Figure 5 ( A ) Lysates of wild - type ( control ) and GRAMD1 TKO ( TKO ) HeLa cells that stably expressed either EGFP or EGFP - GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] were processed by SDS - PAGE and immunoblotted ( IB ) with anti - GFP , anti - GRAMD1b , and anti - actin antibodies . ( B ) Confocal images of live GRAMD1 TKO HeLa cells stably expressing EGFP - GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor , mCherry - tagged GRAM domain of GRAMD1b ( mCherry - GRAM1b ) . Cells were treated with SMase ( 100 mU / ml for 1 hour at 37 ° C ) before imaging . Insets show at higher magnification the regions indicated by white dashed boxes . Note the very weak PM recruitment of mCherry - GRAM1b in cells that stably expressed EGFP - GRAMD1b WT , compared to the strong PM recruitment of mCherry - GRAM1b in cells that stably expressed EGFP - GRAMD1b R189W mutant ( R189W ) or EGFP - GRAMD1b R189W / R191A mutant ( RW / RA ) . Scale bars , 10 µm . ( C ) Quantification of the ratio of PM mCherry - GRAM1b signals to the cytosolic mCherry - GRAM1b signals , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing mCherry - GRAM1b with SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) as shown in ( B ) and Fig EV3B ( mean ± SEM , n = 20 cells for each condition ; data are pooled from two independent experiments ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 . n . s . denotes not significant ) . ( D ) Left : Time course of normalized mCherry signal , as assessed by TIRF microscopy , from GRAMD1 TKO HeLa cells stably expressing either EGFP or EGFP - GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry - GRAM1b . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 31 cells ( EGFP ) , n = 28 cells ( EGFP - GRAMD1b WT ) , n = 23 cells ( EGFP - GRAMD1b R189W ) , n = 27 cells [ EGFP - GRAMD1b R189W / R191A ( RW / RA ) ] ; data are pooled from two independent experiments for each condition ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ( EGFP vs . WT and WT vs . R189W ) , * * P = 0 . 0002 ( WT vs . RW / RA ) ] . ( E ) Wild - type ( control ) and GRAMD1 TKO ( TKO ) HeLa cells that stably expressed either EGFP or EGFP - tagged GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] , were cultured in the medium supplemented with 10 % lipoprotein - deficient serum ( LPDS ) and mevastatin ( 50 µM ) for 16 hours and then treated with SMase ( 100 mU / ml ) for 3 hours at 37 ° C . Top : Lysates of the cells were processed for SDS - PAGE and IB with anti - SREBP - 2 and anti - actin antibodies . Arrows indicate precursor ( P ) and cleaved ( C ) forms of SREBP - 2 . Bottom : The response rate was obtained by normalizing the ratio of the band intensity of the cleaved SREBP - 2 over the total band intensity of cleaved and precursor forms of SREBP - 2 from the cells with SMase treatment by the one from the cells without SMase treatment for each condition . Note that the suppression of SREBP - 2 cleavage is attenuated in GRAMD1 TKO HeLa cells compared to wild - type control HeLa cells . Note also the rescue by expression of wild - type EGFP - GRAMD1b ( WT ) but not by mutant versions of EGFP - GRAMD1b [ R189W , R189W / R191A ( RW / RA ) ] [ mean ± SEM , n = 4 lysates ( independent experiments ) for each condition ; Dunnett ' s multiple comparisons test , * * P = 0 . 0035 ( Control + EGFP vs . TKO + EGFP ) , * * P < 0 . 0001 ( TKO + EGFP vs . TKO + WT ) , n . s . denotes not significant ] . ( F ) Amphotericin B resistance of SMase - treated wild - type ( control ) and GRAMD1 TKO ( TKO ) HeLa cells that stably expressed either EGFP or EGFP - tagged GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] . Left : Cells that had been pre - treated with SMase ( 100 mU / ml ) for 3 hours at 37 ° C were treated with indicated concentration of Amphotericin B for 20 min at 37ºC . After overnight recovery in culture media , cell viability was measured by detecting ATP present in each well via luminescence ( see Materials and Methods ) . The same number of cells were seeded in each well before SMase treatment . Note the reduced viability of cells with increasing amount of Amphotericin B . Right : Quantification of cell viability with increasing amount of Amphotericin B . Note the resistance of wild - type control and GRAMD1 TKO cells that stably expressed EGFP - GRAMD1b ( WT ) compared to GRAMD1 TKO cells or GRAMD1 TKO cells that stably expressed mutant versions of EGFP - GRAMD1b [ R189W , R189W / R191A ( RW / RA ) ] ( mean ± SEM , n = 3 independent experiments for each condition ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysates of wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)] were processed by SDS-PAGE and immunoblotted (IB) with anti-GFP, anti-GRAMD1b, and anti-actin antibodies.(B) Confocal images of live GRAMD1 TKO HeLa cells stably expressing EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor, mCherry-tagged GRAM domain of GRAMD1b (mCherry-GRAM1b). Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note the very weak PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b WT, compared to the strong PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b R189W mutant (R189W) or EGFP-GRAMD1b R189W/R191A mutant (RW/RA). Scale bars, 10 µm.(C) Quantification of the ratio of PM mCherry-GRAM1b signals to the cytosolic mCherry-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing mCherry-GRAM1b with SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (B) and Fig EV3B (mean ± SEM, n = 20 cells for each condition; data are pooled from two independent experiments; Dunnett's multiple comparisons test, **P < 0.0001. n.s. denotes not significant).(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells stably expressing either EGFP or EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry-GRAM1b. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 31 cells (EGFP), n = 28 cells (EGFP-GRAMD1b WT), n = 23 cells (EGFP-GRAMD1b R189W), n = 27 cells [EGFP-GRAMD1b R189W/R191A (RW/RA)]; data are pooled from two independent experiments for each condition; Dunnett's multiple comparisons test, **P < 0.0001 (EGFP vs. WT and WT vs. R189W), **P = 0.0002 (WT vs. RW/RA)].(E) Wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)], were cultured in the medium supplemented with 10% lipoprotein-deficient serum (LPDS) and mevastatin (50 µM) for 16 hours and then treated with SMase (100 mU/ml) for 3 hours at 37°C. Top: Lysates of the cells were processed for SDS-PAGE and IB with anti-SREBP-2 and anti-actin antibodies. Arrows indicate precursor (P) and cleaved (C) forms of SREBP-2. Bottom: The response rate was obtained by normalizing the ratio of the band intensity of the cleaved SREBP-2 over the total band intensity of cleaved and precursor forms of SREBP-2 from the cells with SMase treatment by the one from the cells without SMase treatment for each condition. Note that the suppression of SREBP-2 cleavage is attenuated in GRAMD1 TKO HeLa cells compared to wild-type control HeLa cells. Note also the rescue by expression of wild-type EGFP-GRAMD1b (WT) but not by mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] [mean ± SEM, n = 4 lysates (independent experiments) for each condition; Dunnett's multiple comparisons test, **P = 0.0035 (Control + EGFP vs. TKO + EGFP), **P < 0.0001 (TKO + EGFP vs. TKO + WT), n.s. denotes not significant].(F) Amphotericin B resistance of SMase-treated wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)]. Left: Cells that had been pre-treated with SMase (100 mU/ml) for 3 hours at 37°C were treated with indicated concentration of Amphotericin B for 20 min at 37ºC. After overnight recovery in culture media, cell viability was measured by detecting ATP present in each well via luminescence (see Materials and Methods). The same number of cells were seeded in each well before SMase treatment. Note the reduced viability of cells with increasing amount of Amphotericin B. Right: Quantification of cell viability with increasing amount of Amphotericin B. Note the resistance of wild-type control and GRAMD1 TKO cells that stably expressed EGFP-GRAMD1b (WT) compared to GRAMD1 TKO cells or GRAMD1 TKO cells that stably expressed mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] (mean ± SEM, n = 3 independent experiments for each condition).


