Figure 5 A APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization . The expression levels were tested at different days after transduction . The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line . Data are presented as mean ± SEM ( n = 3 ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : * * P = 0 . 0030 ( S6 naïve vs S6 APOPT1HA ) , * P = 0 . 0165 ( S6 EV vs S6 APOPT1HA ) B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization . The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line . Data are presented as mean ± SEM ( n = 4 ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : * * P = 0 . 0066 ( S6 naïve vs S6 APOPT1GFP ) , * * * P = 0 . 0006 ( S6 EV vs S6 APOPT1GFP ) , * P = 0 . 0169 ( S2 naïve vs S2 APOPT1GFP ) , * * * P = 0 . 0008 ( S2 EV vs S2 APOPT1GFP ) C Western blot analysis of 1D - BNGE of mitoplasts extracted from the indicated cell lines . Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT - CO1 and MT - CO2 . The presence of the assembly intermediates ' MITRAC ' and ' S3 ' was also detected ( arrows ) D Western blot analysis of 2D - BNGE of mitoplasts extracted from the indicated cell lines . Fully assembled COX was immunovisualized with antibodies recognizing MT - CO1 and MT - CO2 . The presence of the assembly intermediates ' MITRAC ' and ' S3 ' was also detected ( arrows ) . Anti - GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein E L - [ 35S ] - Methionine pulse - chase labeling of mtDNA - encoded proteins . After a two - hour exposure with the radioactive label ( pulse ) , cells were cultured in cold medium for the indicated chase times . The graphs show the densitometric quantification of the bands corresponding to MT - CO1 ( left graph ) and MT - CO2 + MT - CO3 ( right graph ) normalized to the ATP6 band over the indicated time points . Graphs represent the values of three biological replicas for each cell line . Data are presented as mean ± SEM ( n = 4 for controls , n = 2 for S2 / S6 / S2 APOPT1GFP / S6 APOPT1GFP ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : MT - CO1 - * * P = 0 . 0039 ( 3 . 5 hours , controls vs S6 ) , * P = 0 . 0202 ( 3 . 5 hours , controls vs S2 ) , * P = 0 . 0163 ( 5 hours , S6vs S6 APOPT1GFP ) , * P = 0 . 0118 ( 5 hours , controls vs S2 ) , * * P = 0 . 0011 ( 20 hours , controls vs S6 ) , * P = 0 . 0343 ( 20 hours , S2 vs S2 APOPT1GFP ) , * * * P = 0 . 0007 ( 20 hours , S6 vs S6 APOPT1GFP ) , * * * P = 0 . 0002 ( 20 hours , controls vs S2 ) , * * * * * P < 0 . 0001 ( 20 hours , controls vs S6 ) , MT - CO2 / MT - CO3 - * P = 0 . 0194 ( 3 . 5 hours , S6 vs S6 APOPTGFP ) , * * * P = 0 . 0003 ( 3 . 5 hours , controls vs S6 ) , * P = 0 . 0375 ( 5 hours , S6 vs S6 APOPT1GFP ) , * P = 0 . 0234 ( 20 hours , S2 vs S2 APOPT1GFP ) , * * * * P < 0 . 0001 ( 20 hours , S6 vs S6 APOPT1GFP ) , * * P = 0 . 0013 ( 20 hours , controls vs S2 ) , * * * * P < 0 . 0001 ( 20 hours , controls vs S6 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The expression levels were tested at different days after transduction. The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line. Data are presented as mean ± SEM (n = 3). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0030 (S6 naïve vs S6 APOPT1HA), *P = 0.0165 (S6 EV vs S6 APOPT1HA ) B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line. Data are presented as mean ± SEM (n = 4). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0066 (S6 naïve vs S6 APOPT1GFP), ***P = 0.0006 (S6 EV vs S6 APOPT1GFP), *P = 0.0169 (S2 naïve vs S2 APOPT1GFP), ***P = 0.0008 (S2 EV vs S2 APOPT1GFP) C Western blot analysis of 1D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows)  D Western blot analysis of 2D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows). Anti-GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein  E L-[35S]-Methionine pulse-chase labeling of mtDNA-encoded proteins. After a two-hour exposure with the radioactive label (pulse), cells were cultured in cold medium for the indicated chase times. The graphs show the densitometric quantification of the bands corresponding to MT-CO1 (left graph) and MT-CO2+MT-CO3 (right graph) normalized to the ATP6 band over the indicated time points. Graphs represent the values of three biological replicas for each cell line. Data are presented as mean ± SEM (n = 4 for controls, n = 2 for S2/S6/S2 APOPT1GFP/S6 APOPT1GFP). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: MT-CO1 - **P = 0.0039 ( 3.5 hours, controls vs S6), *P = 0.0202 (3.5 hours, controls vs S2), *P = 0.0163 (5 hours, S6vs S6 APOPT1GFP), *P = 0.0118 (5 hours, controls vs S2), **P = 0.0011 (20 hours, controls vs S6), *P = 0.0343 (20 hours, S2 vs S2 APOPT1GFP), ***P = 0.0007 (20 hours, S6 vs S6 APOPT1GFP ), ***P = 0.0002 (20 hours, controls vs S2), *****P < 0.0001 (20 hours, controls vs S6), MT-CO2/MT-CO3 - *P = 0.0194 (3.5 hours, S6 vs S6 APOPTGFP), ***P = 0.0003 (3.5 hours, controls vs S6), *P = 0.0375 (5 hours, S6 vs S6 APOPT1GFP), *P = 0.0234 (20 hours, S2 vs S2 APOPT1GFP), ****P < 0.0001 (20 hours, S6 vs S6 APOPT1GFP), **P = 0.0013 (20 hours, controls vs S2), ****P < 0.0001 (20 hours, controls vs S6)


Figure 6 A Western blot analysis of SDS - PAGE of MGM132 - treated 143B APOPT1HA cells . The graph represents the densitometric quantification of the signals for the precursor and mature protein B The upper part of the panel ( Input ) shows Western blot analyses of APOPT1HA in the MGM132 - treated cells . Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor . The bottom part of the panel ( Purified fractions ) shows the analysis of fractions from the same cells immunoprecipitated with anti - HA . Note that the higher molecular weight species are cross - reacting with both anti - HA and anti - ubiquitin C Western blot analysis of SDS - PAGE of total lysates from 143B cells overexpressing tagged APOPT1 ( as indicated ) and exposed to 100 µM H2O2 , as illustrated by the scheme ( H2O2 treatment ) , for the indicated times . The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point . The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2 D Western blot analysis of SDS - PAGE of total lysates from 143B cells overexpressing tagged APOPT1 ( as indicated ) and exposed to 5 µM MitoParaquat ( MitoPQ ) , as illustrated by the scheme ( MitoPQ treatment ) , for the indicated times . The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point . The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A Western blot analysis of SDS-PAGE of MGM132-treated 143B APOPT1HA cells. The graph represents the densitometric quantification of the signals for the precursor and mature protein  B The upper part of the panel (Input) shows Western blot analyses of APOPT1HA in the MGM132-treated cells. Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor. The bottom part of the panel (Purified fractions) shows the analysis of fractions from the same cells immunoprecipitated with anti-HA. Note that the higher molecular weight species are cross-reacting with both anti-HA and anti-ubiquitin  C Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 100 µM H2O2, as illustrated by the scheme (H2O2 treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2  D Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 5 µM MitoParaquat (MitoPQ), as illustrated by the scheme (MitoPQ treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ


Figure 7 A Western blot analysis of SDS - PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times . UT : untreated cells . B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas . C Densitometric quantification of MT - CO1 signal in the non - complemented APOPT1 - less cells ( S6 GFP ) vs . the complemented cells ( S6 APOPT1GFP ) . Three biological replicas ( n = 3 ) were carried out for each cell line . The signals in UT S6 APOPT1 were considered 100 % . The levels of MT - CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells ( * P = 0 . 0202 , two - tailed unpaired Student ' s t - test ) . D Densitometric quantification of MT - CO2 signal in the non - complemented APOPT1 - less cells ( S6 GFP ) vs . the complemented cells ( S6 APOPT1GFP ) . Three biological replicas were carried out for each cell line . The signals in UT S6 APOPT1 were considered 100 %

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times. UT: untreated cells. B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas. C Densitometric quantification of MT-CO1 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas (n=3) were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%. The levels of MT-CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells (*P = 0.0202, two-tailed unpaired Student's t-test). D Densitometric quantification of MT-CO2 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%


Figure 1 ( A , B ) Transmission electron micrographs of negatively stained 2F ( A ) and 3F ( B ) fibrils before sonication and injection into mice . The two fibril polymorphs were grown in vitro from synthetic Aβ40 and show the morphological differences originally reported Fibrils are negatively stained with uranyl acetate . Bars represent 100 nm . ( C ) PK resistance profile of 2F and 3F fibrils . Values are expressed as the mean densitometric value at each PK concentration ( technical triplicates ) ± standard error . Statistical analyses were performed by student ' s t - test ( * p < 0 . 05 ) . D ) Seeding assays using 100 pM of 2F ( blue ) and 3F ( green ) fibrils as seeds as described in Materials and Methods . Curves were generated by measuring ThT emission values at different time points . Reaction kinetics were statistically different as assessed by using the multiple comparisons method to compare pairs of curves ( * * * p < 0 . 0001 ) . Data in graphs is represented as averages depicting standard errors of five technical replicates . F - I ) 2F and 3F fibrils were tested for their reactivity against a variety of luminescent conjugated thiophenes able to discriminate among conformational variants of misfolded proteins . Stock solutions of LCOs ( 1 . 5 mM ) were diluted in distilled water to 15 μM and added to the wells to a final concentration of 0 . 3 μM . All wells contained 10 μM of either 2F or 3F fibrils . The samples were incubated at 37 ° C and the emission spectrum of each probe was collected after 30 minutes by exciting the samples at 440 nm ( HS - 68 ) or 535 nm ( HS - 194 , HS - 208 and HS - 212 ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A, B) Transmission electron micrographs of negatively stained 2F (A) and 3F (B) fibrils before sonication and injection into mice. The two fibril polymorphs were grown in vitro from synthetic Aβ40 and show the morphological differences originally reported Fibrils are negatively stained with uranyl acetate. Bars represent 100 nm.(C) PK resistance profile of 2F and 3F fibrils. Values are expressed as the mean densitometric value at each PK concentration (technical triplicates) ± standard error. Statistical analyses were performed by student's t-test (*p<0.05).D) Seeding assays using 100 pM of 2F (blue) and 3F (green) fibrils as seeds as described in Materials and Methods. Curves were generated by measuring ThT emission values at different time points. Reaction kinetics were statistically different as assessed by using the multiple comparisons method to compare pairs of curves (***p<0.0001). Data in graphs is represented as averages depicting standard errors of five technical replicates.F-I) 2F and 3F fibrils were tested for their reactivity against a variety of luminescent conjugated thiophenes able to discriminate among conformational variants of misfolded proteins. Stock solutions of LCOs (1.5 mM) were diluted in distilled water to 15 μM and added to the wells to a final concentration of 0.3 μM. All wells contained 10 μM of either 2F or 3F fibrils. The samples were incubated at 37°C and the emission spectrum of each probe was collected after 30 minutes by exciting the samples at 440 nm (HS-68) or 535 nm (HS-194, HS-208 and HS-212).


Figure 2 ( A - H ) Representative images of hippocampi from mice treated with 2F - , 3F - and Tg2576 - derived seeds after visualization for Aβ deposits using the 4G8 antibody ( A - D ) or ThS ( E - H ) . CA , cornu ammonis 1 ; DG , dentate gyrus . Scale bar in ( B ) , ( D ) , ( F ) and ( H ) represents 500 µm and applies to all panels . Arrows represent regions of interest ( alveus , DG ) . ( I , J ) Aβ ( I ) and ThS ( J ) burden quantification in hippocampus . Statistical analyses were performed using One - way ANOVA ( * p < 0 . 05 , * * p < 0 . 001 ) . Data in graphs is represented as averages depicting standard errors . Measurements considered five tissue slices per animal , and 4 - 7 animals were included per group .

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 O O O O O O O O O O

Figure 2(A-H) Representative images of hippocampi from mice treated with 2F-, 3F- and Tg2576- derived seeds after visualization for Aβ deposits using the 4G8 antibody (A-D) or ThS (E-H). CA, cornu ammonis 1; DG, dentate gyrus. Scale bar in (B), (D), (F) and (H) represents 500 µm and applies to all panels. Arrows represent regions of interest (alveus, DG).(I, J) Aβ (I) and ThS (J) burden quantification in hippocampus. Statistical analyses were performed using One-way ANOVA (*p<0.05, **p<0.001). Data in graphs is represented as averages depicting standard errors. Measurements considered five tissue slices per animal, and 4-7 animals were included per group.


Figure 3 ( A - H ) Representative pictures of hippocampi from mice treated with different injectate after visualization for micro - and astro - glial associated signals using anti - GFAP ( A - D ) and anti - Iba - 1 ( E - H ) antibodies . Scale bar in ( D ) and ( H ) represents 500 µm and applies to all panels . CA1 - 3 , cornu ammonis 1 - 3 ; DG , dentate gyrus ; so , striatum oriens ; sp , striatum pyramydale ; sr , striatum radiatum . ( I - N ) Anti - GFAP burden in hippocampus ( I ) , dentate gyrus ( J ) and alveus ( K ) for all animal groups was quantified for image analysis . Similarly , anti - Iba - 1 burdens for all experimental and control groups were calculated in hippocampus ( L ) , dentate gyrus ( M ) and alveus ( N ) . Statistical analyses were performed by One - way ANOVA ( * p < 0 . 05 , * * p < 0 . 001 ) . . Data in graphs is represented as averages depicting standard errors . Measurements considered five tissue slices per animal , and 4 - 7 animals were included per group . ( O - R ) Correlations between Aβ burden and glial burden were calculated in the alveus for mice injected with 2F ( O ) and 3F ( P ) fibrils . In the same way , the relationship between glial activation and Aβ burden in the hippocampus was calculated for mice treated with 2F ( Q ) and 3F ( R ) aggregates . Statistical analyses were performed by Pearson correlation coefficients test in which p < 0 . 05 was considered significant .

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 O O O O O O O O O O O O O O O O O O O O O O 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 3(A-H) Representative pictures of hippocampi from mice treated with different injectate after visualization for micro- and astro-glial associated signals using anti-GFAP (A-D) and anti-Iba-1 (E-H) antibodies. Scale bar in (D) and (H) represents 500 µm and applies to all panels. CA1-3, cornu ammonis 1-3; DG, dentate gyrus; so, striatum oriens; sp, striatum pyramydale; sr, striatum radiatum.(I-N) Anti-GFAP burden in hippocampus (I), dentate gyrus (J) and alveus (K) for all animal groups was quantified for image analysis. Similarly, anti-Iba-1 burdens for all experimental and control groups were calculated in hippocampus (L), dentate gyrus (M) and alveus (N). Statistical analyses were performed by One-way ANOVA (*p<0.05, **p<0.001).. Data in graphs is represented as averages depicting standard errors. Measurements considered five tissue slices per animal, and 4-7 animals were included per group.(O-R) Correlations between Aβ burden and glial burden were calculated in the alveus for mice injected with 2F (O) and 3F (P) fibrils. In the same way, the relationship between glial activation and Aβ burden in the hippocampus was calculated for mice treated with 2F (Q) and 3F (R) aggregates. Statistical analyses were performed by Pearson correlation coefficients test in which p<0.05 was considered significant.


Figure 4A ) Panels showing representative epifluorescence microscopy images of the dentate gyrus of mice treated with experimental and control materials and immunostained with both 4G8 ( green ) and anti - GFAP ( red ) antibodies . The third column of panels from left to right depict merged images . Right panels are insets obtained from the merged images ( white punctuated squares ) . Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as " 4G8 " , " GFAP " and " Merge " . Scale bars on the right images ( inset ) represent 50µm . g , granular layer ; h , hilar region ; m , molecular layerB ) A similar analysis as explained in A ) was performed for the alveus . Images were arranged in the same manner as described above . Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as " 4G8 " , " GFAP " and " Merge " . Scale bars on the right images ( inset ) represent 50µm . Alv , alveus ; so , striatum oriens . One tissue slice per animal was used for these analyses .

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

Figure 4A) Panels showing representative epifluorescence microscopy images of the dentate gyrus of mice treated with experimental and control materials and immunostained with both 4G8 (green) and anti-GFAP (red) antibodies. The third column of panels from left to right depict merged images. Right panels are insets obtained from the merged images (white punctuated squares). Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "GFAP" and "Merge". Scale bars on the right images (inset) represent 50µm. g, granular layer; h, hilar region; m, molecular layerB) A similar analysis as explained in A) was performed for the alveus. Images were arranged in the same manner as described above. Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "GFAP" and "Merge". Scale bars on the right images (inset) represent 50µm. Alv, alveus ; so, striatum oriens. One tissue slice per animal was used for these analyses.


Figure 5A ) Panels showing representative pictures of the dentate gyrus of mice treated with experimental and control Aβ seeds and stained with both 4G8 ( green ) and anti - Iba - 1 ( red ) antibodies . The third column of panels from left to right depict merged images . Right panels are insets obtained from the merged images ( white punctuated squares ) . : Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as " 4G8 " , " Iba - 1 " and " Merge " . Scale bars on the right images represent 50µm . ; g , granular layer ; h , hilar region ; m , molecular layer ; B ) A similar analysis as explained in A ) was performed for the alveus . Images were arranged in the same manner as described above . Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as " 4G8 " , " Iba - 1 " and " Merge " . Scale bars on the right images represent 50µm . Alv , alveus ; g , granular layer ; so , striatum oriens . One tissue slice per animal was used for these analyses .

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

Figure 5A) Panels showing representative pictures of the dentate gyrus of mice treated with experimental and control Aβ seeds and stained with both 4G8 (green) and anti-Iba-1 (red) antibodies. The third column of panels from left to right depict merged images. Right panels are insets obtained from the merged images (white punctuated squares). : Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "Iba-1" and "Merge". Scale bars on the right images represent 50µm. ; g, granular layer; h, hilar region; m, molecular layer;B) A similar analysis as explained in A) was performed for the alveus. Images were arranged in the same manner as described above. Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "Iba-1" and "Merge". Scale bars on the right images represent 50µm. Alv, alveus; g, granular layer ; so, striatum oriens. One tissue slice per animal was used for these analyses.


Figure 6A ) 1D 13C ssNMR spectra of fibrils that were prepared in vitro by seeded growth from amyloid - containing extracts of mouse brain homogenates . Mice had been treated with 2F - , 3F - , or Tg2576 - derived seeds . Aβ40 was 15N , 13C - labeled at F19 , V24 , G25 , S26 , A30 , I31 , L34 , and M35 . Vertical dashed lines indicate some of the ssNMR peaks that vary among the three samples . Asterisk indicates a peak from residual sodium dodecyl sulfate in the 2F sample .

