated with Tu . After 4 hours , these cells were washed with PBS and subsequently re - fed with fresh media for indicated periods . The right panels show 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment . Scale bar represents 10 µm . Sustained IRE1α autophosphorylation after wash out of Tu in MITOL - KO MEFs . MEFs were treated with Tu for 4 hours and washed with PBS and re - fed with fresh media for indicated periods , following by immunoblotting anti - IRE1α antibody after Phos - tag SDS - PAGE . Error bars represent SD ( n = 3 ) . MITOL deletion promoted the interaction between IRE1α and BIM under ER stress . MITOL - depleted HEK293 cells ( KO ) were generated using CRISPR - Cas9 system . Cells were co - transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours . Cells were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies .

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Figure 3MITOL KO facilitated IRE1α oligomerization. MEFs were transfected with IRE1α-GFP expression vector for 24 hours prior to Tu treatment for indicated periods (A). The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment (A). To evaluate the oligomerization level of endogenous IRE1α, cells without any transfection were solubilized and separated by sucrose density-gradient centrifugation, followed by immunoblotting with anti-IRE1α antibody (B).IRE1α oligomers were stabilized by MITOL KO. MEFs transfected with IRE1α-GFP were treated with Tu. After 4 hours, these cells were washed with PBS and subsequently re-fed with fresh media for indicated periods. The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment. Scale bar represents 10 µm.Sustained IRE1α autophosphorylation after wash out of Tu in MITOL-KO MEFs. MEFs were treated with Tu for 4 hours and washed with PBS and re-fed with fresh media for indicated periods, following by immunoblotting anti-IRE1α antibody after Phos-tag SDS-PAGE. Error bars represent SD (n=3).MITOL deletion promoted the interaction between IRE1α and BIM under ER stress. MITOL-depleted HEK293 cells (KO) were generated using CRISPR-Cas9 system. Cells were co-transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours. Cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.


Figure 4Interaction between MITOL and IRE1α . Lysates of HEK293 cells were subjected to immunoprecipitation with anti - IRE1α antibody , followed by immunoblotting with indicated antibodies ( A ) . Interaction between MITOL and IRE1α . Lysates of HEK293 cells co - transfected with indicated vectors were subjected to immunoprecipitation , followed by immunoblotting with indicated antibodies ( B ) . Interaction between MITOL and IRE1α . GST - pull - down assay of IRE1α or MITOL was performed on lysates of MEFs , followed by immunoblotting with indicated antibodies ( C , D ) . N - ter : 1 - 90 aa of MITOL , Loop : 158 - 210 aa of MITOL , C - ter : 241 - 278 aa of MITOL , 465 - 977 aa of IRE1α . MITOL ubiquitylated IRE1α . Lysates of HEK293 cells immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies . MITOL ubiquitylated IRE1α . Lysates of HEK293 cells transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies . MITOL ubiquitylated IRE1α . Lysates of MEFs ( G ) transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies . MITOL adds lysine 63 - linked polyubiquitin chains to IRE1α . Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody . IRE1α ubiquitylation was detectedMITOL adds lysine 63 - linked polyubiquitin chains to IRE1α . Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody . IRE1α ubiquitylation was

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Figure 4Interaction between MITOL and IRE1α. Lysates of HEK293 cells were subjected to immunoprecipitation with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies (A).Interaction between MITOL and IRE1α. Lysates of HEK293 cells co-transfected with indicated vectors were subjected to immunoprecipitation, followed by immunoblotting with indicated antibodies (B).Interaction between MITOL and IRE1α. GST-pull-down assay of IRE1α or MITOL was performed on lysates of MEFs, followed by immunoblotting with indicated antibodies (C, D). N-ter: 1-90 aa of MITOL, Loop: 158-210 aa of MITOL, C-ter: 241-278 aa of MITOL, 465-977 aa of IRE1α.MITOL ubiquitylated IRE1α. Lysates of HEK293 cells immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.MITOL ubiquitylated IRE1α. Lysates of HEK293 cells transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.MITOL ubiquitylated IRE1α. Lysates of MEFs (G) transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detectedMITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected