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 ) ) .

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 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 O O O O O O O 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 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 ) .

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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 ) .

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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 ) .

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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 1 ( B ) Electropherograms of DNA sequencing , family pedigrees , and audiograms of two unrelated patients . Electropherograms show a heterozygous c . 3610C > T substitution resulting in a stop codon ( R1204X ) . Arrows in family pedigrees indicate the patients identified in the present study . In audiograms : red and blue indicate right and left ; respectively , solid and dashed lines and square brackets show air conduction hearing and bone conduction hearing ; respectively . The bold line ( at the age of 48 years ) and thin line ( at the age of 50 years ) in the audiogram of patient 1 indicate progressive hearing loss ( green arrow ) .

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Figure 1(B) Electropherograms of DNA sequencing, family pedigrees, and audiograms of two unrelated patients. Electropherograms show a heterozygous c.3610C>T substitution resulting in a stop codon (R1204X). Arrows in family pedigrees indicate the patients identified in the present study. In audiograms: red and blue indicate right and left; respectively, solid and dashed lines and square brackets show air conduction hearing and bone conduction hearing; respectively. The bold line (at the age of 48 years) and thin line (at the age of 50 years) in the audiogram of patient 1 indicate progressive hearing loss (green arrow).


Figure 2GFP - tagged DIA1 , DIA1 ( M1190D ) , and DIA1 ( R1204X ) plasmids were transfected into MDCKmCherry - ESPIN1 cells cultured on membrane inserts . Twenty - four hours after transfection , a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope . Arrows indicate the elongated microvilli visualized by mCherry - ESPIN1 with GFP - hDIA1 ( R1204X ) expression . Scale bars : 5 μm . Representative of 3 experiments .

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Figure 2GFP-tagged DIA1, DIA1(M1190D), and DIA1(R1204X) plasmids were transfected into MDCKmCherry-ESPIN1 cells cultured on membrane inserts. Twenty-four hours after transfection, a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope. Arrows indicate the elongated microvilli visualized by mCherry-ESPIN1 with GFP-hDIA1(R1204X) expression. Scale bars: 5 μm. Representative of 3 experiments.


Figure 3Various GFP - tagged DIA1 plasmids were transfected into HeLa cells , and fixed 24 h after transfection . Fixed cells were stained with Alexa568 - conjugated phalloidin and DAPI ( A , B : representative of 5 experiments ) . ( A ) Stress fiber formation was observed with a confocal laser microscope . Note the induction of stress fibers by GFP - DIA1 ( M1190D ) ( asterisks ) . Arrow and arrowheads indicate a plasma membrane ( PM ) localization of GFP - DIA1 ( M1190D ) and a dot - like localization of GFP - DIA1 ( R1204X ) adjacent to the PM , respectively . Inset in the top panel of DIA1 ( R1204X ) shows the magnified image of the region indicated by rectangle . Scale bars : 10 μm . . Various GFP - tagged DIA1 plasmids were transfected into HeLa cells , and fixed 24 h after transfection . Fixed cells were stained with Alexa568 - conjugated phalloidin and DAPI ( A , B : representative of 5 experiments ) . ( B ) Microvilli with or without GFP - DIA1 ( R1204X ) expression were observed under a confocal laser microscope . Note elongated and thick microvilli ( arrowheads ) in GFP - DIA1 ( R1204X ) - transfected cells , but not in non - transfected cells ( circles ) . 3D movies are available in Movie EV1 ( R1204X ) , Movie EV2 ( WT ) , and Movie EV3 ( ttaa ) . Scale bar : 10 μm .

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Figure 3Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(A) Stress fiber formation was observed with a confocal laser microscope. Note the induction of stress fibers by GFP-DIA1(M1190D) (asterisks). Arrow and arrowheads indicate a plasma membrane (PM) localization of GFP-DIA1(M1190D) and a dot-like localization of GFP-DIA1(R1204X) adjacent to the PM, respectively. Inset in the top panel of DIA1 (R1204X) shows the magnified image of the region indicated by rectangle. Scale bars: 10 μm..Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(B) Microvilli with or without GFP-DIA1(R1204X) expression were observed under a confocal laser microscope. Note elongated and thick microvilli (arrowheads) in GFP-DIA1(R1204X)- transfected cells, but not in non-transfected cells (circles). 3D movies are available in Movie EV1 (R1204X), Movie EV2 (WT), and Movie EV3 (ttaa). Scale bar: 10 μm.


Figure 4 ( B ) Purified GST and GST - tagged DID proteins ( 50 nM ) were mixed with biotin - labeled DADs ( WT ; R1204X ; and M1190D , 50 nM ) in binding buffer . After rotation , streptavidin - coupled magnetic beads were added to the solution , and the mixture was agitated . The material absorbed to the beads was eluted in Laemmli ' s sample buffer , and were subjected to SDS - PAGE , followed by immunoblotting using an HRP - conjugated GST Ab . Comparable levels of input ( GSTproteins ) are confirmed in the right panel . Representative of 5 experiments .

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Figure 4(B) Purified GST and GST-tagged DID proteins (50 nM) were mixed with biotin-labeled DADs (WT; R1204X; and M1190D, 50 nM) in binding buffer. After rotation, streptavidin-coupled magnetic beads were added to the solution, and the mixture was agitated. The material absorbed to the beads was eluted in Laemmli's sample buffer, and were subjected to SDS-PAGE, followed by immunoblotting using an HRP-conjugated GST Ab. Comparable levels of input (GSTproteins) are confirmed in the right panel. Representative of 5 experiments.


Figure 5GFP - tagged DIA1 ( WT ) , DIA1 ( R1204X ) , and DIA1 ( M1190D ) were transfected into XTC cells . XTC cells were spread on the PLL - coated coverslips and time - lapse images were acquired every 300 ms , illuminating the restricted areas near the cell edges , by using a single - molecule speckle microscope . Representative images are shown for DIA1 ( WT ) ( A , Movie EV5 ) , DIA1 ( R1204X ) ( B , Movie EV6 ) , and DIA1 ( M1190D ) ( C , Movie EV7 ) . Circles indicate speckles showing directional movement over a distance of several micrometers . Trajectories of these speckles are also illustrated , and crosses were plotted at the end of the directional movement . Speckles showing directional movement were frequently observed in cells expressing GFP - DIA1 ( R1204X ) ( B ) , whereas such speckles were scarce in cells expressing GFP - DIA1 ( WT ) ( A ) . However , the density of these speckles in GFP - DIA1 ( R1204X ) - expressing cells appeared lower than that in GFP - DIA1 ( M1190D ) - expressing cells , which is considered a fully active mutant ( C ) . To evaluate the frequency of speckles indicating directional movement , we performed a quantitative analysis ( D ) . The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell , which corresponds to the expression level of the GFP - tagged protein . * P = 0 . 0288 and * * P = 0 . 0002 Bonferroni ' s post hoc test following one - way ANOVA ( WT ; n = 5 , R1204X ; n = 6 , M1190D ; n = 6 ) . Time is in seconds . Scale bars : 5 µm .

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Figure 5GFP-tagged DIA1(WT), DIA1(R1204X), and DIA1(M1190D) were transfected into XTC cells. XTC cells were spread on the PLL-coated coverslips and time-lapse images were acquired every 300 ms, illuminating the restricted areas near the cell edges, by using a single-molecule speckle microscope. Representative images are shown for DIA1(WT) (A, Movie EV5), DIA1(R1204X) (B, Movie EV6), and DIA1(M1190D) (C, Movie EV7). Circles indicate speckles showing directional movement over a distance of several micrometers. Trajectories of these speckles are also illustrated, and crosses were plotted at the end of the directional movement. Speckles showing directional movement were frequently observed in cells expressing GFP-DIA1(R1204X) (B), whereas such speckles were scarce in cells expressing GFP-DIA1(WT) (A). However, the density of these speckles in GFP-DIA1(R1204X)-expressing cells appeared lower than that in GFP-DIA1(M1190D)-expressing cells, which is considered a fully active mutant (C). To evaluate the frequency of speckles indicating directional movement, we performed a quantitative analysis (D). The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell, which corresponds to the expression level of the GFP-tagged protein. *P= 0.0288 and **P= 0.0002 Bonferroni's post hoc test following one-way ANOVA (WT; n = 5, R1204X; n = 6, M1190D; n = 6). Time is in seconds. Scale bars: 5 µm.


Figure 6Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) and homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 weeks of age ( A , B ; representative of n = 6 ) . ( A ) Paraffin - embedded 4 μm sections of cochlea were stained with hematoxylin and eosin . No hair cell ( HC ) loss ( either IHC or OHC ) is observed at the basal turn of the Dia1 - / + or Dia1 - / - cochleae . No obvious differences are observed in the spiral ganglion ( SV ) , spiral ligament ( SL ) , stria vascularis ( SV ) , and Reissner ' s membrane ( RM ) in Dia1 - / + , or Dia1 - / - mice compared with control mice . Lower panels ( Scale bars : 20 µm ) are magnified images of the region indicated rectangle in upper panels ( Scale bars : 100 µm ) . Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) and homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 weeks of age ( A , B ; representative of n = 6 ) . ( B ) Basal turns of the dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a nuclear counterstain . No HC loss ( either IHC or OHC ) is observed in Dia1 - / - mice . Scale bars : 20 µm . ( C ) ABR thresholds ( 10 , 20 , 40 kHz ) in control and Dia1 - / - mice at 5 weeks of age ( dB SPL , mean SE ) . No hearing loss was observed in Dia1 - / - mice ( n = 8 ) compared with control mice ( n = 4 ) .

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Figure 6Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(A) Paraffin-embedded 4 μm sections of cochlea were stained with hematoxylin and eosin. No hair cell (HC) loss (either IHC or OHC) is observed at the basal turn of the Dia1-/+ or Dia1-/-cochleae. No obvious differences are observed in the spiral ganglion (SV), spiral ligament (SL), stria vascularis (SV), and Reissner's membrane (RM) in Dia1-/+, or Dia1-/-mice compared with control mice. Lower panels (Scale bars: 20 µm) are magnified images of the region indicated rectangle in upper panels (Scale bars: 100 µm).Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(B) Basal turns of the dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a nuclear counterstain. No HC loss (either IHC or OHC) is observed in Dia1-/-mice. Scale bars: 20 µm.(C) ABR thresholds (10, 20, 40 kHz) in control and Dia1-/-mice at 5 weeks of age (dB SPL, mean SE). No hearing loss was observed in Dia1-/-mice (n = 8) compared with control mice (n = 4).


Figure 7 ( B ) Expression of FLAG - tagged DIA1 ( R1204X ) in the inner ear and heart of TG , but not WT , mice is confirmed by immunoblotting using a FLAG Ab . Representative of 3 experiments . ( C ) Expression levels of FLAG - tagged DIA1 ( R1204X ) relative to endogenous Dia1 in the inner ear , thymus , and heart were determined by immunoblotting using a Dia1 / DIA1 Ab ( inner ear ; n = 4 , thymus and heart ; n = 3 ) , and quantified in the inner ear and thymus ( * P = 0 . 0226 and * * P = 0 . 0016 by two - tailed Student ' s t - test ) . Lysates of HEK293 cells overexpressing equal amounts of GFP - Dia1 and GFP - DIA1 ( determined using a GFP Ab : right end ) are detected with equal affinity by the Dia1 / DIA1 antibody . Comparable loading of proteins is confirmed using an α − tubulin or GAPDH Ab .

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Figure 7(B) Expression of FLAG-tagged DIA1(R1204X) in the inner ear and heart of TG, but not WT, mice is confirmed by immunoblotting using a FLAG Ab. Representative of 3 experiments.(C) Expression levels of FLAG-tagged DIA1(R1204X) relative to endogenous Dia1 in the inner ear, thymus, and heart were determined by immunoblotting using a Dia1/DIA1 Ab (inner ear; n = 4, thymus and heart; n = 3), and quantified in the inner ear and thymus (*P= 0.0226 and **P= 0.0016 by two-tailed Student's t-test). Lysates of HEK293 cells overexpressing equal amounts of GFP-Dia1 and GFP-DIA1 (determined using a GFP Ab: right end) are detected with equal affinity by the Dia1/DIA1 antibody. Comparable loading of proteins is confirmed using an α−tubulin or GAPDH Ab.


Figure 8 ( A ) , Age - related click and pure tone - burst ( 8 , 16 , 24 , 32 kHz ) ABR thresholds ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( 5 weeks , n = 6 ; 10 weeks , n = 16 ; 25 weeks , n = 42 ) and control mice ( 5 weeks , n = 6 ; 10 weeks , n = 16 ; 25 weeks , n = 33 ) . Note the progressive hearing loss of DIA1 ( R1204X ) - TG mice starting at high frequency sounds from 10 weeks . * * P = 0 . 0039 ( 24 kHz ) and * * P < 0 . 0001 ( 32 kHz ) at 10 weeks and * * P < 0 . 0001 ( click ) , * * P < 0 . 0001 ( 8 kHz ) , * * P = 0 . 0001 ( 16 kHz ) , * * P < 0 . 0001 ( 24 kHz ) , and * * P < 0 . 0001 ( 32 kHz ) at 25 weeks by Bonferroni ' s post hoc test following two - way ANOVA . ( B ) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1 ( R1204X ) - TG and control mice at 5 , 10 , and 25 weeks of age . White and red circles show IHC and OHC losses , respectively . Note OHC - dominant HC loss . Scale bars : 5 µm . ( C ) The percentages of remaining OHC and IHC in each turn in DIA1 ( R1204X ) - TG and control mice at 25 weeks of age ( n = 5 ) . Schematic illustration shows the regions of the cochlea divided into three parts : Ap ; apical turn , Md ; middle turn , Bs ; basal turn . * * P = 0 . 0017 ( OHC at Md ) , * * P < 0 . 0001 ( OHC at Bs ) , * P = 0 . 0117 ( IHC at Ap ) , * P = 0 . 0330 ( IHC at Md ) , and * * P = 0 . 0003 ( IHC at Bs ) by Bonferroni ' s post hoc test following two - way ANOVA . ( D ) DPOAE ( f2 frequency at 8 , 12 , 16 , 20 kHz ) amplitude ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( n = 37 ) and control mice ( n = 30 ) at 25 weeks of age . * P = 0 . 0127 , * * P = 0 . 0029 ( at 12 kHz ) , and * * P = 0 . 0013 ( at 16 kHz ) by Bonferroni ' s post hoc test following two - way ANOVA . ( E ) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1 ( R1204X ) - TG and control mice at 25 weeks of age . Note the sparse and short ( arrow ) stereocilia of OHC , and sparse and fused ( arrowheads ) stereocilia of IHC in DIA1 ( R1204X ) - TG mice . Scale bars : 1 µm in OHCs and 5 µm in IHCs .

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

Figure 8(A), Age-related click and pure tone-burst (8, 16, 24, 32 kHz) ABR thresholds (dB SPL, mean SE) in DIA1(R1204X)-TG (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 42) and control mice (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 33). Note the progressive hearing loss of DIA1(R1204X)-TG mice starting at high frequency sounds from 10 weeks. **P= 0.0039 (24 kHz) and **P< 0.0001 (32 kHz) at 10 weeks and **P< 0.0001 (click), **P< 0.0001 (8 kHz), **P= 0.0001 (16 kHz), **P< 0.0001 (24 kHz), and **P< 0.0001 (32 kHz) at 25 weeks by Bonferroni's post hoc test following two-way ANOVA.(B) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1(R1204X)-TG and control mice at 5, 10, and 25 weeks of age. White and red circles show IHC and OHC losses, respectively. Note OHC-dominant HC loss. Scale bars: 5 µm.(C) The percentages of remaining OHC and IHC in each turn in DIA1(R1204X)-TG and control mice at 25 weeks of age (n = 5). Schematic illustration shows the regions of the cochlea divided into three parts: Ap; apical turn, Md; middle turn, Bs; basal turn. **P= 0.0017 (OHC at Md), **P< 0.0001 (OHC at Bs), *P= 0.0117 (IHC at Ap), *P= 0.0330 (IHC at Md), and **P= 0.0003 (IHC at Bs) by Bonferroni's post hoc test following two-way ANOVA.(D) DPOAE (f2 frequency at 8, 12, 16, 20 kHz) amplitude (dB SPL, mean SE) in DIA1(R1204X)-TG (n = 37) and control mice (n = 30) at 25 weeks of age. *P= 0.0127, **P= 0.0029 (at 12 kHz), and **P= 0.0013 (at 16 kHz) by Bonferroni's post hoc test following two-way ANOVA.(E) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1(R1204X)-TG and control mice at 25 weeks of age. Note the sparse and short (arrow) stereocilia of OHC, and sparse and fused (arrowheads) stereocilia of IHC in DIA1(R1204X)-TG mice. Scale bars: 1 µm in OHCs and 5 µm in IHCs.


Figure 1TRIP6 promotes YAP activity by inhibiting LATS1 / 2 ( A ) Full length ( WT ) , the amino - terminal half ( 1 - 277 ) , or the carboxy - terminal half ( 278 - 476 ) of TRIP6 were tested for binding to LATS2 by immunoprecipitation . FLAG - TRIP6 variants were co - expressed with LATS2 - GFP in HEK293 cells , anti - FLAG or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS2 - GFP and FLAG - TRIP6 . Schematic diagram depicts TRIP6 domains ( NES : Nuclear Export Signal ; LIM : LIM domain ; PDZ : PDZ domain binding motif ) . ( B ) Lysates from MCF10A cells were subjected to immunoprecipitation using anti - TRIP6 or control ( IgG ) antibodies , and immune complexes and lysates were probed for TRIP6 and LATS1 . ( C ) FLAG - TRIP6 was tested for binding to various LATS2 - GFP deletion mutants as described in part ( A ) . Schematic diagram of LATS2 shows MOB1 binding domain , and the autophosphorylation ( S872 ) and MST1 / 2 phosphorylation sites ( T1041 ) in the kinase domain . The regions marked in green depict TRIP6 binding sites on LATS2 ( D ) Lysates from HEK293A cells transfected with control or FLAG - TRIP6 plasmid were analyzed by western blotting using the indicated antibodies ( quantification is shown in ( F ) ) . ( E ) Lysates from control ( WT ) or CRISPR generated TRIP6 null ( TRIP6 - KO ) HEK293A cells were analyzed by western blotting using the indicated antibodies ( quantification shown in ( G ) ) . ( F ) The relative levels of LATS1 activating phosphorylation ( pLATS1 - 1079 , 909 ) and YAP S127 inhibitory phosphorylation from ( D ) were measured relative to LATS1 and YAP levels respectively . ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) ( G ) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part ( E ) were quantified ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) . ( H ) GFP - MST2 was expressed with or without FLAG - TRIP6 in HEK293 cells and the levels of MST2 , MST2 activating phosphorylation ( pMST2 - T180 ) , and FLAG - TRIP6 were measured by western blotting with the indicated antibodies . ( Mean ± SD ; n = 3 ; n . s . ≥ 0 . 05 , T - test ) . ( I ) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6 , and YAP target gene expression was analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 , T - test ) . ( J ) The levels of YAP target gene expression was analyzed using RT - qPCR in control ( WT ) and TRIP6 - KO HEK293A cells . ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , T - test ) ( K ) Control ( WT ) and TRIP6 - KO HEK293A cells were stained for YAP and TRIP6 . Merged image shows YAP ( green ) , TRIP6 ( red ) , and DNA ( blue ) . Quantification of at least 100 cells is shown ( Mean ± SD ; n = 3 ; * * * * P ≤ 0 . 0001 , Fisher ' s test ) . Scale bar = 20µm .

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 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 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 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 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 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 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 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 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

Figure 1TRIP6 promotes YAP activity by inhibiting LATS1/2 (A) Full length (WT), the amino-terminal half (1-277), or the carboxy-terminal half (278-476) of TRIP6 were tested for binding to LATS2 by immunoprecipitation. FLAG-TRIP6 variants were co-expressed with LATS2-GFP in HEK293 cells, anti-FLAG or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS2-GFP and FLAG-TRIP6. Schematic diagram depicts TRIP6 domains (NES: Nuclear Export Signal; LIM: LIM domain; PDZ: PDZ domain binding motif).(B) Lysates from MCF10A cells were subjected to immunoprecipitation using anti-TRIP6 or control (IgG) antibodies, and immune complexes and lysates were probed for TRIP6 and LATS1.(C) FLAG-TRIP6 was tested for binding to various LATS2-GFP deletion mutants as described in part (A). Schematic diagram of LATS2 shows MOB1 binding domain, and the autophosphorylation (S872) and MST1/2 phosphorylation sites (T1041) in the kinase domain.The regions marked in green depict TRIP6 binding sites on LATS2 (D) Lysates from HEK293A cells transfected with control or FLAG-TRIP6 plasmid were analyzed by western blotting using the indicated antibodies (quantification is shown in (F)).(E) Lysates from control (WT) or CRISPR generated TRIP6 null (TRIP6-KO) HEK293A cells were analyzed by western blotting using the indicated antibodies (quantification shown in (G)).(F) The relative levels of LATS1 activating phosphorylation (pLATS1-1079, 909) and YAP S127 inhibitory phosphorylation from (D) were measured relative to LATS1 and YAP levels respectively. (Mean ± SD; n=3; **P≤0.01, ***P≤0.001, T-test)(G) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part (E) were quantified (Mean ± SD; n=3; *P≤0.05, T-test).(H) GFP-MST2 was expressed with or without FLAG-TRIP6 in HEK293 cells and the levels of MST2, MST2 activating phosphorylation (pMST2-T180), and FLAG-TRIP6 were measured by western blotting with the indicated antibodies. (Mean ± SD; n=3; n.s.≥0.05, T-test).(I) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6, and YAP target gene expression was analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ****P≤0.0001, T-test).(J) The levels of YAP target gene expression was analyzed using RT-qPCR in control (WT) and TRIP6-KO HEK293A cells. (Mean ± SD; n=3; ***P≤0.001, T-test)(K) Control (WT) and TRIP6-KO HEK293A cells were stained for YAP and TRIP6. Merged image shows YAP (green), TRIP6 (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ****P≤0.0001, Fisher's test). Scale bar=20µm.


Figure 2 ( A ) LATS2 - GFP and Myc - MOB1A were overexpressed in HEK293 cells with or without co - overexpression of full length FLAG - TRIP6 and FLAG - TRIP6 1 - 277 . Myc - MOB1A was immunoprecipitated using anti - Myc antibodies and immune complexes were assayed for Myc - MOB1A and LATS2 - GFP levels . Levels of FLAG - TRIP6 , FLAG - TRIP6 1 - 277 , Myc - MOB1A , and LATS2 - GFP in the lysate are also shown . The levels of LATS2 - GFP in immune complexes relative to the level of Myc - MOB1A are shown in the graph ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test ) ( B ) Competitive binding experiments were done using purified recombinant MBP - LATS2 , GST - TRIP6 , and 6His - MOB1A . MBP - LATS2 bound to maltose beads was incubated with GST - TRIP6 with or without increasing amounts of 6His - MOB1A , and the levels of each protein bound to MBP - LATS2 on the beads at the end of the experiment was determined by western blotting . The levels of input proteins are shown ( lysate ) . The binding of 6His - MOB1A and GST - TRIP6 to MBP alone , and the use BSA as a competitor instead of 6His - MOB1A are shown as controls . The numbers at the bottom are referred to in the text .

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 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

Figure 2(A) LATS2-GFP and Myc-MOB1A were overexpressed in HEK293 cells with or without co-overexpression of full length FLAG-TRIP6 and FLAG-TRIP6 1-277. Myc-MOB1A was immunoprecipitated using anti-Myc antibodies and immune complexes were assayed for Myc-MOB1A and LATS2-GFP levels. Levels of FLAG-TRIP6, FLAG-TRIP6 1-277, Myc-MOB1A, and LATS2-GFP in the lysate are also shown. The levels of LATS2-GFP in immune complexes relative to the level of Myc-MOB1A are shown in the graph (Mean ± SD; n=3; **P≤0.01, T-test)(B) Competitive binding experiments were done using purified recombinant MBP-LATS2, GST-TRIP6, and 6His-MOB1A. MBP-LATS2 bound to maltose beads was incubated with GST-TRIP6 with or without increasing amounts of 6His-MOB1A, and the levels of each protein bound to MBP-LATS2 on the beads at the end of the experiment was determined by western blotting. The levels of input proteins are shown (lysate). The binding of 6His-MOB1A and GST-TRIP6 to MBP alone, and the use BSA as a competitor instead of 6His-MOB1A are shown as controls. The numbers at the bottom are referred to in the text.


Figure 3 ( A ) MCF10A cells were infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 and shTRIP6 - 4 ) and were stained for TRIP6 and LATS1 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) ( quantification of LATS1 localization at cell - cell junctions is shown in Figure EV3D ) . Scale bar = 20µm . ( B ) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells . MCF10A control cells were treated with control siRNA ( siControl ) against fire fly luciferase . Cells were stained for TRIP6 and LATS1 as in ( A ) ( quantification of TRIP6 localization at cell - cell junctions is shown in Figure EV3E ) . Scale bar = 20µm . ( C ) MCF10A cells were grown to high density , and were stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm . ( D ) Cells were grown as in ( C ) , then lysed and anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 . Quantification is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) . ( E ) MCF10A cells grown at high density on PDMS membranes and were stretched ( or not ) at 17 % elongation for 2 hours and lysed while under tension . Anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 . Quantification is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) . ( F ) Cells were treated as in ( E ) , fixed while under tension , and stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm . ( G ) MCF10A cells infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 and shTRIP6 - 4 ) , were grown at high density on PDMS membranes and were stretched or not ( only stretched cells shown ) at 17 % elongation for 2 hours , fixed while under tension , and were stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm . ( H ) Cells were treated as in ( G ) and YAP target gene ( CTGF and Cyr61 ) and TRIP6 expression were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , T - test ) . ( I ) TRIP6 expression levels in ( H ) in control ( shEGFP ) and TRIP6 ( shTRIP6 ) knockdown cells just prior to stretch were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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 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 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 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 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 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 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

Figure 3(A) MCF10A cells were infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4) and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue) (quantification of LATS1 localization at cell-cell junctions is shown in Figure EV3D). Scale bar=20µm.(B) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells. MCF10A control cells were treated with control siRNA (siControl) against fire fly luciferase. Cells were stained for TRIP6 and LATS1 as in (A) (quantification of TRIP6 localization at cell-cell junctions is shown in Figure EV3E). Scale bar=20µm.(C) MCF10A cells were grown to high density, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.(D) Cells were grown as in (C), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).(E) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and lysed while under tension. Anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).(F) Cells were treated as in (E), fixed while under tension, and stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.(G) MCF10A cells infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4), were grown at high density on PDMS membranes and were stretched or not (only stretched cells shown) at 17% elongation for 2 hours, fixed while under tension, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.(H) Cells were treated as in (G) and YAP target gene (CTGF and Cyr61) and TRIP6 expression were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, T-test).(I) TRIP6 expression levels in (H) in control (shEGFP) and TRIP6 (shTRIP6) knockdown cells just prior to stretch were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, T-test).


Figure 4 ( A ) MCF10A cells were either not treated or treated separately with Latrunculin B , Blebbistatin , EGTA , serum starvation , and Y27632 , and were stained for TRIP6 and LATS1 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm . ( B ) Cells were treated as in ( A ) , then lysed and anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 .

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Figure 4(A) MCF10A cells were either not treated or treated separately with Latrunculin B, Blebbistatin, EGTA, serum starvation, and Y27632, and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.(B) Cells were treated as in (A), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6.


Figure 5 ( A ) MCF10A cells were lysed and anti - TRIP6 or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for vinculin and TRIP6 . ( B ) MCF10A cells were either not treated ( control ) or treated separately by growth to high density , serum starvation , Latrunculin B , Blebbistatin , EGTA , or Y27632 treatment , and stained using anti - vinculin antibodies by immunofluorescence . Scale bar = 20µm . ( C ) MCF10A cells grown at high density on PDMS membranes and were stretched ( or not ) at 17 % elongation for 2 hours and fixed while under tension and stained for vinculin . Scale bar = 20µm . ( D ) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho - LATS1 ( T1079 and S909 ) , phospho - YAP ( S127 ) , LATS1 , YAP , vinculin , and tubulin antibodies and the relative levels quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test ) . ( E ) Vinculin was knocked down as described in ( D ) and cells were stained for YAP and vinculin . Merged image shows YAP ( green ) , vinculin ( red ) , and DNA ( blue ) . Quantification of at least 100 cells is shown ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , Fisher ' s test ) . Scale bar = 20µm . ( F ) Vinculin was knocked down as described in ( D ) and the levels of vinculin and YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - test ) . ( G ) Vinculin was knocked down in MCF10A cells as described in ( D ) , grown at high density on PDMS membranes and cells were stretched ( or not ) at 17 % elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - test ) .

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 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 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 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 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 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

Figure 5(A) MCF10A cells were lysed and anti-TRIP6 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin and TRIP6.(B) MCF10A cells were either not treated (control) or treated separately by growth to high density, serum starvation, Latrunculin B, Blebbistatin, EGTA, or Y27632 treatment, and stained using anti-vinculin antibodies by immunofluorescence. Scale bar=20µm.(C) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and fixed while under tension and stained for vinculin. Scale bar=20µm.(D) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho-LATS1 (T1079 and S909), phospho-YAP (S127), LATS1, YAP, vinculin, and tubulin antibodies and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).(E) Vinculin was knocked down as described in (D) and cells were stained for YAP and vinculin. Merged image shows YAP (green), vinculin (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ***P≤0.001, Fisher's test). Scale bar=20µm.(F) Vinculin was knocked down as described in (D) and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).(G) Vinculin was knocked down in MCF10A cells as described in (D), grown at high density on PDMS membranes and cells were stretched (or not) at 17% elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).


Figure 6 ( A ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A cells and cells were stained for LATS1 and TRIP6 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm . ( B ) Vinculin was knocked down as described ( Figure 5D ) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1 , TRIP6 , and vinculin and the relative levels quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test ) . ( C ) MCF10A cells were subjected to different treatments as described ( Figure 4A ) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6 . ( D ) Quantification of TRIP6 - vinculin binding from part ( C ) is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) . ( E , F ) MCF10A cells were treated separately ( or not ) with Latrunculin B ( E ) and by serum starvation ( F ) and LATS1 immune complexes and lysates were probed by western blotting for vinculin , LATS1 , and TRIP6 . Quantification of LATS1 - TRIP6 - vinculin binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) . ( G ) Wild type and T12 mutant of vinculin were co - expressed with LATS2 and FLAG - TRIP6 and anti - FLAG antibody was used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for vinculin , LATS2 and FLAG - TRIP6 . Quantification of LATS2 - TRIP6 - vinculin ( WT or T12 ) binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) . ( H ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A cells grown at high density on PDMS membranes . The membranes were stretched at 17 % elongation for 2 hours and fixed while under tension and stained for TRIP6 . Merged images show TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm .

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Figure 6(A) Vinculin was knocked down as described (Figure 5D) in MCF10A cells and cells were stained for LATS1 and TRIP6. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.(B) Vinculin was knocked down as described (Figure 5D) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1, TRIP6, and vinculin and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).(C) MCF10A cells were subjected to different treatments as described (Figure 4A) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6.(D) Quantification of TRIP6-vinculin binding from part (C) is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).(E, F) MCF10A cells were treated separately (or not) with Latrunculin B (E) and by serum starvation (F) and LATS1 immune complexes and lysates were probed by western blotting for vinculin, LATS1, and TRIP6. Quantification of LATS1-TRIP6-vinculin binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).(G) Wild type and T12 mutant of vinculin were co-expressed with LATS2 and FLAG-TRIP6 and anti-FLAG antibody was used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin, LATS2 and FLAG-TRIP6. Quantification of LATS2-TRIP6-vinculin (WT or T12) binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).(H) Vinculin was knocked down as described (Figure 5D) in MCF10A cells grown at high density on PDMS membranes. The membranes were stretched at 17% elongation for 2 hours and fixed while under tension and stained for TRIP6. Merged images show TRIP6 (red) and DNA (blue). Scale bar=20µm.


Figure 1 ( A ) RNAscope fluorescence in - situ hybridization of Ch25h transcripts ( red ) in the spinal cord of non - immunized mice ( NI ) ( left panels ) and mice 17 days after EAE immunization ( peak disease ) ( 2 right panels ) shown in endothelial cells ( Isolectin B4 ( IsoB4 ) green top panels ) and activated macrophages / microglia ( Ionized calcium biding adaptor molecule 1 ( IBA1 ) green lower panels ) . Nuclei are shown in blue . ( B ) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages / microglia ( MG ) and endothelial cells ( EC ) , n = 6 biological replicates / group . ( E ) Percentage of eGFP + CNS ECs in the same condition as in ( D ) . Symbols indicate individual mice and bars indicate mean ± SD . Ch25hfl / fl NI : n = 4 biological replicates , Ch25hfl / fl EAE : n = 5 biological replicates , Ch25hECKO NI : n = 4 biological replicates , Ch25hECKO EAE : n = 4 biological replicates . Representative results of 1 experiment .

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Figure 1(A) RNAscope fluorescence in-situ hybridization of Ch25h transcripts (red) in the spinal cord of non-immunized mice (NI) (left panels) and mice 17 days after EAE immunization (peak disease) (2 right panels) shown in endothelial cells (Isolectin B4 (IsoB4) green top panels) and activated macrophages/microglia (Ionized calcium biding adaptor molecule 1 (IBA1) green lower panels). Nuclei are shown in blue. (B) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages/microglia (MG) and endothelial cells (EC), n = 6 biological replicates/group.(E) Percentage of eGFP+ CNS ECs in the same condition as in (D). Symbols indicate individual mice and bars indicate mean ± SD. Ch25hfl/fl NI: n = 4 biological replicates, Ch25hfl/fl EAE: n = 5 biological replicates, Ch25hECKO NI: n = 4 biological replicates, Ch25hECKO EAE: n = 4 biological replicates. Representative results of 1 experiment.


Figure 2 ( A ) EAE disease course in Ch25hECKO and Cre negative littermates ( Ch25hfl / fl ) . Top panel : EAE clinical score . Bars indicate mean ± SEM . Bottom panel : Survival analysis of EAE , depicting disease incidence . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph . ( B ) As in ( A ) except that Ch25h fl / fl - Pdgfb - iCreERT2 mice that express Cre recombinase in blood endothelial cells ( Ch25hBECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph . ( C ) As in ( A ) except that Ch25hfl / fl - Prox1 - CreERT2 mice that express Cre recombinase in lymphatic endothelial cells ( Ch25hLECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph . ( D , E ) Histopathological quantifications ( D ) and representatives staining ( E ) of spinal cord sections of immunized Ch25hfl / fl mice ( n = 7 biological replicates ) and Ch25hECKO mice ( n = 10 biological replicates ) at day 21 post - immunization for cellular infiltration ( HE ) . Five sections per mouse were quantified . Scale bars 200 µm ( top panels ) , 100 µm ( bottom panels ) .

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Figure 2(A) EAE disease course in Ch25hECKO and Cre negative littermates (Ch25hfl/fl). Top panel: EAE clinical score. Bars indicate mean ± SEM. Bottom panel: Survival analysis of EAE, depicting disease incidence. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (B) As in (A) except that Ch25h fl/fl- Pdgfb-iCreERT2 mice that express Cre recombinase in blood endothelial cells (Ch25hBECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (C) As in (A) except that Ch25hfl/fl- Prox1-CreERT2 mice that express Cre recombinase in lymphatic endothelial cells (Ch25hLECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.(D, E) Histopathological quantifications (D) and representatives staining (E) of spinal cord sections of immunized Ch25hfl/fl mice (n = 7 biological replicates) and Ch25hECKO mice (n = 10 biological replicates) at day 21 post-immunization for cellular infiltration (HE). Five sections per mouse were quantified. Scale bars 200 µm (top panels), 100 µm (bottom panels).


Figure 3 ( A ) RT - qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells ( pMBMECs ) isolated from Ch25hfl / fl and Ch25hECKO mice . Cells were left unstimulated ( NS ) or stimulated with IL - 1β . n = 5 biological replicates / group . The experiment was performed 3 times . ( F ) RT - qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl / fl mice and stimulated with IL - 1β . n = 4 biological replicates / group . The experiment was realized once . ( G ) RT - qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl / fl mice during EAE ( day 10 after immunization ) . Ch25hECKO n = 3 biological replicates , Ch25hfl / fl n = 4 biological replicates . The experiment was performed once .

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Figure 3(A) RT-qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells (pMBMECs) isolated from Ch25hfl/fl and Ch25hECKO mice. Cells were left unstimulated (NS) or stimulated with IL-1β. n = 5 biological replicates/group. The experiment was performed 3 times.(F) RT-qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl/fl mice and stimulated with IL-1β. n = 4 biological replicates/group. The experiment was realized once.(G) RT-qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl/fl mice during EAE (day 10 after immunization). Ch25hECKO n = 3 biological replicates, Ch25hfl/fl n = 4 biological replicates. The experiment was performed once.


Figure 4 ( C ) 25 - OHC , 7 - keto - 25 - OHC , and 24 ( S ) - OHC concentration in the same conditions as in ( B ) . Bars represent mean ± SD . n = 6 biological replicates / group except for Ch25fl / fl IL - 1β were n = 5 biological replicates . The experiment was performed once . ( D ) 25 - OHC , 7 - keto - 25 - OHC , and 24 ( S ) - OHC levels in spinal cord tissue extracted from Ch25hfl / fl ( n = 8 ) , Ch25hECKO ( n = 3 ) and Ch25hBBBKO ( n = 6 ) at the peak of EAE . ( G ) Prostaglandin concentration ( nM ) in the supernatant of pMBMECs comparing conditions mentioned in ( A ) . n = 5 biological replicates / group and bars represent mean ± SD .

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Figure 4(C) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC concentration in the same conditions as in (B). Bars represent mean ± SD. n = 6 biological replicates/group except for Ch25fl/fl IL-1β were n = 5 biological replicates. The experiment was performed once. (D) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC levels in spinal cord tissue extracted from Ch25hfl/fl (n = 8), Ch25hECKO (n = 3) and Ch25hBBBKO (n = 6) at the peak of EAE.(G) Prostaglandin concentration (nM) in the supernatant of pMBMECs comparing conditions mentioned in (A). n = 5 biological replicates/group and bars represent mean ± SD.


Figure 5 ( C ) Impact of CNS PMN - MDSC and BM - PMN on CD4 + T cell proliferation assessed by CFSE dilution using flow cytometry . PMN - MDSC were FACS - sorted from the CNS ( right panel ) and BM ( left panel ) of WT mice at the peak of EAE . NS = non stimulated . n = 4 biological replicates / group . Bars indicate mean ± SD . CNS PMN - MDSC / CD4 + T cells : representative results of 2 independent experiments ; BM - PMN : 1 experiment . ( H ) Percentage ( upper left panel ) and absolute number ( lower left panel ) of CNS live cells CD45 + CD11b + Ly6CintLy6G + PMN - MDSC ; Percentage of live CD4 + CD44 + Ki67 + cells ( upper right panel ) and ratio ( lower right panel ) of absolute number of PMN - MDSC and proliferating CD4 + T cells in the CNS at the peak of the disease of Ch25hfl / fl ( n = 9 ) and Ch25hECKO mice ( n = 8 ) . Symbols depict individual mice and bars indicate mean ± SD . Combined results of 2 independent experiments . ( I ) EAE disease course in Ch25hBBBKO mice ( n = 11 ) where the Cre - recombinase is expressed in endothelial cells of the CNS and Cre negative littermates ( Ch25hfl / fl : n = 7 and Ch25hfl / wt : n = 2 ) . Bars indicate mean ± SEM . Representative results of 2 independent experiments .

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Figure 5(C) Impact of CNS PMN-MDSC and BM-PMN on CD4+ T cell proliferation assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the CNS (right panel) and BM (left panel) of WT mice at the peak of EAE. NS = non stimulated. n = 4 biological replicates/group. Bars indicate mean ± SD. CNS PMN-MDSC/CD4+ T cells: representative results of 2 independent experiments; BM-PMN: 1 experiment.(H) Percentage (upper left panel) and absolute number (lower left panel) of CNS live cells CD45+CD11b+Ly6CintLy6G+ PMN-MDSC; Percentage of live CD4+CD44+Ki67+ cells (upper right panel) and ratio (lower right panel) of absolute number of PMN-MDSC and proliferating CD4+ T cells in the CNS at the peak of the disease of Ch25hfl/fl (n = 9) and Ch25hECKO mice (n = 8). Symbols depict individual mice and bars indicate mean ± SD. Combined results of 2 independent experiments.(I) EAE disease course in Ch25hBBBKO mice (n = 11) where the Cre-recombinase is expressed in endothelial cells of the CNS and Cre negative littermates (Ch25hfl/fl: n = 7 and Ch25hfl/wt : n = 2). Bars indicate mean ± SEM. Representative results of 2 independent experiments.


Figure 6 ( B ) Survival analysis in the same conditions as in ( A ) . Ch25hfl / fl isotype : n = 7 , Ch25hECKO isotype : n = 8 , Ch25hfl / fl Combo : n = 8 , Ch25hECKO Combo : n = 9 . ( C ) Representative contour plots of PMN - MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in ( A ) . ( D ) As in ( C ) except that statistical analysis and percentage of PMN - MDSC in live cells are shown . Symbols depict individual mice and bars mean ± SD . Ch25hfl / fl Isotype n = 4 , Ch25hECKO isotype n = 4 , Ch25hfl / fl Combo , n = 3 , Ch25hECKO Combo n = 4 . ( ( E ) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry . Ch25hECKO and Ch25hfl / fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE ( day 14 after immunization ) . ( F ) As in ( E ) except that statistical analysis is shown . Symbols depict individual mice , bars mean ± SD . Ch25hfl / lfl isotype n = 7 , Ch25hECKO isotype n = 5 , Ch25hfl / lfl Combo n = 6 , Ch25hECKO Combo n = 5 . The experiment was performed once . (

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Figure 6(B) Survival analysis in the same conditions as in (A). Ch25hfl/fl isotype: n = 7, Ch25hECKO isotype: n = 8, Ch25hfl/fl Combo: n = 8, Ch25hECKO Combo: n = 9.(C) Representative contour plots of PMN-MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in (A). (D) As in (C) except that statistical analysis and percentage of PMN-MDSC in live cells are shown. Symbols depict individual mice and bars mean ± SD. Ch25hfl/fl Isotype n = 4, Ch25hECKO isotype n = 4, Ch25hfl/fl Combo, n = 3, Ch25hECKO Combo n = 4. ((E) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry. Ch25hECKO and Ch25hfl/fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE (day 14 after immunization). (F) As in (E) except that statistical analysis is shown. Symbols depict individual mice, bars mean ± SD. Ch25hfl/lfl isotype n = 7, Ch25hECKO isotype n = 5, Ch25hfl/lfl Combo n = 6, Ch25hECKO Combo n = 5. The experiment was performed once. (