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

Figure 6A) 1D 13C ssNMR spectra of fibrils that were prepared in vitro by seeded growth from amyloid-containing extracts of mouse brain homogenates. Mice had been treated with 2F-, 3F-, or Tg2576-derived seeds. Aβ40 was 15N,13C-labeled at F19, V24, G25, S26, A30, I31, L34, and M35. Vertical dashed lines indicate some of the ssNMR peaks that vary among the three samples. Asterisk indicates a peak from residual sodium dodecyl sulfate in the 2F sample.


Figure 1Detection of NRPB2WT - FLAG , NRPB2P979S - FLAG and NRPB2Y732F - FLAG protein by western blotting in NRPB2WT - FLAG Col - 0 , NRPB2P979S - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 plants . Untagged NRPB2 ( Col - 0 ) was used as a negative control . Histone H3 was used as an internal control and total protein level detected by stain - free blot was used as a loading control . Quantification was done by normalizing to the loading control and anti - H3 blot based on 3 independent replicates . Transmission rate of nrpb2 - 2 allele in nrpb2 - 2 + / - line ( n = 197 ) and nrpb2 - + / - lines combined with homozygous NRPB2P979S - FLAG + / + ( n = 280 ) , NRPB2Y732F - FLAG + / + ( n = 240 ) and NRPB2WT - FLAG + / + ( n = 210 ) , respectively . Fisher ´ s exact test was used as a statistic test , three asterisks denote p < 0 . 001 between samples and n . s . stands for not significant . Image of homozygous mutant nrpb2 - 2 fully complemented by NRPB2WT - FLAG ( top , NRPB2WT + / + nrpb2 - 2 - / - ) and partially complemented by NRPB2Y732F - FLAG ( bottom , NRPB2WT + / + nrpb2 - 2 - / - ) . Plants were grown for 4 weeks in soil . Scale bars represent 1 cm .

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 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 1Detection of NRPB2WT-FLAG, NRPB2P979S-FLAG and NRPB2Y732F-FLAG protein by western blotting in NRPB2WT-FLAG Col-0, NRPB2P979S-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 plants. Untagged NRPB2 (Col-0) was used as a negative control. Histone H3 was used as an internal control and total protein level detected by stain-free blot was used as a loading control. Quantification was done by normalizing to the loading control and anti-H3 blot based on 3 independent replicates.Transmission rate of nrpb2-2 allele in nrpb2-2 +/- line (n=197) and nrpb2- +/- lines combined with homozygous NRPB2P979S-FLAG +/+ (n=280), NRPB2Y732F-FLAG +/+ (n=240) and NRPB2WT-FLAG +/+ (n=210), respectively. Fisher´s exact test was used as a statistic test, three asterisks denote p<0.001 between samples and n.s. stands for not significant.Image of homozygous mutant nrpb2-2 fully complemented by NRPB2WT-FLAG (top, NRPB2WT +/+ nrpb2-2 -/-) and partially complemented by NRPB2Y732F-FLAG (bottom, NRPB2WT +/+ nrpb2-2 -/-). Plants were grown for 4 weeks in soil. Scale bars represent 1 cm.


Figure 2Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo . In brief , Arabidopsis seedlings of NRPB2WT - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days . Flagellin peptides ( flagellin 22 ) were added into media and treated samples were collected in a 0 minute ( no treatment ) , 2 minutes and 4 minutes time course . The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes . See Methods for technical details . Nascent RNA profile of AT5G41750 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represent 0 . 5 kb . Nascent RNA profile of AT4G19520 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represents 0 . 5 kb .

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 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 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

Figure 2Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo. In brief, Arabidopsis seedlings of NRPB2WT-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days. Flagellin peptides (flagellin 22) were added into media and treated samples were collected in a 0 minute (no treatment), 2 minutes and 4 minutes time course. The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes. See Methods for technical details.Nascent RNA profile of AT5G41750. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represent 0.5 kb.Nascent RNA profile of AT4G19520. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represents 0.5 kb.


Figure 3plaNET - seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal at the region of promoter - proximal stalling . Metagene profile of plaNET - seq mean signal of RNAPII in a 1 Kb window centered at the + 1 nucleosome in Arabidopsis genes ( n = 25474 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from - 25 bins to + 25 bins around + 1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two - sided Mann - Whitney U - test , p = 5 . 20e - 10 . RNAPII stalling index calculated for all the genes with plaNET - Seq FPKM ≥ 10 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) ( n = 6596 ) . Medians of the stalling index are 1 . 891 and 1 . 222 for NRPB2WT and NRPB2Y732F , respectively . * * * denotes p - value < 0 . 001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Metagene profile of plaNET - seq mean signal over whole genes ( length from 0 . 5 Kb to 5 Kb , scaled to 500 bins , n = 27042 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Metagene profile of plaNET - seq mean signal of RNAPII in exons ( length from 50 bp to 300 bp , scaled to 100 bins , n = 73925 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in exons , p < 1e - 16 . Metagene profile of plaNET - seq mean signal of RNAPII in introns ( 50 bp to 300 bp , scaled to 100 bins , n = 102260 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in introns , p < 1e - 16 .

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

Figure 3plaNET-seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal at the region of promoter-proximal stalling.Metagene profile of plaNET-seq mean signal of RNAPII in a 1 Kb window centered at the +1 nucleosome in Arabidopsis genes (n=25474) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from -25 bins to +25 bins around +1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two-sided Mann-Whitney U-test, p=5.20e-10.RNAPII stalling index calculated for all the genes with plaNET-Seq FPKM ≥ 10 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red) (n=6596). Medians of the stalling index are 1.891 and 1.222 for NRPB2WT and NRPB2Y732F, respectively. *** denotes p-value <0.001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Metagene profile of plaNET-seq mean signal over whole genes (length from 0.5 Kb to 5 Kb, scaled to 500 bins, n=27042) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).Metagene profile of plaNET-seq mean signal of RNAPII in exons (length from 50 bp to 300 bp, scaled to 100 bins, n=73925) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons. A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in exons, p<1e-16. Metagene profile of plaNET-seq mean signal of RNAPII in introns (50 bp to 300 bp, scaled to 100 bins, n=102260) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in introns, p<1e-16.


Figure 4Bar charts showing the fractions of 3 ' and 5 ' splicing intermediate reads from plaNET - seq in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb . The fraction of RNA - seq intronic reads calculated for all genes ( n = 24912 ) in NRPB2WT + / + nrpb2 - 2 - / - and NRPB2Y732F + / + nrpb2 - 2 - / - . Two - sided Mann - Whitney U test : * * * * denotes p - value < 2 . 2e - 16 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Differentially expressed ( DE ) exons and introns in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - based on RNA - seq results . Numbers of DE exons and introns were shown in plot . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) exons and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * denotes p - value < 0 . 01 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) introns and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * * * denotes p - value < 0 . 0001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

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 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 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 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

Figure 4Bar charts showing the fractions of 3' and 5' splicing intermediate reads from plaNET-seq in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb.The fraction of RNA-seq intronic reads calculated for all genes (n=24912) in NRPB2WT +/+ nrpb2-2 -/- and NRPB2Y732F +/+ nrpb2-2 -/-. Two-sided Mann-Whitney U test: **** denotes p-value<2.2e-16. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Differentially expressed (DE) exons and introns in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/- based on RNA-seq results. Numbers of DE exons and introns were shown in plot. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) exons and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. ** denotes p-value <0.01 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) introns and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. **** denotes p-value <0.0001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Figure 5plaNET - seq signal of RNAPII at 3 ' end of AT2G21410 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal peaks at PAS stalling region . Metagene profile of plaNET - seq mean signal of RNAPII in a 1 kb window centered at PAS ( n = 24448 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from PAS to + 100 bins between NRPB2WT and NRPB2Y732F were assessed by Two - sided Mann - Whitney U - test , p = 1 . 53e - 06 . Histogram of transcriptional read - through length ( nt ) from PAS of protein - coding gene ( plaNET - seq FPKM > 5 , n = 9316 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Box plot shows the RNAPII transcriptional read - through length from PAS of protein - coding genes ( plaNET - seq FPKM > 5 n = 9316 ) called based on statistic model ( see Methods ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Median of read - through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt . Two - sided Mann - Whitney U - test : * * * denotes p = 9 . 9e - 62 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Metagene plot of RNAPII signal by plaNET - seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes ( n = 5753 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrow denotes the direction of transcriptional read - through . Pink dashed line rectangle indicates the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction . Metagene plot of RNAPII signal by plaNET - seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in " tail to tail " orientation ( n = 1384 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrows denote the directions of transcriptional read - through from both PASs . Pink dashed line rectangles indicate the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction .

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 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 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 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

Figure 5plaNET-seq signal of RNAPII at 3' end of AT2G21410 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal peaks at PAS stalling region.Metagene profile of plaNET-seq mean signal of RNAPII in a 1 kb window centered at PAS (n=24448) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from PAS to +100 bins between NRPB2WT and NRPB2Y732F were assessed by Two-sided Mann-Whitney U-test, p = 1.53e-06.Histogram of transcriptional read-through length (nt) from PAS of protein-coding gene (plaNET-seq FPKM>5, n=9316) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).Box plot shows the RNAPII transcriptional read-through length from PAS of protein-coding genes (plaNET-seq FPKM>5 n=9316) called based on statistic model (see Methods) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Median of read-through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt. Two-sided Mann-Whitney U-test: *** denotes p = 9.9e-62. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Metagene plot of RNAPII signal by plaNET-seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes (n=5753) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrow denotes the direction of transcriptional read-through. Pink dashed line rectangle indicates the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction. Metagene plot of RNAPII signal by plaNET-seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in "tail to tail" orientation (n=1384) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrows denote the directions of transcriptional read-through from both PASs. Pink dashed line rectangles indicate the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction.


Figure 1A ) ELISA quantification of PD - L1 , pTau 181 and Aβx - 42 in the CSF of control and AD patients ( n = 10 + / - SEM , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , PD - L1 : t = 2 . 47 df = 18 , Aβx - 42 : t = 4 . 83 df = 18 , pTau 181 : t = 5 , 768 df = 18 ) . B , C ) ( B ) Colocalization of PD - L1 and GFAP in AD and ( C ) in control patients ( MX - O4 = amyloid deposits , upper picture : bar = 100 µm , lower picture : bar = 30 µm ) . D ) Immunohistochemical detection of PD - L1 in 9 - month - old female APP / PS1 mice ( bar = 50 µM ) . E ) Immunohistochemical detection of PD - L1 in 4 - month - old female APP / PS1 mice ( 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 O O O O O 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

Figure 1A) ELISA quantification of PD-L1, pTau 181 and Aβx-42 in the CSF of control and AD patients (n=10 +/-SEM, Student's t-test, * p<0.05, *** p<0.001, PD-L1: t=2.47 df = 18, Aβx-42: t=4.83 df=18, pTau 181: t=5,768 df=18).B, C) (B) Colocalization of PD-L1 and GFAP in AD and (C) in control patients (MX-O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar=30 µm). D) Immunohistochemical detection of PD-L1 in 9-month-old female APP/PS1 mice (bar = 50 µM). E) Immunohistochemical detection of PD-L1 in 4-month-old female APP/PS1 mice (bar = 20 µm).


Figure 2A ) C6 cells expressing PD - L1 - myc treated with the γ - secretase inhibitor DAPT for 18 h . PD - L1 was detected using a myc - antibody . B ) Immunoblot of HEK293 cells expressing PD - L1 - myc and presenilin 1 ( hPS1 ) or a presenilin 1 dominant negative mutant ( hPS1 D246A ) . D ) 18 h 1 μM PMA treatment of C6 cells expressing PD - L1 - myc . Secretion of PD - L1 in the conditioned medium ( CM ) was detected using antibody AF1019 . E ) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg / ml TNFα / 100 U / μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019 .

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

Figure 2A) C6 cells expressing PD-L1-myc treated with the γ-secretase inhibitor DAPT for 18 h. PD-L1 was detected using a myc-antibody.B) Immunoblot of HEK293 cells expressing PD-L1-myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).D) 18 h 1 μM PMA treatment of C6 cells expressing PD-L1-myc. Secretion of PD-L1 in the conditioned medium (CM) was detected using antibody AF1019.E) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNFα / 100 U/μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019.


Figure 3A ) Colocalization of PD - 1 and CD11b ( MX - 04 = methoxy - XO4 , stains amyloid deposits ) in AD by immunohistochemistry ( bar = 50 µm ) . B ) Colocalization of PD - 1 and Iba1 or PD - 1 and Aβ in a female APP / PS1 mouse at 9 months of age by immunohistochemistry ( bar = 20 µm ) . E ) ELISA analysis of TNFα secretion by wild type ( wt ) and PD - 1 - / - microglia induced by 0 . 5 µM Aβ1 - 42 for 6 h in vitro ( n = 3 biological replicates , one - way ANOVA ( DF = 1 , F = 8 . 29 , p = 0 . 021 ) , Tukey ' s HSD , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR . F - H ) Microglia from methoxy - XO4 - injected , 8 - month - old female , wild type ( wt ) and APP / PS1 mice were analyzed ( F ) for PD - 1 ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 13 . 99 , p = 0 . 0013 ) , Tukey ' s HSD , * * p < 0 . 01 ) , ( G ) for CD11b ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 30 . 06 , p = 5 . 47x10 - 05 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and ( H ) CD45 by flow cytometry ( n = 5 biological replicates per group , one - way ANOVA ( DF = 1 , F = 24 . 96 , p = 0 . 00013 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and normalized to the expression of wild type mice ( represented by dashed lines ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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

Figure 3A) Colocalization of PD-1 and CD11b (MX-04=methoxy-XO4, stains amyloid deposits) in AD by immunohistochemistry (bar=50 µm). B) Colocalization of PD-1 and Iba1 or PD-1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar=20 µm). E) ELISA analysis of TNFα secretion by wild type (wt) and PD-1-/- microglia induced by 0.5 µM Aβ1-42 for 6 h in vitro (n=3 biological replicates, one-way ANOVA (DF = 1, F = 8.29, p= 0.021), Tukey's HSD, * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.F-H) Microglia from methoxy-XO4-injected, 8-month-old female, wild type (wt) and APP/PS1 mice were analyzed (F) for PD-1 (n=5 biological replicates per group, one-way ANOVA (DF = 2, F = 13.99, p=0.0013), Tukey's HSD, ** p<0.01), (G) for CD11b (n=5 biological replicates per group, one- way ANOVA (DF = 2, F = 30.06, p=5.47x10-05), Tukey's HSD, *** p<0.001) and (H) CD45 by flow cytometry (n=5 biological replicates per group, one-way ANOVA (DF = 1, F = 24.96, p=0.00013), Tukey's HSD, *** p<0.001) and normalized to the expression of wild type mice (represented by dashed lines). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


Figure 4A , B ) ELISA of the ( A ) RIPA soluble fraction of female APP / PS1 and APP / PS1 PD - 1 - / - mice for Aβx - 40 ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 6 , p = 0 . 45 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( B ) of the SDS soluble fraction ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 32 . 8 , p = 2x10 - 5 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) . D , E ) Mice were analyzed for ( D ) percentage of the covered areas ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 905 , p = 0 . 90 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( E ) the number of plaques per mm2 ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 4 . 08 , p = 0 . 054 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) . G ) Time needed to reach the hidden platform ( latency in seconds ) in the Morris water maze test ( mean + / - SEM of biological replicates with n = 12 for wt , n = 5 for PD - 1 - / - , n = 7 for APP / PS1 , and n = 8 for APP / PS1 PD - 1 - / - , one - way ANOVA ( DF = 3 , F = 17 . 72 , p = 1x10 - 9 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) .

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 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 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

Figure 4A, B) ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aβx-40 (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001).D, E) Mice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=0.905, p=0.90), Tukey's HSD, ** p<0.01, *** p<0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=4.08, p=0.054), Tukey's HSD, ** p<0.01, *** p<0.001).G) Time needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean +/-SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=7 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, one-way ANOVA (DF=3, F=17.72, p=1x10-9), Tukey's HSD, * p<0.05, *** p<0.001).


Figure 5A ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type or PD - 1 - / - microglia for up to 6 h ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate ) . B ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type microglia cultured in astrocyte - conditioned medium ( ACM ) from wild type or PD - L1 - / - astroglial cultures ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate ) D ) Quantification of microglial in vivo phagocytosis in female APP / PS1 and APP / PS1 PD - 1 - / - mice at 8 . 5 month of age after intraperitoneal injection of methoxy - XO4 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , Student ' s t - test ( t = 2 . 1808 , df = 6 . 9829 ) , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR . E - G ) Relative expression of the cell surface marker ( E ) CD36 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , ANOVA ( DF = 1 , F = 0 . 002 , p = 0 . 94 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) , ( F ) CD11b ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) and ( G ) CD45 on methoxy - XO4 + versus methoxy - XO4 - microglia in APP / PS1 and APP / PS1 PD - 1 - / - mice normalized to methoxy - X04 - negative cells from APP / PS1 mice ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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 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 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

Figure 5A) In vitro phagocytosis of FAM-Aβ1-42 by wild type or PD-1-/- microglia for up to 6 h (mean +/-SEM of a technical quadruplicate, Student's t-test, ** p<0.01, *** p<0.001, representative result of an n=2 biological replicate). B) In vitro phagocytosis of FAM-Aβ1-42 by wild type microglia cultured in astrocyte-conditioned medium (ACM) from wild type or PD-L1-/- astroglial cultures (mean +/-SEM of a technical quadruplicate, Student's t-test, * p<0.05, *** p<0.001, representative result of an n=2 biological replicate) D) Quantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD-1-/- mice at 8.5 month of age after intraperitoneal injection of methoxy-XO4 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, Student's t-test (t = 2.1808, df = 6.9829), * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.E-G) Relative expression of the cell surface marker (E) CD36 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, ANOVA (DF=1, F= 0.002, p=0.94), Tukey's HSD, * p<0.05, *** p<0.001), (F) CD11b (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) and (G) CD45 on methoxy-XO4+ versus methoxy-XO4- microglia in APP/PS1 and APP/PS1 PD-1-/- mice normalized to methoxy-X04-negative cells from APP/PS1 mice (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


Figure 6B ) Different expression of inflammasome - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia . C ) Different expression of complement system - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia .

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

Figure 6B) Different expression of inflammasome-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini- Hochberg adjusted) in PD-1-/- vs. wild type microglia. C) Different expression of complement system-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini-Hochberg adjusted) in PD-1-/- vs. wild type microglia.


Figure 1Immunoblot analysis of p62 and LC3 I / II in KRIT1 wt and KRIT1 - KO endothelial cells . Actin was used as a loading control . Quantification of total LC3 on actin is reported ( * P = 0 . 02712 ) . The results are representative of three independent experiments . Representative images of p62 dots in KRIT1 wt and KRIT1 - KO endothelial cells . Scale bar , 20 μm . Magnifications in insets . Right , quantitative analysis of p62 distribution of dots is reported ( four independent experiments ; n = 35 cells per group ) . * P = 0 . 00542 ( dotted ) ; * P = 0 . 00014 ( nuclear ) . Immunoblot analysis of p62 and LC3 I / II in KRIT1 - KO and KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) MEFs . Left , immunoblot showing KRIT1 levels in KRIT1 - KO and KO + KRIT1 cells . Right , immunoblots for p62 and LC3 I / II . Actin was used as a loading marker . Quantification of total LC3 on actin is reported ( * P = 0 . 01248 ) . The results are representative of three independent experiments . Representative images of p62 dots in KO + KRIT1 ( top ) and KRIT1 - KO cells ( bottom ) . Scale bar , 50 μm . Magnifications in insets . Right , quantitative analysis of the number of p62 dots per cell is shown ( four independent experiments ; n = 50 cells per group ) . * P = 7 . 18e − 14 . Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblots for p62 and LC3 I / II . Actin was used as a loading marker . Quantification of total LC3 on actin is reported ( * P = 0 . 03071 ) . The results are representative of three independent experiments . Immunoblot analysis of EA . hy926 cells transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblot for p62 and LC3 I / II . Actin was used as a loading marker . Quantification of total LC3 on actin is reported ( * P = 0 . 02527 ) . The results are representative of three independent experiments . Immunofluorescence analysis of p62 ( green ) and ProteoStat Aggresome staining detection reagent ( red ) in KRIT1 wt and KRIT1 - KO lung endothelial cells . The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence . Magnification in insets . Scale bar , 50 μm . The images are representative of four independent experiments . Immunofluorescence analysis of p62 ( green ) and ProteoStat Aggresome staining detection reagent ( red ) in KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) and KRIT1 - KO MEFs . The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence . Magnification in insets . Scale bar , 50 μm . The images are representative of four independent experiments .

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 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 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 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 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

Figure 1Immunoblot analysis of p62 and LC3 I/II in KRIT1 wt and KRIT1-KO endothelial cells. Actin was used as a loading control. Quantification of total LC3 on actin is reported (*P = 0.02712). The results are representative of three independent experiments.Representative images of p62 dots in KRIT1 wt and KRIT1-KO endothelial cells. Scale bar, 20 μm. Magnifications in insets. Right, quantitative analysis of p62 distribution of dots is reported (four independent experiments; n = 35 cells per group). *P = 0.00542 (dotted); *P = 0.00014 (nuclear).Immunoblot analysis of p62 and LC3 I/II in KRIT1-KO and KRIT1-KO re-expressing KRIT1 (KO+KRIT1) MEFs. Left, immunoblot showing KRIT1 levels in KRIT1-KO and KO+KRIT1 cells. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.01248). The results are representative of three independent experiments.Representative images of p62 dots in KO+KRIT1 (top) and KRIT1-KO cells (bottom). Scale bar, 50 μm. Magnifications in insets. Right, quantitative analysis of the number of p62 dots per cell is shown (four independent experiments; n = 50 cells per group). *P = 7.18e−14.Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.03071). The results are representative of three independent experiments.Immunoblot analysis of EA.hy926 cells transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblot for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.02527). The results are representative of three independent experiments.Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1 wt and KRIT1-KO lung endothelial cells. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.


Figure 2Immunoblot analysis with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , and total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 wt and KRIT1 - KO endothelial cells were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments . Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1 - KO endothelial cells . Where indicated , cells were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments . Immunoblot analysis of p62 , LC3 I / II , and actin in KRIT1 wt and KRIT1 - KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . The results are representative of three independent experiments . Immunoblot analysis with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , and total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) and KRIT1 - KO MEFs were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments . Immunoblot analysis of phosphorylated ULK1 ( Ser 757 ) , total ULK1 , and actin in KRIT1 KO + KRIT1 , and KRIT1 KO MEFs . Where indicated , cells were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments . Immunoblot analysis of p62 , actin , LC3 I / II in KO + KRIT1 and KRIT1 - KO cells . Where indicated , cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h . The results are representative of three independent experiments . KRIT1 wt and KRIT1 - KO endothelial cells were transiently transfected with mRFP - GFP - LC3 . Where indicated , the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . The differences in the autophagic flux were evaluated by counting the yellow LC3 I / II dots / cell ( RFP + GFP + ) and red LC3 dots / cell ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 5 . 74e − 5 ( red dots , WT ctrl vs . WT Tor1 ) ; * P = 9 . 62e − 5 ( red dots , WT ctrl vs . WT xesto ) ; * P = 0 . 00727 ( red dots , WT ctrl vs . KO ctrl ) ; # P = 0 . 00046 ( red dots , KO ctrl vs . KO Tor1 ) . The data are expressed as the mean ± s . e . m . KO + KRIT1 and KRIT1 - KO MEFs were transiently transfected with the mRFP - GFP - LC3 tandem construct . Where indicated , the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . The differences in the autophagic flux were evaluated by counting the yellow LC3 I / II dots / cell ( RFP + GFP + ) and red LC3 dots / cell ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 0 . 00023 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 Tor1 ) ; * P = 0 . 00045 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 xesto ) ; # P = 3 . 08e − 6 ( red dots , KO ctrl vs . KO Tor1 ) ; # # P = 6 . 73e − 5 ( yellow dots , KO + KRIT1 ctrl vs . KO ctrl ) . The data are expressed as the mean ± s . e . m . of four independent experiments .

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

Figure 2Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m.KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 0.00023 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 Tor1); *P = 0.00045 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 xesto); #P = 3.08e−6 (red dots, KO ctrl vs. KO Tor1); ##P = 6.73e−5 (yellow dots, KO+KRIT1 ctrl vs. KO ctrl). The data are expressed as the mean ± s.e.m. of four independent experiments.