Figure 6 ( B ) Quantification of the ratio of PM signals to the cytosolic signals of wild - type EGFP - GRAM1b ( WT ) and mutant versions of EGFP - GRAM1b ( R166L , Q173I , F182I , T184V , F186L , G187L , A188S ) , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells , expressing indicated constructs , as shown in ( C ) and Fig EV4A ( mean ± SEM , n = 10 cells for each condition ; data are pooled from one experiment ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ) . ( D ) Confocal images of live wild - type ( control ) and GRAMD1 TKO HeLa cells , expressing either wild - type EGFP - GRAM1b ( WT ) or mutant EGFP - GRAM1b ( G187L ) , with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) . Insets show at higher magnification the regions indicated by white dashed boxes . Note the recruitment of mutant EGFP - GRAM1b ( G187L ) to the PM even in wild - type HeLa cells at rest , which was further enhanced by SMase treatment . Scale bars , 10 µm . ( E ) Quantification of the ratio of PM signals to the cytosolic signals of wild - type EGFP - GRAM1b ( WT ) and mutant EGFP - GRAM1b ( G187L ) , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells , expressing indicated constructs , with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) , as shown in ( D ) ( mean ± SEM , n = 10 cells for each condition ; data are pooled from one experiment ) . ( F ) A confocal image of live GRAMD1 TKO HeLa cells expressing mutant EGFP - GRAM1b ( G187L ) . Cells were cultured in the medium supplemented with 10 % lipoprotein deficient serum ( LPDS ) and mevastatin ( 50 µM ) for 16 hours to deplete accessible cholesterol before imaging . An inset shows at higher magnification the region indicated by a white dashed box . Note the absence of PM recruitment ( compare to ( D ) ) . Scale bars , 10 µm . ( G ) Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 TKO ( TKO ) HeLa cells expressing either wild - type EGFP - GRAM1b ( WT ) or mutant EGFP - GRAM1b ( G187L ) as indicated . Cells were cultured in the medium supplemented with 10 % lipoprotein deficient serum ( LPDS ) and mevastatin ( 50 µM ) for 16 hours before imaging . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 36 cells ( WT ) , n = 37 cells ( G187L ) , data are pooled from two independent experiments for each condition ; two - tailed unpaired Student ' s t - test , * * P = 0 . 0003 ] . Note the rapid PM recruitment of G187L mutant compared to WT . ( H , I ) Liposome sedimentation assays of wild - type GRAM1b ( WT ) and mutant GRAM1b ( G187L ) . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as shown . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining [ mean ± SEM , n = 3 independent experiments for all the conditions ; ( H ) Left ( 10 % PS ) : Holm - Sidak t - test for multiple comparisons , * P = 0 . 022679 at 40 % cholesterol , * * P = 0 . 009195 at 50 % cholesterol . Right ( 20 % PS ) : Holm - Sidak t - test for multiple comparisons , * P = 0 . 023163 at 30 % cholesterol , * * P = 0 . 000327 at 40 % cholesterol , * P = 0 . 017194 at 50 % cholesterol . ( I ) Holm - Sidak t - test for multiple comparisons , n . s . denotes not significant , * * P < 0 . 000001 at 50 % DOPC , 50 % cholesterol , * P = 0 . 011271 at 40 % DOPC , 60 % cholesterol ] . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol .

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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(B) Quantification of the ratio of PM signals to the cytosolic signals of wild-type EGFP-GRAM1b (WT) and mutant versions of EGFP-GRAM1b (R166L, Q173I, F182I, T184V, F186L, G187L, A188S), as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells, expressing indicated constructs, as shown in (C) and Fig EV4A (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment; Dunnett's multiple comparisons test, **P < 0.0001).(D) Confocal images of live wild-type (control) and GRAMD1 TKO HeLa cells, expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L), with or without SMase treatment (100 mU/ml for 1 hour at 37°C). Insets show at higher magnification the regions indicated by white dashed boxes. Note the recruitment of mutant EGFP-GRAM1b (G187L) to the PM even in wild-type HeLa cells at rest, which was further enhanced by SMase treatment. Scale bars, 10 µm.(E) Quantification of the ratio of PM signals to the cytosolic signals of wild-type EGFP-GRAM1b (WT) and mutant EGFP-GRAM1b (G187L), as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells, expressing indicated constructs, with or without SMase treatment (100 mU/ml for 1 hour at 37°C), as shown in (D) (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment).(F) A confocal image of live GRAMD1 TKO HeLa cells expressing mutant EGFP-GRAM1b (G187L). Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours to deplete accessible cholesterol before imaging. An inset shows at higher magnification the region indicated by a white dashed box. Note the absence of PM recruitment (compare to (D)). Scale bars, 10 µm.(G) Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO (TKO) HeLa cells expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L) as indicated. Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours before imaging. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 36 cells (WT), n = 37 cells (G187L), data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P = 0.0003]. Note the rapid PM recruitment of G187L mutant compared to WT.(H, I) Liposome sedimentation assays of wild-type GRAM1b (WT) and mutant GRAM1b (G187L). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all the conditions; (H) Left (10% PS): Holm-Sidak t-test for multiple comparisons, *P = 0.022679 at 40% cholesterol, **P = 0.009195 at 50% cholesterol. Right (20% PS): Holm-Sidak t-test for multiple comparisons, *P = 0.023163 at 30% cholesterol, **P = 0.000327 at 40% cholesterol, *P = 0.017194 at 50% cholesterol. (I) Holm-Sidak t-test for multiple comparisons, n.s. denotes not significant, **P < 0.000001 at 50% DOPC, 50% cholesterol, *P = 0.011271 at 40% DOPC, 60% cholesterol]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol.


Figure 7 ( A ) Effects of mutation of G187 on the property of the GRAM domain of GRAMD1b ( GRAM1b ) to sense transient expansion of the accessible pool of PM cholesterol . Indicated EGFP - tagged mutant versions of GRAM1b were expressed in GRAMD1 TKO HeLa cells and imaged under confocal microscopy with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) or with SMase treatment followed by 5 min treatment with Methyl - β - cyclodextrin ( MCD ) ( 5 mM ) to assess their PM recruitment . The mean values ( n = 20 cells from two independent experiments for untreated and SMase - treated conditions ; n = 10 cells from one experiment for SMase & MCD - treated conditions ) of the ratio of PM signals to the cytosolic signals , as assessed by line scan analysis , are presented for each condition as a heatmap . Amino acids are ranked according to Goldman , Engelman and Steitz ( GES ) hydrophobicity scale with the most hydrophobic amino acid on the top . ( B ) Liposome tethering by purified GRAMD1b proteins . Left : Representative time course of liposome tethering , as assessed by turbidity of mixtures containing both LPM and LER together with either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b G187L proteins ( G187L ) , or buffer alone as indicated . Experiments and data analysis were performed as in Fig 4 . Right : Values of turbidity corresponding to the end of the experiment as indicated by the arrow ( mean ± SEM , n = 3 independent experiments ; Dunnett ' s multiple comparisons test , * P = 0 . 0143 ) . ( C ) DHE transfer between liposomes by purified GRAMD1b proteins . Left : Representative time course of DHE transfer from LPM to LER mediated by either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b G187L proteins ( G187L ) , or buffer alone as indicated . Methyl - β - cyclodextrin ( MCD ) at 1 mM was used to determine DHE equilibration . Experiments and data analysis were performed as in Fig 4 . Right : Values of DHE transfer rate of wild - type GRAMD1b ( WT ) and mutant GRAMD1b ( G187L ) , as estimated by the FRET - based lipid transfer assay ( mean ± SEM , n = 3 independent experiments ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0014 ) . ( D ) Confocal images of live GRAMD1 TKO HeLa cells that stably expressed either EGFP control or EGFP - GRAMD1b constructs as indicated . Cells were stained with recombinant mCherry - D4H proteins ( 10 mg / ml ) ( accessible PM cholesterol biosensor ) for 15 min at room temperature before imaging . Insets show at higher magnification the regions indicated by white dashed boxes . Note that expression of EGFP - GRAMD1b - WT , but not EGFP - GRAMD1b - R189W , reduced binding of mCherry - D4H to the PM , in GRAMD1 TKO HeLa cells . Further reduction in mCherry - D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP - GRAMD1b - G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP - GRAMD1b - WT . Scale bars , 10 µm . ( E ) Values of mCherry - D4H signals at the PM after background subtraction , as assessed by confocal microscopy and line scan analysis of GRAMD1 TKO HeLa cells that stably expressed either EGFP or indicated EGFP - GRAMD1b constructs as shown in ( D ) [ mean ± SEM , n = 30 cells for all the conditions ; data are pooled from three independent experiments ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ] .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7(A) Effects of mutation of G187 on the property of the GRAM domain of GRAMD1b (GRAM1b) to sense transient expansion of the accessible pool of PM cholesterol. Indicated EGFP-tagged mutant versions of GRAM1b were expressed in GRAMD1 TKO HeLa cells and imaged under confocal microscopy with or without SMase treatment (100 mU/ml for 1 hour at 37°C) or with SMase treatment followed by 5 min treatment with Methyl-β-cyclodextrin (MCD) (5 mM) to assess their PM recruitment. The mean values (n = 20 cells from two independent experiments for untreated and SMase-treated conditions; n = 10 cells from one experiment for SMase & MCD-treated conditions) of the ratio of PM signals to the cytosolic signals, as assessed by line scan analysis, are presented for each condition as a heatmap. Amino acids are ranked according to Goldman, Engelman and Steitz (GES) hydrophobicity scale with the most hydrophobic amino acid on the top.(B) Liposome tethering by purified GRAMD1b proteins. Left: Representative time course of liposome tethering, as assessed by turbidity of mixtures containing both LPM and LER together with either wild-type GRAMD1b proteins (WT), mutant GRAMD1b G187L proteins (G187L), or buffer alone as indicated. Experiments and data analysis were performed as in Fig 4. Right: Values of turbidity corresponding to the end of the experiment as indicated by the arrow (mean ± SEM, n = 3 independent experiments; Dunnett's multiple comparisons test, *P = 0.0143).(C) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b G187L proteins (G187L), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Experiments and data analysis were performed as in Fig 4. Right: Values of DHE transfer rate of wild-type GRAMD1b (WT) and mutant GRAMD1b (G187L), as estimated by the FRET-based lipid transfer assay (mean ± SEM, n = 3 independent experiments; two-tailed unpaired Student's t-test, **P < 0.0014).(D) Confocal images of live GRAMD1 TKO HeLa cells that stably expressed either EGFP control or EGFP-GRAMD1b constructs as indicated. Cells were stained with recombinant mCherry-D4H proteins (10 mg/ml) (accessible PM cholesterol biosensor) for 15 min at room temperature before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note that expression of EGFP-GRAMD1b-WT, but not EGFP-GRAMD1b-R189W, reduced binding of mCherry-D4H to the PM, in GRAMD1 TKO HeLa cells. Further reduction in mCherry-D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-WT. Scale bars, 10 µm. (E) Values of mCherry-D4H signals at the PM after background subtraction, as assessed by confocal microscopy and line scan analysis of GRAMD1 TKO HeLa cells that stably expressed either EGFP or indicated EGFP-GRAMD1b constructs as shown in (D) [mean ± SEM, n = 30 cells for all the conditions; data are pooled from three independent experiments; Dunnett's multiple comparisons test, **P < 0.0001].


Figure 1A Heatmap of all the DE genes in choroid plexus between the AB and SPF mice . The color scale of the heatmap represents the scaled log2 normalized gene expression . Data information : AB , antibiotics - treated ; SPF , specific pathogen - free ;

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Figure 1A Heatmap of all the DE genes in choroid plexus between the AB and SPF mice. The color scale of the heatmap represents the scaled log2 normalized gene expression. Data information: AB, antibiotics-treated; SPF, specific pathogen-free;


Figure 2A Representative images of immunostainings for ZO - 1 , OCLN , CDH1 and CLDN1 in choroid plexus of SPF , AB , ABR , GF and GFR mice . Scale bar : 50 μm . B - D Quantification of continuous length ( B ) , maximal length ( C ) , and expressed area ( D ) of the TJ immunostainings displayed in ( A ) ( n = 5 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . AB , antibiotics - treated ; ABR , recolonized AB ; GF , germ - free ; GFR , recolonized GF ; SPF , specific pathogen - free ; TJ , tight junction . E Representative western blot showing the IgG levels in CSF of SPF , AB , ABR , GF and GFR mice . F Quantitative analysis of IgG in CSF via western blot in ( E ) ( n = 4 - 6 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . AB , antibiotics - treated ; ABR , recolonized AB ; CSF , cerebrospinal fluid ; GF , germ - free ; GFR , recolonized GF ; SPF , specific pathogen - freeG Assessment of the blood - CSF barrier permeability to 4 kDa FITC - dextran ( n = 5 - 9 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . AB , antibiotics - treated ; ABR , recolonized AB ; CSF , cerebrospinal fluid ; GF , germ - free ; GFR , recolonized GF ; SPF , specific pathogen -

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Figure 2A Representative images of immunostainings for ZO-1, OCLN, CDH1 and CLDN1 in choroid plexus of SPF, AB, ABR, GF and GFR mice. Scale bar: 50 μm. B-D Quantification of continuous length (B), maximal length (C), and expressed area (D) of the TJ immunostainings displayed in (A) (n=5, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. AB, antibiotics-treated; ABR, recolonized AB; GF, germ-free; GFR, recolonized GF; SPF, specific pathogen-free; TJ, tight junction.E Representative western blot showing the IgG levels in CSF of SPF, AB, ABR, GF and GFR mice. F Quantitative analysis of IgG in CSF via western blot in (E) (n=4-6, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. AB, antibiotics-treated; ABR, recolonized AB; CSF, cerebrospinal fluid; GF, germ-free; GFR, recolonized GF; SPF, specific pathogen-freeG Assessment of the blood-CSF barrier permeability to 4 kDa FITC-dextran (n=5-9, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. AB, antibiotics-treated; ABR, recolonized AB; CSF, cerebrospinal fluid; GF, germ-free; GFR, recolonized GF; SPF, specific pathogen-free


Figure 3C , D Relative abundance of propionic acid ( C ) and butyric acid ( D ) in mice fecal samples ( n = 4 - 9 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * * p < 0 . 01 . AB , antibiotics - treated ; SPF , specific pathogen - free .

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Figure 3C, D Relative abundance of propionic acid (C) and butyric acid (D) in mice fecal samples (n=4-9, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. **p < 0.01. AB, antibiotics-treated; SPF, specific pathogen-free.


Figure 4B , C TEER ( B ) and assessment of the 70 kDa FITC - dextran paracellular permeability ( C ) of primary CPE cells treated as showed in ( A ) ( n = 3 , technical duplicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . CPE , choroid plexus epithelial ; LPS , lipopolysaccharides ; TEER , trans - epithelial electrical resistanceD Representative images of immunostainings for ZO - 1 and OCLN in primary CPE cells following treatment for 6 h with 1 μM sodium propionate or 1 μM sodium butyrate , with 100 ng / ml LPS stimulation . Scale bar : 50 μm . E The percentage of cells with intact the TJ immunostainings in displayed ( D ) ( n = 3 , technical duplicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . CPE , choroid plexus epithelial LPS , lipopolysaccharides TJ , tight junction . G Representative images of immunostainings for ZO - 1 and OCLN in choroid plexus of sodium propionate or sodium butyrate - treated mice . Scale bar : 50 μm . H - J Quantification of continuous length ( H ) , maximal length ( I ) , and expressed area ( J ) of TJ immunostainings in ( G ) ( n = 5 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . TJ , tight junction . L Representative western blot showing the IgG levels in CSF of sodium propionate or sodium butyrate - treated mice with disrupted gut microbiota via antibiotics treatment . Data information : CSF , cerebrospinal

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 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 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 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

Figure 4B,C TEER (B) and assessment of the 70 kDa FITC-dextran paracellular permeability (C) of primary CPE cells treated as showed in (A) (n=3, technical duplicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CPE, choroid plexus epithelial; LPS, lipopolysaccharides; TEER, trans-epithelial electrical resistanceD Representative images of immunostainings for ZO-1 and OCLN in primary CPE cells following treatment for 6 h with 1 μM sodium propionate or 1 μM sodium butyrate, with 100 ng/ml LPS stimulation. Scale bar: 50 μm. E The percentage of cells with intact the TJ immunostainings in displayed (D) (n=3, technical duplicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CPE, choroid plexus epithelial LPS, lipopolysaccharides TJ, tight junction.G Representative images of immunostainings for ZO-1 and OCLN in choroid plexus of sodium propionate or sodium butyrate-treated mice. Scale bar: 50 μm. H-J Quantification of continuous length (H), maximal length (I), and expressed area (J) of TJ immunostainings in (G) (n=5, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. TJ, tight junction.L Representative western blot showing the IgG levels in CSF of sodium propionate or sodium butyrate-treated mice with disrupted gut microbiota via antibiotics treatment. Data information: CSF, cerebrospinal fluid


Figure 5A Representative images of immunostainings for OCLN , CDH1 and ZO - 1 in choroid plexus of sham and vagotomized mice . Scale bar : 50 μm . B - D Quantification of continuous length ( B ) , maximal length ( C ) , and expressed area ( D ) of TJ immunostainings in ( A ) ( n = 4 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . TJ , tight junction . H Representative images of immunostainings for OCLN , CDH1 and ZO - 1 in choroid plexus of sodium propionate or sodium butyrate - treated vagotomized mice . Scale bar : 50 μm . I - K Quantification of continuous length ( I ) , maximal length ( J ) , and expressed area ( K ) of the TJ immunostainings displayed in ( H ) ( n = 4 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . TJ , tight junction .

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 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

Figure 5A Representative images of immunostainings for OCLN, CDH1 and ZO-1 in choroid plexus of sham and vagotomized mice. Scale bar: 50 μm. B-D Quantification of continuous length (B), maximal length (C), and expressed area (D) of TJ immunostainings in (A) (n=4, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons *p < 0.05, **p < 0.01, ***p < 0.001. TJ, tight junction.H Representative images of immunostainings for OCLN, CDH1 and ZO-1 in choroid plexus of sodium propionate or sodium butyrate-treated vagotomized mice. Scale bar: 50 μm. I-K Quantification of continuous length (I), maximal length (J), and expressed area (K) of the TJ immunostainings displayed in (H) (n=4, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons *p < 0.05, **p < 0.01, ***p < 0.001. TJ, tight junction.


Figure 6B Representative images of immunostainings for OCLN , CDH1 , ZO - 1 and CLDN1 in choroid plexus of sodium propionate or sodium butyrate - treated AppNL - G - F mice . Scale bar : 50 μm . C - E Quantification of continuous length ( C ) , maximal length ( D ) , and expressed area ( E ) of theTJ immunostainings displayed in ( B ) ( n = 5 - 6 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . I Representative images of 6E10 staining in brain of sodium propionate or sodium butyrate - treated AppNL - G - F mice . Scale bar : 1 mm . J Quantification of Aβ plaque area and number in whole sagittal section of the brain ( n = 3 mice ; one section per mouse ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 6B Representative images of immunostainings for OCLN, CDH1, ZO-1 and CLDN1 in choroid plexus of sodium propionate or sodium butyrate-treated AppNL-G-F mice. Scale bar: 50 μm. C-E Quantification of continuous length (C), maximal length (D), and expressed area (E) of theTJ immunostainings displayed in (B) (n=5-6, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.I Representative images of 6E10 staining in brain of sodium propionate or sodium butyrate-treated AppNL-G-F mice. Scale bar: 1 mm. J Quantification of Aβ plaque area and number in whole sagittal section of the brain (n=3 mice; one section per mouse). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.


Figure 7A Representative images of immunostainings for IBA1 and 6E10 . Scale bar : 200 μm ( up ) and 20 μm ( down ) . D Quantification of summarized intersects , ramification index and ending radius by Sholl analysis from immunostained IBA1 signals ( n = 5 - 6 , biological replicates ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 . F Plaques were divided into small ( < 250 μm2 ) , medium ( 250 - 600 μm2 ) , and large ( > 600 μm2 ) , and the number of microglia per plaque was quantified ( n = 5 - 6 mice ; 3 - 5 plaques per mouse ) . Data information : Bars represent mean ± SEM . Statistics were performed with one - way ANOVA Bonferroni ' s post hoc test for multiple comparisons . * p < 0 . 05 , * * p < 0 . 01 .

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Figure 7A Representative images of immunostainings for IBA1 and 6E10. Scale bar: 200 μm (up) and 20 μm (down).D Quantification of summarized intersects, ramification index and ending radius by Sholl analysis from immunostained IBA1 signals (n=5-6, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons.*p < 0.05, **p < 0.01.F Plaques were divided into small (< 250 μm2), medium (250-600 μm2), and large (> 600 μm2), and the number of microglia per plaque was quantified (n=5-6 mice; 3-5 plaques per mouse). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons.*p < 0.05, **p < 0.01.


Figure 1 ( A ) Detection of Fstl1 protein in ischemic myocardium of WT mice . After MI induction , the heart was harvested at the indicated time points , and separated to ischemic and remote ( non - ischemic ) areas . Fstl1 protein was detected by western blotting ( n = 3 , each time point ) . Statistical analysis was performed by ordinary one way ANOVA . Post Hoc test was performed by Dunnett test . ( B ) Serum Fstl1 was detected by western blotting at the indicated time points . Ponceau S staining of serum reveals equivalent amount of loaded protein ( n = 4 , each time point ) . Error bars represent mean ± SEM . Statistical analysis was performed by ordinary one way ANOVA . Post Hoc test was performed by Dunnett test .

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Figure 1(A) Detection of Fstl1 protein in ischemic myocardium of WT mice. After MI induction, the heart was harvested at the indicated time points, and separated to ischemic and remote (non-ischemic) areas. Fstl1 protein was detected by western blotting (n=3, each time point). Statistical analysis was performed by ordinary one way ANOVA. Post Hoc test was performed by Dunnett test.(B) Serum Fstl1 was detected by western blotting at the indicated time points. Ponceau S staining of serum reveals equivalent amount of loaded protein (n=4, each time point). Error bars represent mean ± SEM. Statistical analysis was performed by ordinary one way ANOVA. Post Hoc test was performed by Dunnett test.


Figure 2 ( A ) DABimmunohistochemistry staining of Fstl1 with ischemic heart at day 3 after LAD ligation . Arrows indicate a subset of Fstl1 positive cells . Counter staining was performed with hematoxylin . Scale bar indicates 50μm . ( B ) Immunofluorescent microscopy staining of Fstl1 ( green ) and sarcomeric actin ( red ) . Fstl1 protein was detected in interstitial areas of the ischemic and border zone but not in cardiomyocytes . Scale bar indicates 100μm . ( C ) Immunofluorescent microscopy staining of Fstl1 ( green ) and S100a4 ( red ) in the ischemic area . Arrows indicate Fstl1 and S100a4 double positive cells . Scale bar indicates 20μm . ( D ) Immunofluorescent microscopy staining of Fstl1 ( green ) and α - smooth muscle actin ( red ) . Arrows indicate Fstl1 and α - SMA double positive cells . Scale bar indicates 20μm . Nuclei were stained by DAPI .

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Figure 2(A) DABimmunohistochemistry staining of Fstl1 with ischemic heart at day 3 after LAD ligation. Arrows indicate a subset of Fstl1 positive cells. Counter staining was performed with hematoxylin. Scale bar indicates 50μm.(B) Immunofluorescent microscopy staining of Fstl1 (green) and sarcomeric actin (red). Fstl1 protein was detected in interstitial areas of the ischemic and border zone but not in cardiomyocytes. Scale bar indicates 100μm.(C) Immunofluorescent microscopy staining of Fstl1 (green) and S100a4 (red) in the ischemic area. Arrows indicate Fstl1 and S100a4 double positive cells. Scale bar indicates 20μm.(D) Immunofluorescent microscopy staining of Fstl1 (green) and α-smooth muscle actin (red). Arrows indicate Fstl1 and α-SMA double positive cells. Scale bar indicates 20μm. Nuclei were stained by DAPI.


Figure 3Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( A ) S100a4 expression is induced in the ischemic area ( IA ) . qPCR analysis of mRNA expression of S100a4 in ischemic heart and sham operated heart of WT mice and cfKO mice . Heart samples were harvested at 7 days after the surgery . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . Error bars represent mean ± SEM ( n = 16 and 15 for WT and cfKO sham group , n = 15 and 14 for WT and cfKO MI group , respectively ) . Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( B ) qPCR analysis of mRNA expression of Fstl1 and Tgfβ1 in sham and post - MI heart . Error bars represent mean ± SEM ( n = 16 and 15 for WT and cfKO sham group , n = 15 and 14 for WT and cfKO MI group , respectively ) . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( C ) Western blot analysis of Fstl1 protein expression in ischemic and sham - operated hearts at day 7 after the surgery . Quantified values of Fstl1 protein in WT and cfKO mouse hearts normalized by GAPDH band intensity are shown . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . Error bars represent mean ± SEM ( n = 5 for each sham group and n = 6 for each MI group ) . Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( D ) Fstl1 protein expression in isolated cardiac fibroblasts from cfKO and littermate WT neonatal mice . Cell lysate and its media from cells cultured for 24 hours without FBS were assessed by western blotting . Error bars represent mean ± SEM ( n = 3 per group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . The experiments were performed twice independently .

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Figure 3Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (A) S100a4 expression is induced in the ischemic area (IA). qPCR analysis of mRNA expression of S100a4 in ischemic heart and sham operated heart of WT mice and cfKO mice. Heart samples were harvested at 7 days after the surgery. Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. Error bars represent mean ± SEM (n=16 and 15 for WT and cfKO sham group, n=15 and 14 for WT and cfKO MI group, respectively).Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (B) qPCR analysis of mRNA expression of Fstl1 and Tgfβ1 in sham and post-MI heart. Error bars represent mean ± SEM (n=16 and 15 for WT and cfKO sham group, n=15 and 14 for WT and cfKO MI group, respectively). Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test.Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (C) Western blot analysis of Fstl1 protein expression in ischemic and sham-operated hearts at day 7 after the surgery. Quantified values of Fstl1 protein in WT and cfKO mouse hearts normalized by GAPDH band intensity are shown. Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. Error bars represent mean ± SEM (n=5 for each sham group and n=6 for each MI group).Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (D) Fstl1 protein expression in isolated cardiac fibroblasts from cfKO and littermate WT neonatal mice. Cell lysate and its media from cells cultured for 24 hours without FBS were assessed by western blotting. Error bars represent mean ± SEM (n=3 per group). Statistical analysis was performed by unpaired t-test (2-tailed). The experiments were performed twice independently.


Figure 4 ( A ) Mouse survival curve after MI and sham surgery . The mortality of the WT and Fstl1 - cfKO mice after MI surgery was 27 . 1 % and 46 . 7 % , respectively . Log - rank ( Mantel - Cox ) test was used for statistical analysis ( n = 16 for WT sham , n = 15 for Fstl1 - cfKO sham , n = 59 for WT MI and n = 30 for Fstl1 - cfKO MI ) . ( B ) Representative macroscopic images of cardiac rupture . Left ; impending LV rupture found in a cfKO mouse post - MI when sampling . Right ; evidence of hemothorax that was diagnosed as cardiac rupture in deceased mice . ( C ) Representative microscopic images of ruptured heart stained by H & amp ; amp ; E . Penetrating hematoma due to a tear from the LV endocardium to the epicardium . Left ; an image obtained with a low magnification lens . Scale bar indicates 200μm . Right ; higher magnification image . Scale bar indicates 100μm . Arrow indicates the tear site . H indicates hematoma .

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Figure 4(A) Mouse survival curve after MI and sham surgery. The mortality of the WT and Fstl1-cfKO mice after MI surgery was 27.1% and 46.7%, respectively. Log-rank (Mantel-Cox) test was used for statistical analysis (n=16 for WT sham, n=15 for Fstl1-cfKO sham, n=59 for WT MI and n=30 for Fstl1-cfKO MI).(B) Representative macroscopic images of cardiac rupture. Left; impending LV rupture found in a cfKO mouse post-MI when sampling. Right; evidence of hemothorax that was diagnosed as cardiac rupture in deceased mice.(C) Representative microscopic images of ruptured heart stained by H&amp;E. Penetrating hematoma due to a tear from the LV endocardium to the epicardium. Left; an image obtained with a low magnification lens. Scale bar indicates 200μm. Right; higher magnification image. Scale bar indicates 100μm. Arrow indicates the tear site. H indicates hematoma.


Figure 5 ( A ) Transcript mRNA expression of Col1a1 and Fn1 in sham and post - MI hearts . Hearts were obtained at day 7 after surgery . Error bars represent mean ± SEM ( n = 13 - 16 for each group ) . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . ( B ) Protein expression of collagen I , fibronectin and αSMA in the infarcted and sham hearts at day 7 after surgery , as assessed by western blotting . Quantified values of proteins of interest were normalized relative to the tubulin band intensity . Error bars represent mean ± SEM . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) for fibronectin and αSMA and non - parametric t - test ( 2 - tailed ) for collagen I ( fibronectin and collagen I : n = 5 per group , αSMA : n = 3 and 4 for WT - IA and cfKO - IA , respectively ) . ( C ) α - smooth muscle actin - positive myofibroblasts ( red ) were stained in histology sections from infarcted hearts of WT and cfKO mice . Samples were harvested at day 7 after MI . Counter staining was performed by hematoxylin . Scale bar indicates 100μm for top and middle images , 50µm for bottom images . Error bars represent mean ± SEM . Statistical analysis was performed by Mann - Whitney test ( n = 16 for WT and n = 14 for cfKO ) . ( D ) Representative images of Picro Sirius Red staining of infarcted heart at 7 days after MI surgery . Images were taken by standard light ( top ) and polarized light ( bottom ) microscopes . Scale bar indicates 100μm . Polarized collagen fibers were sorted to orange - red ( middle ) and green - yellow ( bottom ) fibers by Image J software . Error bars represent mean ± SEM . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) ( n = 11 for WT and n = 13 for cfKO ) .

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Figure 5(A) Transcript mRNA expression of Col1a1 and Fn1 in sham and post-MI hearts. Hearts were obtained at day 7 after surgery. Error bars represent mean ± SEM (n=13-16 for each group). Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test.(B) Protein expression of collagen I, fibronectin and αSMA in the infarcted and sham hearts at day 7 after surgery, as assessed by western blotting. Quantified values of proteins of interest were normalized relative to the tubulin band intensity. Error bars represent mean ± SEM. Statistical analysis was performed by unpaired t-test (2-tailed) for fibronectin and αSMA and non-parametric t-test (2-tailed) for collagen I (fibronectin and collagen I: n=5 per group, αSMA: n=3 and 4 for WT-IA and cfKO-IA, respectively).(C) α-smooth muscle actin-positive myofibroblasts (red) were stained in histology sections from infarcted hearts of WT and cfKO mice. Samples were harvested at day 7 after MI. Counter staining was performed by hematoxylin. Scale bar indicates 100μm for top and middle images, 50µm for bottom images. Error bars represent mean ± SEM. Statistical analysis was performed by Mann-Whitney test (n=16 for WT and n=14 for cfKO). (D) Representative images of Picro Sirius Red staining of infarcted heart at 7 days after MI surgery. Images were taken by standard light (top) and polarized light (bottom) microscopes. Scale bar indicates 100μm. Polarized collagen fibers were sorted to orange-red (middle) and green-yellow (bottom) fibers by Image J software. Error bars represent mean ± SEM. Statistical analysis was performed by unpaired t-test (2-tailed) (n=11 for WT and n=13 for cfKO).


Figure 6 ( A ) Immunofluorescence microscopy staining of Fst1 ( green ) and α - SMA ( red ) in NRCFbs at 24 hours after recombinant TGF - β1 ( 10ng / ml ) stimulation . Nuclei were stained by DAPI . Scale bar indicates 50μm . ( B ) Time course changes of Fstl1 protein expression in NRCFbs after stimulation of recombinant TGF - β1 ( 10ng / ml ) . Protein expression of Fstl1 was detected by immunoblotting . Tubulin was used as a loading control ( n = 3 for each time point ) . Two independent experiments were performed . ( C ) Transcript level of Fstl1 , S100a4 and Acta2 mRNA in NRCFbs was determined by qPCR analysis . The samples were harvested at 24 hours after stimulation with recombinant TGF - β1 ( 10ng / ml ) or control vehicle . Error bars represent mean ± SEM ( Fstl1 and S100a4 : n = 6 for each group , Acta2 : n = 3 for each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) for Fstl1 and S100a4 , and non - parametric unpaired t - test ( 2 - tailed ) for Acta2 . Two independent experiments were performed .

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Figure 6(A) Immunofluorescence microscopy staining of Fst1 (green) and α-SMA (red) in NRCFbs at 24 hours after recombinant TGF-β1 (10ng/ml) stimulation. Nuclei were stained by DAPI. Scale bar indicates 50μm.(B) Time course changes of Fstl1 protein expression in NRCFbs after stimulation of recombinant TGF-β1 (10ng/ml). Protein expression of Fstl1 was detected by immunoblotting. Tubulin was used as a loading control (n=3 for each time point). Two independent experiments were performed.(C) Transcript level of Fstl1, S100a4 and Acta2 mRNA in NRCFbs was determined by qPCR analysis. The samples were harvested at 24 hours after stimulation with recombinant TGF-β1 (10ng/ml) or control vehicle. Error bars represent mean ± SEM (Fstl1 and S100a4: n=6 for each group, Acta2: n=3 for each group). Statistical analysis was performed by unpaired t-test (2-tailed) for Fstl1 and S100a4, and non-parametric unpaired t-test (2-tailed) for Acta2. Two independent experiments were performed.


Figure 7 ( A ) NRCFbs at passage 1 were stimulated with recombinant Fstl1 ( 50ng / ml ) or vehicle after cultured in serum - reduced conditions ( FBS 0 . 5 % ) for 24 hours . The samples were harvested at the indicated time points after stimulation . The expression of ERK1 / 2 and tubulin were detected by immunoblotting . Error bars represent mean ± SEM . Statistical analysis was performed by one - way ANOVA and Tukey multi comparison test ( n = 3 for each time point ) . Three independent experiments were performed . ( B ) Fstl1 induced phosphorylation of ERK1 / 2 was ablated by pretreatment with MEK inhibitor PD98059 . NRCFbs were treated by PD98059 ( 5nM ) for 30min and then stimulated with recombinant Fstl1 protein ( 50ng / ml ) for 15min . Error bars represent mean ± SEM . Statistical analysis was performed by one - way ANOVA and Tukey multi comparison test ( n = 3 for each time point ) . Two independent experiments were performed . ( C ) Representative images of NRCFbs scratch assay ( left ) and quantified cell migration ( right ) . NRCFbs were transfected by Fstl1 siRNA or siRNA non - targeting negative control for 12 hours followed by culturing in 0 . 5 % FBS media for 24 hours . The confluent cell sheet was scratched and cell migration was assessed at 6 hours after the scratch . Scale bar indicates 100µm . Error bars represent mean ± SEM ( n = 10 for each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . Two independent experiments were performed . ( D ) Fstl1 - stimulation of cell migration was reversed by PD98059 . Endogenous Fstl1 in NRCFbs was ablated by Fstl1 siRNA . Serum - deprived NRCFbs were treated with PD98059 ( 5nM ) for 30 min and then stimulated by Fstl1 ( 50ng / ml ) or vehicle . Cell migration was assessed at 6 hours after Fstl1 stimulation . Error bars represent mean ± SEM ( n = 7 - 9 for each group ) . Statistical analysis was performed by Kruskal - Wallis test and Dunnett ' s T3 test . Two independent experiments were performed . ( E ) Morphological changes of NRCFbs after recombinant Fstl1stimulation was assessed by immunocytochemistry . 24 hours serum starved NRCFbs was stimulated by Fstl1 ( 50ng / ml ) or vehicle for 3 hours . PD98059 ( 5nM ) or DMSO was added 30min prior to Fstl1 stimulation . Cells were stained with Alexa Fluor 488 - conjugated phalloidin antibody and nuclei were stained by DAPI . Arrow shows lamellipodium of the cell . Scale bar indicates 50μm .

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 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 O O O O O 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

Figure 7(A) NRCFbs at passage 1 were stimulated with recombinant Fstl1 (50ng/ml) or vehicle after cultured in serum-reduced conditions (FBS 0.5%) for 24 hours. The samples were harvested at the indicated time points after stimulation. The expression of ERK1/2 and tubulin were detected by immunoblotting. Error bars represent mean ± SEM. Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test (n=3 for each time point). Three independent experiments were performed.(B) Fstl1 induced phosphorylation of ERK1/2 was ablated by pretreatment with MEK inhibitor PD98059. NRCFbs were treated by PD98059 (5nM) for 30min and then stimulated with recombinant Fstl1 protein (50ng/ml) for 15min. Error bars represent mean ± SEM. Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test (n=3 for each time point). Two independent experiments were performed.(C) Representative images of NRCFbs scratch assay (left) and quantified cell migration (right). NRCFbs were transfected by Fstl1 siRNA or siRNA non-targeting negative control for 12 hours followed by culturing in 0.5% FBS media for 24 hours. The confluent cell sheet was scratched and cell migration was assessed at 6 hours after the scratch. Scale bar indicates 100µm. Error bars represent mean ± SEM (n=10 for each group). Statistical analysis was performed by unpaired t-test (2-tailed). Two independent experiments were performed.(D) Fstl1-stimulation of cell migration was reversed by PD98059. Endogenous Fstl1 in NRCFbs was ablated by Fstl1 siRNA. Serum-deprived NRCFbs were treated with PD98059 (5nM) for 30 min and then stimulated by Fstl1 (50ng/ml) or vehicle. Cell migration was assessed at 6 hours after Fstl1 stimulation. Error bars represent mean ± SEM (n=7-9 for each group). Statistical analysis was performed by Kruskal-Wallis test and Dunnett's T3 test. Two independent experiments were performed.(E) Morphological changes of NRCFbs after recombinant Fstl1stimulation was assessed by immunocytochemistry. 24 hours serum starved NRCFbs was stimulated by Fstl1 (50ng/ml) or vehicle for 3 hours. PD98059 (5nM) or DMSO was added 30min prior to Fstl1 stimulation. Cells were stained with Alexa Fluor 488-conjugated phalloidin antibody and nuclei were stained by DAPI. Arrow shows lamellipodium of the cell. Scale bar indicates 50μm.


Figure 8 ( A , B ) Fibroblast proliferation was assessed by Edu incorporation assay . Endogenous Fstl1 was ablated by siRNA . NRCFbs were cultured in 0 . 5 % FBS condition for 48 hours to synchronize the cell cycle . EdU ( 10μM as the final concentration ) was added into media at 4 hours before harvest . Error bars represent mean ± SEM ( n = 5 , each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . Two independent experiments were performed . ( B ) Effect of exogenous Fstl1 on cardiac fibroblast proliferation . Cells were cultured in FBS 0 . 5 % media for 24 hours . Recombinant Fstl1 ( 50ng / ml ) or vehicle were added to 2 % FBS - containing media and cultured for 48 hours . EdU was added into media at 4 hours before harvest . Error bars represent mean ± SEM ( n = 7 , each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . Two independent experiments were performed . ( C ) Fstl1 promoted fibroblasts proliferation was diminished by PD98059 . PD98059 ( 5nM ) was added 30 min prior to recombinant Fstl1 stimulation ( 50ng / ml ) . The cells were cultured for 48 hours , and EdU was added into media at 4 hours before harvest . Error bars represent mean ± SEM ( n = 4 - 10 , per treatment group ) . Statistical analysis was performed by one - way ANOVA and Tukey multi comparison test . Two independent experiments were performed .

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 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 8(A, B) Fibroblast proliferation was assessed by Edu incorporation assay. Endogenous Fstl1 was ablated by siRNA. NRCFbs were cultured in 0.5% FBS condition for 48 hours to synchronize the cell cycle. EdU (10μM as the final concentration) was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=5, each group). Statistical analysis was performed by unpaired t-test (2-tailed). Two independent experiments were performed. (B) Effect of exogenous Fstl1 on cardiac fibroblast proliferation. Cells were cultured in FBS 0.5% media for 24 hours. Recombinant Fstl1 (50ng/ml) or vehicle were added to 2% FBS-containing media and cultured for 48 hours. EdU was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=7, each group). Statistical analysis was performed by unpaired t-test (2-tailed). Two independent experiments were performed.(C) Fstl1 promoted fibroblasts proliferation was diminished by PD98059. PD98059 (5nM) was added 30 min prior to recombinant Fstl1 stimulation (50ng/ml). The cells were cultured for 48 hours, and EdU was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=4-10, per treatment group). Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test. Two independent experiments were performed.


Figure 1 ( D ) Immunocytochemistry of β - catenin in primary WT neurons treated with anti - Shank3 antibody or IgG . Notice the nuclear accumulation of β - catenin upon anti - Shank3 antibody treatment . Arrows point to β - catenin - negative nucleus . Arrowheads point to β - catenin - positive nucleus . : Bar = 50 μm in ( D ) . mice ( F ) Western - blotting of β - catenin , Axin2 and p - GSK3β ( S9 ) in WT or Shank3 - / - primary neurons . mice and 5 batches of cells ( F ) per group . Mean ratio ± SEM . Two - Tailed unpaired T - test . * P < 0 . 05 . * * P < 0 . 01 . * * * P < 0 . 001 . WT , wild type . KO , Shank3 - / - mice . ( G ) Western - blotting of SFRP1 and p - β - catenin ( S33 ) in the total proteins , and TCF7L1 in the nuclear protein of WT or Shank3 - / - ACC . 3 in mice Mean ratio ± SEM . Two - Tailed unpaired T - test . * P < 0 . 05 . * * P < 0 . 01 . * * * P < 0 . 001 . WT , wild type . KO , Shank3 - / - mice . ( H ) Western - blotting of Axin2 and p - GSK3β ( S9 ) in the total proteins , and β - catenin ( S33 ) in the nuclear protein of ACC of WT or VPA - treated mice . 3 in H ) mice Mean ratio ± SEM . Two - Tailed unpaired T - test . * P < 0 . 05 . * * P < 0 . 01 . * * * P < 0 . 001 . Con , control .

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 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 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

Figure 1(D) Immunocytochemistry of β-catenin in primary WT neurons treated with anti-Shank3 antibody or IgG. Notice the nuclear accumulation of β-catenin upon anti-Shank3 antibody treatment. Arrows point to β-catenin-negative nucleus. Arrowheads point to β-catenin-positive nucleus. : Bar = 50 μm in (D). mice(F) Western-blotting of β-catenin, Axin2 and p-GSK3β(S9) in WT or Shank3-/- primary neurons. mice and 5 batches of cells (F) per group. Mean ratio ± SEM. Two-Tailed unpaired T-test. *P<0.05. **P<0.01. ***P<0.001. WT, wild type. KO, Shank3-/- mice.(G) Western-blotting of SFRP1 and p-β-catenin(S33) in the total proteins, and TCF7L1 in the nuclear protein of WT or Shank3-/- ACC. 3 in mice Mean ratio ± SEM. Two-Tailed unpaired T-test. *P<0.05. **P<0.01. ***P<0.001. WT, wild type. KO, Shank3-/- mice.(H) Western-blotting of Axin2 and p-GSK3β(S9) in the total proteins, and β-catenin(S33) in the nuclear protein of ACC of WT or VPA-treated mice. 3 in H) mice Mean ratio ± SEM. Two-Tailed unpaired T-test. *P<0.05. **P<0.01. ***P<0.001. Con, control.


Figure 2 ( D ) Western - blotting of ADH1 , ALDH3 and ENO1 in the ACC of WT and Shank3 - / - mice . Notice the up - regulation of ADH1 , ALDH3 and ENO1 in Shank3 - / - ACC . 3 - 4 mice in ( D ) P < 0 . 05 . * * P < 0 . 01 . * * * P < 0 . 001 . WT , wild type . KO , Shank3 - / - . ( F ) ECAR assay of primary WT and Shank3 - / - neurons . Notice the higher ECAR values of Shank3 - / - neurons upon glucose and oligomycin stimulation . 5 batches of cells * P < 0 . 05 . * * P < 0 . 01 . * * * P < 0 . 001 . WT , wild type . KO , Shank3 - / ( G ) LC - MS measurement of lactic acid and pyruvic acid in the ACC of control and VPA - treated mice . Lactic acid is increased in VPA - treated mice . Two - Tailed unpaired T - test . Welch ' s t - test Pyruvic acid analysis in G * P < 0 . 05 . * * P < 0 . 01 . * * * P < 0 . 001 . Con , control .

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 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

Figure 2(D) Western-blotting of ADH1, ALDH3 and ENO1 in the ACC of WT and Shank3-/- mice. Notice the up-regulation of ADH1, ALDH3 and ENO1 in Shank3-/- ACC. 3-4 mice in (D) P<0.05. **P<0.01. ***P<0.001. WT, wild type. KO, Shank3-/-.(F) ECAR assay of primary WT and Shank3-/- neurons. Notice the higher ECAR values of Shank3-/- neurons upon glucose and oligomycin stimulation. 5 batches of cells *P<0.05. **P<0.01. ***P<0.001. WT, wild type. KO, Shank3-/(G) LC-MS measurement of lactic acid and pyruvic acid in the ACC of control and VPA-treated mice. Lactic acid is increased in VPA-treated mice. Two-Tailed unpaired T-test. Welch's t-test Pyruvic acid analysis in G *P<0.05. **P<0.01. ***P<0.001. Con, control.


Figure 3 ( C , D ) Spine density and spine subtypes in the ACC of control and β - catenin over - expressing mice . Notice the reduction of total spine density , stubby and mushroom - like spines in β - catenin over - expressing mice . 20 μm in ( C Two - Tailed unpaired T - test ( Spine analysis excepting mushoom analysis in D . Mann - Whitney U test ( Mushroom analysis in D . Patch - clamp recording of mEPSC in the ACC of control and β - catenin over - expressing mice . Both the frequency and average amplitudes of mEPSC were reduced in Wnt over - activated ACC . , 9 neurons from 3 mice in H ) Social novelty and social preference scores in , H Amplitude of mEPSC in H . ( I ) 3 - chamber assay of control and β - catenin over - expressing mice . 9 - 10 mice ( I - K ) Social novelty and social preference scores in I , Two - tailed paired T - Test ( Time in compartment in I ) . ECAR assay of primary cultured control neurons ( GFP + EX3 ) and β - catenin over - expressing neurons ( CaMKII - Cre + EX3 ) . 5 batches of cells ECAR in Max ECAR in M .

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 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 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

Figure 3(C, D) Spine density and spine subtypes in the ACC of control and β-catenin over-expressing mice. Notice the reduction of total spine density, stubby and mushroom-like spines in β-catenin over-expressing mice. 20 μm in (C Two-Tailed unpaired T-test (Spine analysis excepting mushoom analysis in D. Mann-Whitney U test (Mushroom analysis in D.Patch-clamp recording of mEPSC in the ACC of control and β-catenin over-expressing mice. Both the frequency and average amplitudes of mEPSC were reduced in Wnt over-activated ACC. , 9 neurons from 3 mice in H) Social novelty and social preference scores in , H Amplitude of mEPSC in H.(I) 3-chamber assay of control and β-catenin over-expressing mice. 9-10 mice (I-K) Social novelty and social preference scores in I, Two-tailed paired T-Test (Time in compartment in I).ECAR assay of primary cultured control neurons (GFP+EX3) and β-catenin over-expressing neurons (CaMKII-Cre+EX3). 5 batches of cells ECAR in Max ECAR in M.


Figure 4 ( A ) Typical images of Golgi staining and reconstructed neurons in the ACC of Shank3 - / - mice treated with 2 - DG ( KO + 2 - DG ) or vehicle ( KO + Veh ) . Sholl analysis of pyramidal neurons in the ACC of Shank3 - / - mice treated with 2 - DG or vehicle . Bar = 50 μm in ( A ) N = 24 - 28 neurons from 4 mice in KO , Shank3 - / - . Veh , vehicle . ( G ) Western - blotting of GluR1 , PSD95 and Homer1 in the total proteins of Shank3 - / - ACC with or without 2 - DG treatment . 3 mice in ( G ) KO , Shank3 - / - . Veh , vehicle . resident - intruder assay of Shank3 - / - mice treated with 2 - DG or vehicle . Notice the improvement of social preference and social interaction by 2 - DG treatment . 10 - 12 mice in KO , Shank3 - / - . Veh , vehicle .

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 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

Figure 4(A) Typical images of Golgi staining and reconstructed neurons in the ACC of Shank3-/- mice treated with 2-DG (KO+2-DG) or vehicle (KO+Veh). Sholl analysis of pyramidal neurons in the ACC of Shank3-/- mice treated with 2-DG or vehicle. Bar = 50 μm in (A) N = 24-28 neurons from 4 mice in KO, Shank3-/-. Veh, vehicle.(G) Western-blotting of GluR1, PSD95 and Homer1 in the total proteins of Shank3-/- ACC with or without 2-DG treatment. 3 mice in (G) KO, Shank3-/-. Veh, vehicle.resident-intruder assay of Shank3-/- mice treated with 2-DG or vehicle. Notice the improvement of social preference and social interaction by 2-DG treatment. 10-12 mice in KO, Shank3-/-. Veh, vehicle.


Figure 5 ( A ) ECAR assay of primary Shank3 - / - neurons treated with or without XAV939 . Notice the lower levels of glycolysis in XAV939 treated cells . ( B ) ECAR assay of primary Shank3 - / - neurons treated with or without ICG - 001 . N = 4 - 5 batches of cells in Two - Tailed unpaired t - test * P < 0 . 05 . * * P < 0 . 01 . KO , Shank3 - / - . ( E ) Double - immunostaining of ENO1 / Axin2 in WT neurons , Shank3 - / - neurons , and XAV939 treated Shank3 - / - neurons . Notice the colocalization of Axin2 / ENO1 - immunoreactivity in Shank3 - / - neurons and the membrane localization of Axin2 - immunoreactivity in XAV939 treated Shank3 - / - neurons . Arrows point to membrane Axin2 - immunoreactivity . Bar = 5 μm in E . WT , wild type . KO , Shank3 - / - . ( G ) Protein CO - IP of Axin2 / ENO1 in WT and Shank3 - / - neurons treated with or without XAV939 . Notice the strong interaction of Axin2 / ENO1 in Shank3 - / - neurons and diminished interaction upon XAV939 treatment . per group and 3 samples from 9 mice per group in One - way ANOVA with Tukey ' s multiple - comparison test WT , wild type . KO , Shank3 - / - .

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 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 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

Figure 5(A) ECAR assay of primary Shank3-/- neurons treated with or without XAV939. Notice the lower levels of glycolysis in XAV939 treated cells. (B) ECAR assay of primary Shank3-/- neurons treated with or without ICG-001. N = 4-5 batches of cells in Two-Tailed unpaired t-test *P<0.05. **P<0.01. KO, Shank3-/-.(E) Double-immunostaining of ENO1/Axin2 in WT neurons, Shank3-/- neurons, and XAV939 treated Shank3-/- neurons. Notice the colocalization of Axin2/ENO1-immunoreactivity in Shank3-/- neurons and the membrane localization of Axin2-immunoreactivity in XAV939 treated Shank3-/- neurons. Arrows point to membrane Axin2-immunoreactivity. Bar = 5 μm in E. WT, wild type. KO, Shank3-/-.(G) Protein CO-IP of Axin2/ENO1 in WT and Shank3-/- neurons treated with or without XAV939. Notice the strong interaction of Axin2/ENO1 in Shank3-/- neurons and diminished interaction upon XAV939 treatment. per group and 3 samples from 9 mice per group in One-way ANOVA with Tukey's multiple-comparison test WT, wild type. KO, Shank3-/-.


Figure 6Spine length , spine density and spine subtypes of Shank3 - / - ACC neurons treated with or without XAV939 . Notice the increase of total spine density , stubby and mushroom - like spines and the decrease of average spine length in XAV939 treated Shank3 - / - mice . 32 spines from 3 mice in Filopodia analysis in F . Spines analysis excepting filopodia analysis in F . KO , Shank3 - / - . ( H ) Western - blotting of p - PSD95 and Homer1 in WT and Shank3 - / - ACC treated with or without XAV939 . 4 - 6 mice in One - way ANOVA with Tukey ' s multiple - comparison test WT , wild type . KO , Shank3 - / - . ( I , J ) Western - blotting of GluR1 in the total and membrane protein of WT and Shank3 - / - neurons treated with or without XAV939 . Notice the rescuing effects of XAV939 on the expression of GluR1 in Shank3 - / - neurons . 4 - 6 mice in and 3 samples from 12 mice in ( J ) per group . One - way ANOVA with Tukey ' s multiple - comparison test WT , wild type . KO , Shank3 - / - .

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 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 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 6Spine length, spine density and spine subtypes of Shank3-/- ACC neurons treated with or without XAV939. Notice the increase of total spine density, stubby and mushroom-like spines and the decrease of average spine length in XAV939 treated Shank3-/- mice. 32 spines from 3 mice in Filopodia analysis in F. Spines analysis excepting filopodia analysis in F. KO, Shank3-/-.(H) Western-blotting of p-PSD95 and Homer1 in WT and Shank3-/- ACC treated with or without XAV939. 4-6 mice in One-way ANOVA with Tukey's multiple-comparison test WT, wild type. KO, Shank3-/-.(I, J) Western-blotting of GluR1 in the total and membrane protein of WT and Shank3-/- neurons treated with or without XAV939. Notice the rescuing effects of XAV939 on the expression of GluR1 in Shank3-/- neurons. 4-6 mice in and 3 samples from 12 mice in (J) per group. One-way ANOVA with Tukey's multiple-comparison test WT, wild type. KO, Shank3-/-.


Figure 7Calcium response of WT and Shank3 - / - neurons treated with or without XAV939 . Notice that XAV939 treatment significantly restored the amplitude , rising and decay time of calcium response in Shank3 - / - neurons . Bar = 15 μm in ( A ) and 30 μm in magnified images of ( A ) . N = 39 neurons from 3 batches of cells in Kruskal - Wallis H test with Dunn ' s multiple - comparison test KO , Shank3 - / - . Veh , vehicle . Calcium response of WT and Shank3 - / - neurons treated with or without XAV939 . Notice that XAV939 treatment significantly restored the amplitude , rising and decay time of calcium response in Shank3 - / - neurons . N = 39 neurons from 3 batches of cells in Dunn ' s multiple - comparison test KO , Shank3 - / - . Veh , vehicle . ( D ) Patch - clamp recording of mEPSCs in Shank3 - / - ACC treated with or without XAV939 . Both the frequency and amplitude of Shank3 - / - neurons were increased by XAV939 . , 7 - 8 neurons from 3 mice in ( D ) KO , Shank3 - / - . Veh , vehicle . resident - intruder assays of Shank3 - / - mice treated with or without XAV939 . Notice the improvement of social preference , social novelty and social interaction of Shank3 - / - mice by XAV939 treatment . 8 - 10 mice KO , Shank3 - / - . Veh , vehicle .

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 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

Figure 7Calcium response of WT and Shank3-/- neurons treated with or without XAV939. Notice that XAV939 treatment significantly restored the amplitude, rising and decay time of calcium response in Shank3-/- neurons. Bar = 15 μm in (A) and 30 μm in magnified images of (A). N = 39 neurons from 3 batches of cells in Kruskal-Wallis H test with Dunn's multiple-comparison test KO, Shank3-/-. Veh, vehicle.Calcium response of WT and Shank3-/- neurons treated with or without XAV939. Notice that XAV939 treatment significantly restored the amplitude, rising and decay time of calcium response in Shank3-/- neurons. N = 39 neurons from 3 batches of cells in Dunn's multiple-comparison test KO, Shank3-/-. Veh, vehicle.(D) Patch-clamp recording of mEPSCs in Shank3-/- ACC treated with or without XAV939. Both the frequency and amplitude of Shank3-/- neurons were increased by XAV939. , 7-8 neurons from 3 mice in (D) KO, Shank3-/-. Veh, vehicle.resident-intruder assays of Shank3-/- mice treated with or without XAV939. Notice the improvement of social preference, social novelty and social interaction of Shank3-/- mice by XAV939 treatment. 8-10 mice KO, Shank3-/-. Veh, vehicle.


Figure 8 ( B ) Western - blotting of β - catenin , p - β - catenin ( S33 ) , GSK - 3β , p - GSK - 3β ( S9 ) and TCF7L1 in neurons derived from WT or Shank3 mutant human ESCs . Notice the similar change of Wnt signaling components as in Shank3 - / - mice . N = 3 - 4 batches of cells in Mean ratio ± SEM . Two - tailed unpaired t - test ( Wnt components expression excepting TCF7L1 and GSK3β in Mann - Whitney U test ( TCF7L1 expression in B ) . Welch ' s t - test ( GSK3β expression in B ) . WT , wild type . KO , Shank3 - ( C ) Protein CO - IP assay of Axin2 / ENO1 in WT and Shank3 mutant human NPCs . ( D ) Protein CO - IP assay of Axin2 / ENO1 in WT and Shank3 mutant human neurons . Notice the strong interaction of Axin2 / ENO1 Shank3 mutant human neurons . N = 3 - 4 batches of cells in WT , wild type . KO , Shank3 - / NPC , neural progenitor cell . ( H ) ECAR assay of naive human neurons , VPA - pretreated human neurons with or without XAV939 treatment . Notice the higher levels of glycolysis in VPA - pretreated human neurons and the inhibitory effects of XAV939 . , 4 - 5 batches of cells in per group .

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Figure 8(B) Western-blotting of β-catenin, p-β-catenin(S33), GSK-3β, p-GSK-3β(S9) and TCF7L1 in neurons derived from WT or Shank3 mutant human ESCs. Notice the similar change of Wnt signaling components as in Shank3-/- mice. N = 3-4 batches of cells in Mean ratio ± SEM. Two-tailed unpaired t-test (Wnt components expression excepting TCF7L1 and GSK3β in Mann-Whitney U test (TCF7L1 expression in B). Welch's t-test (GSK3β expression in B). WT, wild type. KO, Shank3-(C) Protein CO-IP assay of Axin2/ENO1 in WT and Shank3 mutant human NPCs. (D) Protein CO-IP assay of Axin2/ENO1 in WT and Shank3 mutant human neurons. Notice the strong interaction of Axin2/ENO1 Shank3 mutant human neurons. N = 3-4 batches of cells in WT, wild type. KO, Shank3-/ NPC, neural progenitor cell.  (H) ECAR assay of naive human neurons, VPA-pretreated human neurons with or without XAV939 treatment. Notice the higher levels of glycolysis in VPA-pretreated human neurons and the inhibitory effects of XAV939. , 4-5 batches of cells in per group.


Figure 1The syntheses of the ligands PR - 924 , 9 , 14 , 16 , 17 and 18 have been described previously ( de Bruin et al , 2014 ) . All ligands share the peptide scaffold shown in the upper left corner with P1 , P2 and P3 side chains and an N - cap ; they are equipped with a l - Ala ( stereochemistry ( S ) ) or d - Ala ( stereochemistry ( R ) ) residue at P3 ( red ) and either an N - acylmorpholine or a 3 - methyl - 1H - indene group ( green ) . Furthermore , compounds differ in their P2 residue ( either methoxytyrosine or tryptophane ; blue ) . Inhibitor 9 , also termed LU - 015i , is endowed with an exceptional cyclohexyl residue ( gray ) , which is associated with increased selectivity for human β5i .

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Figure 1The syntheses of the ligands PR-924, 9, 14, 16, 17 and 18 have been described previously (de Bruin et al, 2014). All ligands share the peptide scaffold shown in the upper left corner with P1, P2 and P3 side chains and an N-cap; they are equipped with a l-Ala (stereochemistry (S)) or d-Ala (stereochemistry (R)) residue at P3 (red) and either an N-acylmorpholine or a 3-methyl-1H-indene group (green). Furthermore, compounds differ in their P2 residue (either methoxytyrosine or tryptophane; blue). Inhibitor 9, also termed LU-015i, is endowed with an exceptional cyclohexyl residue (gray), which is associated with increased selectivity for human β5i.


Figure 2A . Inhibition of human β5c and β5i by ONX 0914 and PR - 924 . IC50 values are deduced from the fitted data . B . IC50 values [ µM ] for all compounds towards the yeast , human and mouse β5 subunits .

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Figure 2A. Inhibition of human β5c and β5i by ONX 0914 and PR-924. IC50 values are deduced from the fitted data.B. IC50 values [µM] for all compounds towards the yeast, human and mouse β5 subunits.


Figure 3E . Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 ° C or 37 ° C . Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity . F . Surface illustration of the yCP . The β5 and β6 subunits are depicted in dark gray , with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue , respectively . The β5 ' and β6 ' subunits of the second β - ring are hidden on the back of 20S proteasome .

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Figure 3E. Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 °C or 37 °C. Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity.F. Surface illustration of the yCP. The β5 and β6 subunits are depicted in dark gray, with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue, respectively. The β5' and β6' subunits of the second β-ring are hidden on the back of 20S proteasome.