Figure 3Cd44 , PAI1 ( also known as Serpine1 ) , and Id1 mRNA expression levels in KRIT1 wt and KRIT1 - KO endothelial cells were assessed by quantitative real - time PCR . Where indicated , KRIT1 wt and KRIT1 - KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h . The data are expressed as the mean ± s . e . m . Cd44 : * P = 0 . 02848 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 02605 ( KO ctrl vs . KO Tor1 ) . PAI1 : * P = 0 . 04446 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 03996 ( KO ctrl vs . KO Tor1 ) . Id1 : * P = 0 . 00266 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 01554 ( KO ctrl vs . KO Tor1 ) . n = 3 independent experiments . Immunoblot analysis of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , and actin in KRIT1 - KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h . The results are representative of three independent experiments . Immunoblot analysis of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , N - cadherin , alpha - smooth muscle actin ( alpha - SMA ) , and actin in HUVECs transfected with control siRNA or ATG7 siRNA . Formation of capillary - like structures measured by tube formation assays . HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h . Representative phase - contrast microscopy ( Scale bar , 100 μm ) and calcein - fluorescent ( Scale bar , 50 μm ) images were reported . All data are presented as percentage ± s . e . m from three different experiments performed in duplicate . * P = 1 . 29e − 11 . Immunoblot analysis of p62 , LC3I / II , and rapamycin in CCM3 wt and CCM3 - KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . The results are representative of three independent experiments . Representative immunostaining of retina sections from wt and a model of inducible and endothelial - specific CCM3 - KO ( CCM3 - ECKO ) mice at postnatal day 14 . Endothelium was stained with isolectin B4 ( ISOB4 ) ( blue ) . A , artery ; V , vein . p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3 - ECKO animals ( scale bar : 200 μm ) . Scale bar of magnifications : 100 μm . Representative immunostaining of brain sections from wt and a model of inducible and endothelial - specific CCM3 - knockout mice ( CCM3 - ECKO ) at postnatal day 9 . p62 aggregates can be observed in the proximity of CCM lesions ( arrows ) . Cell nuclei ( DAPI ) are in blue . Scale bar , 30 μm . Smaller panel shows the magnifications of blood vessels ( green ) . Scale bar , 10 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 3Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments.Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), and actin in KRIT1-KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h. The results are representative of three independent experiments.Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), N-cadherin, alpha-smooth muscle actin (alpha-SMA), and actin in HUVECs transfected with control siRNA or ATG7 siRNA.Formation of capillary-like structures measured by tube formation assays. HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h. Representative phase-contrast microscopy (Scale bar, 100 μm) and calcein-fluorescent (Scale bar, 50 μm) images were reported. All data are presented as percentage ± s.e.m from three different experiments performed in duplicate. *P = 1.29e−11.Immunoblot analysis of p62, LC3I/II, and rapamycin in CCM3 wt and CCM3-KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.Representative immunostaining of retina sections from wt and a model of inducible and endothelial-specific CCM3-KO (CCM3-ECKO) mice at postnatal day 14. Endothelium was stained with isolectin B4 (ISOB4) (blue). A, artery; V, vein. p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3-ECKO animals (scale bar: 200 μm). Scale bar of magnifications: 100 μm.Representative immunostaining of brain sections from wt and a model of inducible and endothelial-specific CCM3-knockout mice (CCM3-ECKO) at postnatal day 9. p62 aggregates can be observed in the proximity of CCM lesions (arrows). Cell nuclei (DAPI) are in blue. Scale bar, 30 μm. Smaller panel shows the magnifications of blood vessels (green). Scale bar, 10 μm.


Figure 4p62 immunohistochemistry ( IHC ) staining in humanbrain tissue . A , B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62 . Scale bars : ( A ) 200 μm ; ( B ) 100 μm . p62immunohistochemistry ( IHC ) staining in humanbrain tissue . C - F Two different representative samples of CCM lesions with a thin , single layer brain endothelium displaying either moderate ( C , D ) or marked ( E , F ) positive perinuclear " pearl necklace - like " immunostaining for p62 granules . ( C , D ) , case n ° 4 ( p62 + + ) , and ( E , F ) , case n ° 8 ( p62 + + + ) are representative of CCM cases listed in Appendix Table S1 . Scale bars : ( C , E ) 200 μm ; ( D , F ) 100 μm . Arrows indicate endothelial p62 positive staining . p62immunohistochemistry ( IHC ) staining in humanbrain tissue . G - I Hematoxylin and eosin ( H & amp ; amp ; E ) staining ( G ) and p62immunohistochemistry analysis ( H , I ) of a CCM surgical sample ( case n ° 6 in Appendix Table S1 ) containing normal vessels in the peri - lesional area , which served as an internal control . Notice marked p62 - positive staining in endothelial cells lining a CCM lesion ( H , arrows ) and p62 - negative staining in endothelial cells lining a normal peri - lesional vessel ( I , arrows ) . Scale bars : ( G ) 300 μm ; ( H , I ) 100 μm . Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells .

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 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 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

Figure 4p62 immunohistochemistry (IHC) staining in humanbrain tissue.A, B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62. Scale bars: (A) 200 μm; (B) 100 μm.p62immunohistochemistry (IHC) staining in humanbrain tissue.C-F Two different representative samples of CCM lesions with a thin, single layer brain endothelium displaying either moderate (C, D) or marked (E, F) positive perinuclear "pearl necklace-like" immunostaining for p62 granules. (C, D), case n° 4 (p62++), and (E, F), case n° 8 (p62+++) are representative of CCM cases listed in Appendix Table S1. Scale bars: (C, E) 200 μm; (D, F) 100 μm. Arrows indicate endothelial p62 positive staining.p62immunohistochemistry (IHC) staining in humanbrain tissue.G-I Hematoxylin and eosin (H&amp;E) staining (G) and p62immunohistochemistry analysis (H, I) of a CCM surgical sample (case n° 6 in Appendix Table S1) containing normal vessels in the peri-lesional area, which served as an internal control. Notice marked p62-positive staining in endothelial cells lining a CCM lesion (H, arrows) and p62-negative staining in endothelial cells lining a normal peri-lesional vessel (I, arrows). Scale bars: (G) 300 μm; (H, I) 100 μm. Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells.


Figure 1Depletion of RBM14 induces ectopic formation of centriolar protein complexesA - E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) ( A ) , Centrobin ( green ) and C - Nap1 ( magenta ) ( B ) , centrin - 2 ( green ) and CPAP ( magenta ) ( C ) , acetylated - tubulin ( green ) and CP110 ( magenta ) ( D ) , and centrin - 2 ( green ) and γ - tubulin ( magenta ) ( E ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) . Note that we counted the number of C - Nap1 foci after anaphase in mitosis when C - Nap1 signals recover from the reduction in prometaphase .

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Figure 1Depletion of RBM14 induces ectopic formation of centriolar protein complexesA-E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase.