Figure 2Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP . Neurons were fixed at DIV7 and immunostained with anti - AC3 antibody to detect the primary cilium . GFP - positive cells represent infected cells . Scale bar 20 µm , and 10 µm in the enlarged image . Western blot comparing MeCP2 levels in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons . Data are normalized to total protein content , visualized by a TGX stain - free technology . MECP2 expression in null neurons induced a significant recovery of ciliogenesis . ( C ) The graph represents the percentage of ciliated cells , counting n = 50 WT and n = 100 null cells of two different preparations ( mean ± S . E . , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post - hoc test ) . Statistical analysis reported a significant interaction ( F ( 1 , 135 ) = 8 . 264 ) , a significant genotype effect ( F ( 1 , 135 ) = 37 . 59 ) and a significant infection effect ( F ( 1 , 135 ) = 26 . 89 ) . ( D ) The graph represents the mean ± S . E . of cilium length in WT and Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses , counting n = 28 cells of two different preparations ( * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post - hoc test ) . Two - way ANOVA reported a significant interaction ( F ( 1 , 103 ) = 6 . 405 ) , a significant genotype effect ( F ( 1 , 103 ) = 35 ) and a significant infection effect ( F ( 1 . 103 ) = 4 . 039 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP. Neurons were fixed at DIV7 and immunostained with anti-AC3 antibody to detect the primary cilium. GFP-positive cells represent infected cells. Scale bar 20 µm, and 10 µm in the enlarged image.Western blot comparing MeCP2 levels in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons. Data are normalized to total protein content, visualized by a TGX stain-free technology.MECP2 expression in null neurons induced a significant recovery of ciliogenesis. (C) The graph represents the percentage of ciliated cells, counting n=50 WT and n=100 null cells of two different preparations (mean ± S.E., ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Statistical analysis reported a significant interaction (F (1,135) = 8.264), a significant genotype effect (F (1,135) = 37.59) and a significant infection effect (F (1,135) = 26.89). (D) The graph represents the mean ± S.E. of cilium length in WT and Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses, counting n=28 cells of two different preparations (**p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Two-way ANOVA reported a significant interaction (F (1,103) = 6.405), a significant genotype effect (F (1,103) = 35) and a significant infection effect (F (1.103) = 4.039).


Figure 3MECP2 - silenced or control hTERT - RPE - 1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h . Cells were stained with anti - Smo and anti - acetylated α - tubulin antibodies . The percentages of Smo - positive cilia were calculated . The graph shows the average of three independent experiments ( mean ± S . E . , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post hoc test ) . Two - way ANOVA demonstrated a significant silencing effect ( F ( 1 , 12 ) = 160 ; p < 0 . 0001 ) , a significant SAG treatment ( F ( 2 , 12 ) = 259 . 4 ; p < 0 . 0001 ) and a significant interaction ( F ( 2 , 12 ) = 36 . 85 ; p < 0 . 0001 ) . WT or Mecp2 null MEF cells were treated with SAG ( 200 and 500 nM ) for 24 h and the percentages of Smo - positive cilia were calculated . The graph shows the average of three independent experiments ( mean ± S . E . , * p < 0 . 05 , by two - way ANOVA followed by Bonferroni post hoc test ) . Two - way ANOVA indicated a significant genotype effect ( F ( 1 , 12 ) = 11 . 22 ; p < 0 . 01 ) and a significant SAG treatment ( F ( 2 , 12 ) = 11 . 7 ; p < 0 . 0001 ) . Gli1 protein levels were measured by Western blot in untreated ( UT ) and 100 nM SAG - treated WT and Mecp2 null MEF cells ( n = 5 / 6 ) . Representative bands of Gli1 , Mecp2 and α - tubulin are depicted above the histogram , which shows the mean ± S . E of the percentage of Gli1 expression levels , compared to the untreated WT samples ( * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA , followed by Bonferroni post hoc test ) . Two - way ANOVA reported a significant interaction ( F ( 1 , 18 ) = 6 . 652 ; p < 0 . 05 ) , a significant genotype effect ( F ( 1 , 18 ) = 15 . 95 ; p < 0 . 001 ) and a significant treatment ( F ( 1 , 18 ) = 20 . 15 ; p < 0 . 001 ) . Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons , following SAG treatment ( 200 and 400 nM ) . Representative bands of Gli1 , Mecp2 and α - tubulin are depicted above the histogram , which shows the mean ± S . E of the percentage of Gli1 expression levels , compared to the untreated WT samples ( * p < 0 . 05 , * * * p < 0 . 001 , by two - way ANOVA , followed by Bonferroni post hoc test ) . Two - way ANOVA indicated a significant interaction ( F ( 2 , 20 ) = 3 . 788 ; p < 0 . 05 ) , a significant genotype effect ( F ( 1 , 20 ) = 4 . 362 ; p < 0 . 05 ) and a significant treatment ( F ( 2 , 20 ) = 13 . 5 ; p < 0 . 001 ) . Gli1 mRNA expression levels upon SAG exposure in MEF cells ( n = 7 WT and n = 9 null samples ) ( F ; SAG 100 nM ) and cortical neurons ( n = 15 WT and n = 12 null samples ) ( G ; SAG 400 nM ) . The graphs show the mean ± S . E . of the fold change derived from at least three independent experiments ( mean ± S . E . , * * p < 0 . 01 , by two - way ANOVA , followed by Bonferroni post hoc test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3MECP2-silenced or control hTERT-RPE-1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies. The percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *** p<0.001, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA demonstrated a significant silencing effect (F (1, 12) = 160; p<0.0001), a significant SAG treatment (F (2, 12) = 259.4; p<0.0001) and a significant interaction (F (2, 12) = 36.85; p<0.0001).WT or Mecp2 null MEF cells were treated with SAG (200 and 500 nM) for 24 h and the percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant genotype effect (F (1, 12) = 11.22; p<0.01) and a significant SAG treatment (F (2, 12) = 11.7; p<0.0001).Gli1 protein levels were measured by Western blot in untreated (UT) and 100 nM SAG-treated WT and Mecp2 null MEF cells (n=5/6). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (**p<0.01, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant interaction (F (1, 18) = 6.652; p<0.05), a significant genotype effect (F (1,18) = 15.95; p<0.001) and a significant treatment (F (1, 18) = 20.15; p<0.001).Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons, following SAG treatment (200 and 400 nM). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (*p<0.05, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant interaction (F (2, 20) = 3.788; p<0.05), a significant genotype effect (F (1, 20) = 4.362; p<0.05) and a significant treatment (F (2, 20) = 13.5; p<0.001).Gli1 mRNA expression levels upon SAG exposure in MEF cells (n=7 WT and n=9 null samples) (F; SAG 100 nM) and cortical neurons (n= 15 WT and n=12 null samples) (G; SAG 400 nM). The graphs show the mean ± S.E. of the fold change derived from at least three independent experiments (mean ± S.E., **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test).


Figure 4Representative immunostaining of Arl13b ( red ) and γ - tubulin ( green ) in the P14 mouse cortical layers . Nuclei were stained with DAPI ( blue ) . Scale bar 10 µm . Primary cilium length was measured in the cortex of Mecp2 null mice Average cilium length was measured in the cortex ( mean ± S . E . , n = 4 WT and n = 3 Mecp2 null ) ( B ; * p < 0 . 05 , by Mann - Whitney test ) and for each cortical layer ( C ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , by two - way ANOVA followed by Bonferroni post hoc test ) . In C , cilium length is analysed in separate cortical layers while in B , the values indicate the average of cilium lengths for each layer . Representative immunostaining of Arl13b ( green ) in the internal ( IGL ) and external ( EGL ) granular layers of Mecp2 null ( n = 3 ) and heterozygous ( het ) cerebella ( n = 3 ) at P7 , and the corresponding WT ( n = 3 males and n = 3 females ) . Nuclei were stained with DAPI ( blue ) . Scale bar 5 µm . Primary cilium length was measured in the cerebellum of Mecp2 null and heterozygous mice . Histograms show the mean ± S . E . of cilium length ( n = 3 males WT , n = 3 females WT , n = 3 Mecp2 null and Mecp2 het ; * * p < 0 . 01 , * * * p < 0 . 001 , by Mann - Whitney test ) in Mecp2 null ( E ) and heterozygous ( F ) mice , compared to the corresponding WT . Gli1 protein levels were measured by Western blot in WT and Mecp2 null cerebellum ( n = 7 WT and n = 6 Mecp2 null ) . Representative bands of Gli1 and α - tubulin are depicted beside the histogram , which shows the mean ± S . E of the percentage of Gli1 expression levels , compared to WT animals ( * p < 0 . 05 , by Student ' s t test ) . The mRNA expression levels of Gli1 and its downstream genes ( Cyclin D1 and D2 ) were analyzed in WT and Mecp2 null cerebella ( n = 8 WT and n = 7 Mecp2 null ) . The graph shows the fold change of transcripts compared to WT ( mean ± S . E . , * p < 0 . 05 , by Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Representative immunostaining of Arl13b (red) and γ-tubulin (green) in the P14 mouse cortical layers. Nuclei were stained with DAPI (blue). Scale bar 10 µm.Primary cilium length was measured in the cortex of Mecp2 null mice Average cilium length was measured in the cortex (mean ± S.E., n=4 WT and n=3 Mecp2 null) (B; *p<0.05, by Mann-Whitney test) and for each cortical layer (C; ***p<0.001, **p<0.01, *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). In C, cilium length is analysed in separate cortical layers while in B, the values indicate the average of cilium lengths for each layer.Representative immunostaining of Arl13b (green) in the internal (IGL) and external (EGL) granular layers of Mecp2 null (n=3) and heterozygous (het) cerebella (n=3) at P7, and the corresponding WT (n=3 males and n=3 females). Nuclei were stained with DAPI (blue). Scale bar 5 µm.Primary cilium length was measured in the cerebellum of Mecp2 null and heterozygous mice. Histograms show the mean ± S.E. of cilium length (n=3 males WT, n=3 females WT, n=3 Mecp2 null and Mecp2 het; **p<0.01, ***p<0.001, by Mann-Whitney test) in Mecp2 null (E) and heterozygous (F) mice, compared to the corresponding WT.Gli1 protein levels were measured by Western blot in WT and Mecp2 null cerebellum (n=7 WT and n=6 Mecp2 null). Representative bands of Gli1 and α-tubulin are depicted beside the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to WT animals (*p<0.05, by Student's t test).The mRNA expression levels of Gli1 and its downstream genes (Cyclin D1 and D2) were analyzed in WT and Mecp2 null cerebella (n=8 WT and n=7 Mecp2 null). The graph shows the fold change of transcripts compared to WT (mean ± S.E., *p<0.05, by Student's t test).


Figure 5WT and Mecp2 null MEF cells were starved for 48 h and then treated with the HDAC6 inhibitor tubacin ( TUB ) ( 1 μM ) for 48 h or with DMSO ( VEH ) or left untreated ( UT ) . Cells were fixed , stained for anti - acetylated α - tubulin and cilia were counted . The graph shows the percentage of ciliated cells out of three independent experiments ( mean ± S . E . , * p < 0 . 05 , * * p < 0 . 01 , by two - way ANOVA followed by Bonferroni post hoc test ) . Two - way ANOVA reported a significant genotype effect ( F ( 1 , 10 ) = 17 . 94 ; p < 0 . 01 ) , a significant tubacin treatment ( F ( 2 , 10 ) = 4 . 425 ; p < 0 . 05 ) and a significant interaction ( F ( 2 , 10 ) = 7 . 266 ; p < 0 . 05 ) . Primary cortical neurons were treated at DIV5 with DMSO ( VEH ) or tubacin ( TUB , 1 μM ) for 48 h , or left untreated ( UT ) , fixed at DIV7 and stained with anti - AC3 antibody . The graph shows the percentage of ciliated cells obtained from three independent experiments counting n = 300 UT cells , n = 216 VEH treated cells , n = 267 TUB treated cells for each genotype ( mean ± S . E . , * p < 0 . 05 , * * p < 0 . 01 , by two - way ANOVA , followed by Bonferroni post hoc test ) . Two - way ANOVA reported a significant genotype effect ( F ( 1 , 15 ) = 23 . 42 ; p < 0 . 001 ) , a significant treatment effect ( F ( 2 , 15 ) = 11 . 25 ; p < 0 . 01 ) and a significant interaction ( F ( 2 , 15 ) = 4 . 853 ; p < 0 . 05 ) . MECP2 silenced or control hTERT - RPE - 1 cells were starved for 48 h , treated with tubacin ( TUB , 1 µM ) for 24 h and then exposed to 200 nM SAG for 24 h in the presence of tubacin . Cells were stained with anti - Smo and anti - acetylated α - tubulin antibodies and the percentages of Smo - positive cilia were calculated . The graph shows the averages of three independent experiments ( mean ± S . E . , * * p < 0 . 01 , by three - way ANOVA followed by Tukey ' s post hoc test ) . Statistical analysis reported a significant difference between genotype ( F ( 1 , 2 ) = 21 . 47 ; p < 0 . 001 ) , a significant SAG effect ( F ( 2 , 2 ) = 154 . 5 ; p < 0 . 0001 ) and a significant tubacin effect ( F ( 1 , 2 ) = 25 . 18 ; p < 0 . 001 ) . In details , a significant interaction between genotype and SAG treatment ( F ( 2 , 2 ) = 5 . 413 ; p < 0 . 05 ) and between SAG and tubacin treatment ( F ( 2 , 2 ) = 6 . 939 ; p < 0 . 01 ) were reported , demonstrating respectively a difference between CTRL and MeCP2 depleted cells in response to SAG and a different SAG effect when cells were treated with tubacin . Representative immunostaining of Smo ( green ) and acetylated α - tubulin ( red ) in control ( CTRL ) and silenced ( siMeCP2 ) hTERT - RPE - 1 cells , after stimulation with 200 nM SAG in the presence or absence of the HDAC6 inhibitor tubacin ( TUB ) . Scale bar 50 µm , and 20 µm in the enlarged image .

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

Figure 5WT and Mecp2 null MEF cells were starved for 48 h and then treated with the HDAC6 inhibitor tubacin (TUB) (1 μM) for 48 h or with DMSO (VEH) or left untreated (UT). Cells were fixed, stained for anti-acetylated α-tubulin and cilia were counted. The graph shows the percentage of ciliated cells out of three independent experiments (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F(1, 10) = 17.94; p<0.01), a significant tubacin treatment (F(2, 10) = 4.425; p<0.05) and a significant interaction (F(2, 10) = 7.266; p<0.05).Primary cortical neurons were treated at DIV5 with DMSO (VEH) or tubacin (TUB, 1 μM) for 48 h, or left untreated (UT), fixed at DIV7 and stained with anti-AC3 antibody. The graph shows the percentage of ciliated cells obtained from three independent experiments counting n=300 UT cells, n=216 VEH treated cells, n=267 TUB treated cells for each genotype (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F (1, 15) = 23.42; p<0.001), a significant treatment effect (F (2, 15) = 11.25; p<0.01) and a significant interaction (F (2, 15) = 4.853; p<0.05).MECP2 silenced or control hTERT-RPE-1 cells were starved for 48 h, treated with tubacin (TUB, 1 µM) for 24 h and then exposed to 200 nM SAG for 24 h in the presence of tubacin. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies and the percentages of Smo-positive cilia were calculated. The graph shows the averages of three independent experiments (mean ± S.E., **p<0.01, by three-way ANOVA followed by Tukey's post hoc test). Statistical analysis reported a significant difference between genotype (F (1, 2) = 21.47; p<0.001), a significant SAG effect (F (2, 2) = 154.5; p<0.0001) and a significant tubacin effect (F (1, 2) = 25.18; p<0.001). In details, a significant interaction between genotype and SAG treatment (F (2, 2) = 5.413; p<0.05) and between SAG and tubacin treatment (F (2, 2) = 6.939; p<0.01) were reported, demonstrating respectively a difference between CTRL and MeCP2 depleted cells in response to SAG and a different SAG effect when cells were treated with tubacin.Representative immunostaining of Smo (green) and acetylated α-tubulin (red) in control (CTRL) and silenced (siMeCP2) hTERT-RPE-1 cells, after stimulation with 200 nM SAG in the presence or absence of the HDAC6 inhibitor tubacin (TUB). Scale bar 50 µm, and 20 µm in the enlarged image.


Figure 6Representative images of MAP2 positive WT and Mecp2 null neurons ( DIV7 ) . At DIV5 neurons were treated with tubacin ( TUB , 1µM for 48 h ) , Aurora A inhibitor TC - S 7010 ( TC - S , 7 nM for 48 h ) or left untreated and morphologically analyzed by Sholl analysis plug - in . Scale bar 20 µm . The graph reports the mean ± S . E . of the number of intersections measured 85 to 145 nm from the soma calculated by Sholl analysis for WT ( n = 39 neurons ) and Mecp2 null neurons , when left untreated ( n = 57 neurons ) or treated with tubacin ( n = 53 neurons ) or TC - S 7010 ( n = 38 neurons ) . Data from WT and null neurons were compared by Mann - Whitney test ( * * p < 0 . 01 ) ; drug effects on null cells were analyzed by one - way ANOVA followed by Dunn ' s post hoc test ( * p < 0 . 05 , * * p < 0 . 01 ) . Representative images of WT and Mecp2 null neurons ( DIV14 ) immunostained for MAP2 ( white ) , Synapsin1 / 2 ( green ) and Shank2 ( red ) . Mecp2 null cells were treated at DIV12 with tubacin ( TUB , 1µM for 48 h ) or Aurora A inhibitor TC - S 7010 ( TC - S , 7 nM for 48 h ) . Scale bar 15 µm , and 5 µm in the enlarged image . The graphs represent the mean ± S . E . of the Synapsin1 / 2 and the Shank2 puncta density in WT ( n = 67 neurons ) and Mecp2 null neurons , when untreated ( n = 51 neurons ) or treated with tubacin ( n = 59 neurons ) or TC - S 7010 ( n = 55 neurons ) . Neurons derived from at least 6 different biological replicates . Data from WT and null neurons were compared by Mann - Whitney test ( * * p < 0 . 01 , * * * p < 0 . 001 ) ; drug effects on null cells were analyzed by one - way ANOVA followed by Dunn ' s post hoc test ( * p < 0 . 05 , * * * p < 0 . 00 1 ) . The graph depicts the mean ± S . E . of the number of co - localized pre - and post - synaptic puncta ( n = 20 neurons deriving from 3 different biological samples ) . Data from WT and null neurons were compared by Mann - Whitney test ( * * * p < 0 . 001 ) ; drug effects on null cells were analyzed by one - way ANOVA followed by Dunn ' s post hoc test ( * p < 0 . 05 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Representative images of MAP2 positive WT and Mecp2 null neurons (DIV7). At DIV5 neurons were treated with tubacin (TUB, 1µM for 48 h), Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h) or left untreated and morphologically analyzed by Sholl analysis plug-in. Scale bar 20 µm. The graph reports the mean ± S.E. of the number of intersections measured 85 to 145 nm from the soma calculated by Sholl analysis for WT (n=39 neurons) and Mecp2 null neurons, when left untreated (n=57 neurons) or treated with tubacin (n=53 neurons) or TC-S 7010 (n=38 neurons). Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, **p<0.01).Representative images of WT and Mecp2 null neurons (DIV14) immunostained for MAP2 (white), Synapsin1/2 (green) and Shank2 (red). Mecp2 null cells were treated at DIV12 with tubacin (TUB, 1µM for 48 h) or Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h). Scale bar 15 µm, and 5 µm in the enlarged image.The graphs represent the mean ± S.E. of the Synapsin1/2 and the Shank2 puncta density in WT (n=67 neurons) and Mecp2 null neurons, when untreated (n=51 neurons) or treated with tubacin (n=59 neurons) or TC-S 7010 (n=55 neurons). Neurons derived from at least 6 different biological replicates. Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01, ***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, ***p<0.001). The graph depicts the mean ± S.E. of the number of co-localized pre- and post-synaptic puncta (n=20 neurons deriving from 3 different biological samples). Data from WT and null neurons were compared by Mann-Whitney test (***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05).


Figure 7Representative immunostaining of primary cilia and MeCP2 in cultured human fibroblasts from healthy controls ( CTRL , n = 3 ) and RTT patients . Fibroblasts were starved for 24 h and then stained for acetylated α - tubulin ( green ) , MeCP2 ( red ) and DAPI ( blue ) . Scale bar 50 µm . Histograms indicate ( mean ± S . E . ) the percentage of MeCP2 positive cellsHistograms indicate ( mean ± S . E . ) the percentage of ciliated cells in control and RTT fibroblastsThe effect of tubacin ( TUB ) treatment ( 250 nM , 24 h ) on primary cilium length of controls ( CTRL ) and RTT fibroblasts is reported . The graph indicates the mean ± S . E of the cilium length analyzed in untreated ( n = 130 CTRL ; n = 73 705delG ; n = 57 Q244X ; n = 68 R255X ) and treated cells ( n = 25 CTRL ; n = 26 705delG ; n = 26 Q244X ; n = 49 R255X ) ( * * * p < 0 . 001 vs untreated CTRL , § p < 0 . 05 vs corresponding mutated untreated fibroblasts by two - way ANOVA , followed by Bonferroni post hoc test ) . Statistical analysis reported a significant treatment effect ( F ( 1 , 429 ) = 26 . 56 ; p < 0 . 0001 ) and a significant genotype ( F ( 3 , 429 ) = 42 . 85 ; 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 B-PANEL_START 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7Representative immunostaining of primary cilia and MeCP2 in cultured human fibroblasts from healthy controls (CTRL, n=3) and RTT patients. Fibroblasts were starved for 24 h and then stained for acetylated α-tubulin (green), MeCP2 (red) and DAPI (blue). Scale bar 50 µm.Histograms indicate (mean ± S.E.) the percentage of MeCP2 positive cellsHistograms indicate (mean ± S.E.) the percentage of ciliated cells in control and RTT fibroblastsThe effect of tubacin (TUB) treatment (250 nM, 24 h) on primary cilium length of controls (CTRL) and RTT fibroblasts is reported. The graph indicates the mean ± S.E of the cilium length analyzed in untreated (n=130 CTRL; n=73 705delG; n=57 Q244X; n=68 R255X) and treated cells (n=25 CTRL; n=26 705delG; n=26 Q244X; n=49 R255X) (***p<0.001 vs untreated CTRL, §p<0.05 vs corresponding mutated untreated fibroblasts by two-way ANOVA, followed by Bonferroni post hoc test). Statistical analysis reported a significant treatment effect (F (1,429) = 26.56; p<0.0001) and a significant genotype (F(3,429) = 42.85; p<0.001).


Figure 1Human DCs were incubated with a non - cytolytic dose ( 0 . 2 μg / ml ) of purified PLY for 45 min . Immunofluorescence staining shows that PLY ( green ) co - localize with MRC - 1 in DCs and EEA - 1 ( purple , early endosomes ) . All scale bars , 5 μm . Images are representative of three independent experiments . Human DCs were incubated with a non - cytolytic dose ( 0 . 2 μg / ml ) of purified LLO for 45 min . Immunofluorescence staining shows that LLO ( green ) co - localize with MRC - 1 ( red ) in DCs and EEA - 1 ( purple , early endosomes ) . All scale bars , 5 μm . Images are representative of three independent experiments . Human DCs were incubated with a non - cytolytic dose ( 0 . 2 μg / ml ) of purified SLO for 45 min . Immunofluorescence staining shows that SLO ( green ) co - localize with MRC - 1 ( red ) in DCs and EEA - 1 ( purple , early endosomes ) . All scale bars , 5 μm . Images are representative of three 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified PLY for 45 min. Immunofluorescence staining shows that PLY (green) co-localize with MRC-1 in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified LLO for 45 min. Immunofluorescence staining shows that LLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified SLO for 45 min. Immunofluorescence staining shows that SLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.


Figure 3 ( A ) ELISA showing the dose - dependent binding of plate - bound MRC - 1 peptides P2 , P3 , and the control peptides CP1 and CP2 to PLY ( 0 - 0 . 5 μM ) . BSA was used as negative control to show the binding specificity . Data are mean ± s . e . m . of two independent experiments , each containing three replicates per condition . ( B ) Hemolysis assay ( n = 4 ) of 1 μg / ml purified PLY in the presence of increasing concentrations of MRC - 1 peptides , P2 , scrambled P2 , P3 and control peptide CP2 ( 1 - 1000 μM ) . Data represent mean ± s . e . m . * denotes P < 0 . 05 by one - way ANOVA with Dunnett ' s post hoc test for multiple comparisons . Exact P values are shown in Appendix Table S4 . ( C ) LDH cytotoxicity assay in human THP - 1 macrophages stimulated with purified PLY , LLO or SLO ( 0 . 5 μg / ml ) in the presence or absence of 100 μM peptides P2 , scrambled P2 , P3 or control peptide CP2 for 18 h . Cholesterol ( 100 μM ) was used as positive control to inhibit hemolysis . Data are mean ± s . e . m from 4 independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 . ( D ) Binding of FITC - labelled peptides P2 and CP2 to wild - type pneumococci , TIGR4 ( T4 ) and isogenic PLY mutant ( T4Δply ) was visualized by fluorescence microscopy . Scale bars , 10 μm . In magnified images , scale bars , 1 μm . Images are representative of three independent experiments . ( E ) The hemolytic activity of wild - type pneumococci , TIGR4 ( T4 ) and PLY mutant , T4Δply in the presence of 100 μM peptide P2 and CP2 . Data are the mean ± s . e . m . of three independent experiments . * * * denotes P < 0 . 001 by one - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA showing the dose-dependent binding of plate-bound MRC-1 peptides P2, P3, and the control peptides CP1 and CP2 to PLY (0-0.5 μM). BSA was used as negative control to show the binding specificity. Data are mean ± s.e.m. of two independent experiments, each containing three replicates per condition.(B) Hemolysis assay (n=4) of 1 μg/ml purified PLY in the presence of increasing concentrations of MRC-1 peptides, P2, scrambled P2, P3 and control peptide CP2(1-1000 μM). Data represent mean ± s.e.m. * denotes P < 0.05 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4.(C) LDH cytotoxicity assay in human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, scrambled P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from 4 independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.(D) Binding of FITC-labelled peptides P2 and CP2 to wild-type pneumococci, TIGR4 (T4) and isogenic PLY mutant (T4Δply) was visualized by fluorescence microscopy. Scale bars, 10 μm. In magnified images, scale bars, 1 μm. Images are representative of three independent experiments.(E) The hemolytic activity of wild-type pneumococci, TIGR4 (T4) and PLY mutant, T4Δply in the presence of 100 μM peptide P2 and CP2. Data are the mean ± s.e.m. of three independent experiments. *** denotes P < 0.001 by one-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


Figure 4 ( A ) IL - 8 released by human THP - 1 macrophages stimulated with purified PLY , LLO or SLO ( 0 . 5 μg / ml ) in the presence or absence of 100 μM peptides P2 , P3 or control peptide CP2 for 18 h . Cholesterol ( 100 μM ) was used as positive control to inhibit hemolysis . Data are mean ± s . e . m from three independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 . ( C ) 3D volume images of the GFP - lung epithelial models at 1 h and 3 h post stimulation with 1 μg / ml PLY in the presence or absence of 100 μM peptide P2 or the control peptide CP2 . Images are representative of two independent experiments with n = 3 models / condition . ( D ) Invasion of wild - type pneumococci T4 ( TIGR4 ) or its isogenic PLY mutant T4Δply into the lung epithelial models ( n = 3 / condition ) in the presence or absence of 100 μM peptide P2 or the control peptide CP2 at 2h post infection was measured using CFU viability assay following gentamicin killing of extracellular bacteria . Anti - PLY was used as control to test the effect of blocking PLY . Data in d and e are mean ± s . e . m . of n = 3 models / condition from two independent experiments . % bacterial entry = ( bacteria uptaken / input ) x100 . * * denotes P < 0 . 01 by one - way ANOVA with Dunnett ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 . ( E ) Human DCs were infected with type 4 and type 2 pneumococci , T4 and D39 , respectively , at MOI of 10 in the presence or absence of 100 μM peptides , P2 or CP2 , and intracellular bacteria were counted at 3 h post infection following gentamicin killing of extracellular bacteria . Cytochalasin D ( 0 . 5 mM ) was used as negative control to inhibit phagocytosis . Anti - PLY was used as control to test the effect of blocking PLY . Data are mean ± s . e . m . of three independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 . ( F ) DCs were infected with the unencapsulated pneumococcal strain T4R in the presence of 100 μM peptides , P2 or CP2 at MOI of 10 for 2 h . Immunofluorescence microscopy images show that in DCs treated with peptide P2 ( but not the control peptide CP2 ) , intracellular T4R ( green ) do not co - localize with MRC - 1 ( red ) , but with the autophagy protein LC3B ( cyan ) . Images are representative of three independent experiments . Scale bars , 10 μm . In magnified images , scale bars , 5 μm . Arrows indicate regions of co - localization of intracellular T4R with MRC - 1 and LC3B . ( G ) Quantification of percentage of intracellular S . pneumoniae ( n = 50 ) in infected DCs that co - localize with MRC - 1 and LC3B . Data are mean ± s . e . m from two independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . P values are shown in Appendix Table S4 .

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

Figure 4(A) IL-8 released by human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from three independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.(C) 3D volume images of the GFP-lung epithelial models at 1 h and 3 h post stimulation with 1 μg/ml PLY in the presence or absence of 100 μM peptide P2 or the control peptide CP2. Images are representative of two independent experiments with n=3 models/condition.(D) Invasion of wild-type pneumococci T4 (TIGR4) or its isogenic PLY mutant T4Δply into the lung epithelial models (n=3/condition) in the presence or absence of 100 μM peptide P2 or the control peptide CP2 at 2h post infection was measured using CFU viability assay following gentamicin killing of extracellular bacteria. Anti-PLY was used as control to test the effect of blocking PLY. Data in d and e are mean ± s.e.m. of n=3 models/condition from two independent experiments. % bacterial entry = (bacteria uptaken/input) x100. ** denotes P < 0.01 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.(E) Human DCs were infected with type 4 and type 2 pneumococci, T4 and D39, respectively, at MOI of 10 in the presence or absence of 100 μM peptides, P2 or CP2, and intracellular bacteria were counted at 3 h post infection following gentamicin killing of extracellular bacteria. Cytochalasin D (0.5 mM) was used as negative control to inhibit phagocytosis. Anti-PLY was used as control to test the effect of blocking PLY. Data are mean ± s.e.m. of three independent experiments. **** denotes P < 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.(F) DCs were infected with the unencapsulated pneumococcal strain T4R in the presence of 100 μM peptides, P2 or CP2 at MOI of 10 for 2 h. Immunofluorescence microscopy images show that in DCs treated with peptide P2 (but not the control peptide CP2), intracellular T4R (green) do not co-localize with MRC-1 (red), but with the autophagy protein LC3B (cyan). Images are representative of three independent experiments. Scale bars, 10 μm. In magnified images, scale bars, 5 μm. Arrows indicate regions of co-localization of intracellular T4R with MRC-1 and LC3B. (G) Quantification of percentage of intracellular S. pneumoniae (n=50) in infected DCs that co-localize with MRC-1 and LC3B. Data are mean±s.e.m from two independent experiments. **** denotes P <0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. P values are shown in Appendix Table S4.


Figure 5 ( A ) Survival percentage of 3 - 4 dpf zebrafish embryos ( n ≥ 156 ) upon infection with S . pneumoniae T4 alone or its isogenic PLY mutant , T4Δply . Injection with E3 growth medium served as mock control . * * * denotes P < 0 . 0005 and * * * * denotes P < 0 . 0001 by Mantel Cox test . Exact P values are shown in Appendix Table S4 . ( B ) Zebrafish survival percentage upon infection with T4 alone or together with peptide P2 or CP2 or P2 - conjugated CaP NPs ( P2 - NPs ) . * * * denotes P < 0 . 0005 and * * * * denotes P < 0 . 0001 by Mantel Cox test . Exact P values are shown in Appendix Table S4 . ( C ) Survival of mice ( n = 10 ) upon intranasal infection with 2x106 CFU of S . pneumoniae T4 together with peptide P2 or CP2 or P2 - NPs over 3 days post infection . Infected mice were checked twice daily in the morning at 9 am ( early check ) and in evening at 7 pm ( late check ) for clinical symptoms . Unloaded NPs and the isogenic PLY mutant strain ( T4Δply ) served as negative controls . * denotes P < 0 . 05 and * * denotes P < 0 . 005 by Mantel Cox test . Exact P values are shown in Appendix Table S4 . Bacterial load in the lungs of infected mice ( n = 10 ) were measured post sacrifice . Data represent mean ± s . e . m . * denotes P < 0 . 05 , * * denotes P < 0 . 01 and * * * * denotes P < 0 . 0001 by one - way ANOVA with Bonferroni ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 . levels of pro - inflammatory cytokines TNF - α in the lungs of infected mice ( n = 10 ) were measured post sacrifice . Data represent mean ± s . e . m . * denotes P < 0 . 05 , * * denotes P < 0 . 01 and * * * * denotes P < 0 . 0001 by one - way ANOVA with Bonferroni ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 . levels of pro - inflammatory cytokines IL - 12 in the lungs of infected mice ( n = 10 ) were measured post sacrifice . Data represent mean ± s . e . m . * denotes P < 0 . 05 , * * denotes P < 0 . 01 and * * * * denotes P < 0 . 0001 by one - way ANOVA with Bonferroni ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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

Figure 5(A) Survival percentage of 3-4 dpf zebrafish embryos (n ≥156) upon infection with S. pneumoniae T4 alone or its isogenic PLY mutant, T4Δply. Injection with E3 growth medium served as mock control. *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4.(B) Zebrafish survival percentage upon infection with T4 alone or together with peptide P2 or CP2 or P2-conjugated CaP NPs (P2-NPs). *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4.(C) Survival of mice (n=10) upon intranasal infection with 2x106 CFU of S. pneumoniae T4 together with peptide P2 or CP2 or P2-NPs over 3 days post infection. Infected mice were checked twice daily in the morning at 9 am (early check) and in evening at 7 pm (late check) for clinical symptoms. Unloaded NPs and the isogenic PLY mutant strain (T4Δply) served as negative controls. * denotes P < 0.05 and ** denotes P < 0.005 by Mantel Cox test. Exact P values are shown in Appendix Table S4.Bacterial load in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.levels of pro-inflammatory cytokines TNF-α in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.levels of pro-inflammatory cytokines IL-12 in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


Figure 3 ( A ) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors and matched normal samples across 23 tumor types from TCGA with normal tissue data available . The boxplot indicates the median ( central band ) and the interquartile range ( boxes ) of the expression value and whiskers indicates values that outside of the middle 50 % ( interquartile ) . The barplot on the top shows the number of normal tissue samples for each corresponding tumor type . Asterisks indicate significant differences by student ' s t - test after Benjamini - Hochberg multiple testing correction ( FDR < 0 . 05 ) .

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

Figure 3(A) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors and matched normal samples across 23 tumor types from TCGA with normal tissue data available. The boxplot indicates the median (central band) and the interquartile range (boxes) of the expression value and whiskers indicates values that outside of the middle 50% (interquartile). The barplot on the top shows the number of normal tissue samples for each corresponding tumor type. Asterisks indicate significant differences by student's t-test after Benjamini-Hochberg multiple testing correction (FDR < 0.05).


Figure 6 ( B ) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine ( Rv ) for 12 , 24 , 48 , and 72 hours . Thapsigargin ( Tg ) is shown as positive control . ( D ) Xbp1 splicing in B16 melanoma cells and eight B16 : MEF fused clonal cell lines . ( E ) Detection of mRNA expression ( by RT - PCR ) of UPR - associated genes ( Ern1 , Af4 , Ddit3 and Atf6 ) in tumor cells treated with Reversine ( Rv ) at 4µM ( DLD1 cells ) or 0 . 5µM ( SKOV3 cells ) for 6 hours . Data points refer to triplicate samples collected at the same time point , run in duplicate , and expressed as means ± SD . ( F ) Western Blot analysis of the activation of IRE1 , PERK and ATF branches of the UPR in DLD1 and SKOV3 cells treated or not with Rv at 4µM for 24H ( DLD1 cells ) or 0 . 5µM for 3 days ( SKOV3 cells ) . Thapsigargin ( Tg ) was used as positive control . ( G ) Western Blot analysis of phosphorylation of GCN2 and PKR defining the integrated stress response ( ISR ) in DLD1 and SKOV3 cells treated or not with Rv as in F ) . Cells starved for 24 hours then treated for 30 min with Calyculin - A ( Cal - A ) were used as positive control .

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

Figure 6(B) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine (Rv) for 12, 24, 48, and 72 hours. Thapsigargin (Tg) is shown as positive control.(D) Xbp1 splicing in B16 melanoma cells and eight B16:MEF fused clonal cell lines.(E) Detection of mRNA expression (by RT-PCR) of UPR-associated genes (Ern1, Af4, Ddit3 and Atf6) in tumor cells treated with Reversine (Rv) at 4µM (DLD1 cells) or 0.5µM (SKOV3 cells) for 6 hours. Data points refer to triplicate samples collected at the same time point, run in duplicate, and expressed as means ± SD.(F) Western Blot analysis of the activation of IRE1, PERK and ATF branches of the UPR in DLD1 and SKOV3 cells treated or not with Rv at 4µM for 24H (DLD1 cells) or 0.5µM for 3 days (SKOV3 cells). Thapsigargin (Tg) was used as positive control.(G) Western Blot analysis of phosphorylation of GCN2 and PKR defining the integrated stress response (ISR) in DLD1 and SKOV3 cells treated or not with Rv as in F). Cells starved for 24 hours then treated for 30 min with Calyculin-A (Cal-A) were used as positive control.


Figure 7 ( A ) Xbp1 splicing analysis ( left panel ) and quantification ( right panel ) of bone marrow - derived macrophages ( BMDM ) cultured in conditioned medium ( CM ) of Rv - treated DLD1 and SKOV3 cells or respective control medium ( culture medium of cancer cells not treated with Rv ) , and CM of fused B16 melanoma cells or their nonfused parental cells . ( B , C ) mRNA expression ( RT - PCR ) of Il6 ( B ) and Arg1 ( C ) in BMDM cultured in the CM of Rv - treated SKOV3 cells ( 0 . 5µM for 3 days ) or control cells , and CM of fused B16 cells or their nonfused parental cells . Data points refer to triplicate experiments expressed as means ± SD . ( E ) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti - CD3 / anti - CD28 Dynabeads in the presence of CM from Rv - treated SKOV3 cells or control SKOV3 cells ( cultured without Rv ) . Column graphs represent the percentage expression of IFNγ ( upper panel ) and Granzyme B ( lower panel ) . Percentages in parenthesis refer to change from control . Data representative triplicate samples at the same time point repeated at least twice . Data are expressed as means ± SD . ( F ) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti - CD3 / anti - CD28 Dynabeads in complete medium spiked with Rv ( 0 . 5µM ) . Column graphs represent the percentage expression of IFNγ ( upper panel ) and Granzyme B ( lower panel ) . Data representative triplicate samples at the same time point repeated at least twice . Data are expressed as means ± SD . ( G ) IFNγ ( left ) and Granzyme B ( right ) production by human T cells activated by anti - CD3 / anti - CD28 Dynabeads cultured in CM from fused B16 melanoma cells or their parental non - fused cells . Percentages in parenthesis refer to change from parental non - fused cells . Data representative triplicate samples at the same time point repeated at least twice . Data are expressed as means ± SD .

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

Figure 7(A) Xbp1 splicing analysis (left panel) and quantification (right panel) of bone marrow-derived macrophages (BMDM) cultured in conditioned medium (CM) of Rv-treated DLD1 and SKOV3 cells or respective control medium (culture medium of cancer cells not treated with Rv), and CM of fused B16 melanoma cells or their nonfused parental cells.(B, C) mRNA expression (RT-PCR) of Il6 (B) and Arg1 (C) in BMDM cultured in the CM of Rv-treated SKOV3 cells (0.5µM for 3 days) or control cells, and CM of fused B16 cells or their nonfused parental cells. Data points refer to triplicate experiments expressed as means ± SD.(E) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in the presence of CM from Rv-treated SKOV3 cells or control SKOV3 cells (cultured without Rv). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Percentages in parenthesis refer to change from control. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.(F) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in complete medium spiked with Rv (0.5µM). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.(G) IFNγ (left) and Granzyme B (right) production by human T cells activated by anti-CD3/anti-CD28 Dynabeads cultured in CM from fused B16 melanoma cells or their parental non-fused cells. Percentages in parenthesis refer to change from parental non-fused cells. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.


Figure 1Tau levels in the cortex ( A , C - E ) and hippocampus ( B , F - H ) of 4 - 10 - month - old mice were determined by western blot analysis . ( A , B ) Representative western blots of cortical ( A ) and hippocampal ( B ) homogenates from mice of the indicated genotypes show levels of hTau ( HT7 ) and total tau ( Tau5 ) . Actin served as a loading control . ( C - H ) Quantitation of western blot signals for full - length tau detected with the mTau / hTau cross - reactive antibodies Tau5 ( C , F ) or EP2456Y ( D , G ) or the hTau - specific antibody Tau12 ( E , H ) . n = 3 - 8 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed unpaired t test ( E , H ) or by Welch ' s one - way ANOVA with post - hoc two - tailed Welch ' s t test ( C , D , F , G ) . P values were Holm - adjusted for multiple comparisons . Values are means ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Tau levels in the cortex (A, C-E) and hippocampus (B, F-H) of 4-10-month-old mice were determined by western blot analysis. (A, B) Representative western blots of cortical (A) and hippocampal (B) homogenates from mice of the indicated genotypes show levels of hTau (HT7) and total tau (Tau5). Actin served as a loading control. (C-H) Quantitation of western blot signals for full-length tau detected with the mTau/hTau cross-reactive antibodies Tau5 (C, F) or EP2456Y (D, G) or the hTau-specific antibody Tau12 (E, H). n = 3-8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (E, H) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (C, D, F, G). P values were Holm-adjusted for multiple comparisons. Values are means ± SEM.


Figure 2Tau expression in the cortex ( A , C , E ) and hippocampus ( B , D , F ) of 4 - 10 - month - old mice was determined by quantitative RT - PCR ( A , B ) . ( A , B ) hTau mRNA levels in tissue homogenates were determined by quantitative RT - PCR . Tau expression in the cortex ( A , C , E ) and hippocampus ( B , D , F ) of 4 - 10 - month - old mice was determined by western blot analysis ( C - F ) . ( C , D ) Western blots from Figure 1A and B were enhanced to better reveal tau fragments of ~ 40 kDa . ( E , F ) Quantitation of western blot signals for tau fragments of ~ 40 kDa detected with Tau5 in tissue homogenates . n = 3 - 4 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed unpaired t test ( A , B ) or by Welch ' s one - way ANOVA with post - hoc two - tailed Welch ' s t test ( E , F ) . P values were Holm - adjusted . Values are means ± SEM .

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

Figure 2Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by quantitative RT-PCR (A, B). (A, B) hTau mRNA levels in tissue homogenates were determined by quantitative RT-PCR.Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by western blot analysis (C-F).(C, D) Western blots from Figure 1A and B were enhanced to better reveal tau fragments of ~40 kDa. (E, F) Quantitation of western blot signals for tau fragments of ~40 kDa detected with Tau5 in tissue homogenates. n = 3-4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (A, B) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (E, F). P values were Holm-adjusted. Values are means ± SEM.


Figure 3Coronal brain sections from 2 - 4 - month - old mice of the indicated genotypes were immunostained with different tau antibodies . ( A - I ) Sections of hemibrain ( A - C ) , whole hippocampus ( D - F ) , and CA1 region ( G - I ) immunostained for hTau ( HT7 ) . ( J - O ) Hippocampal sections immunostained with phosphorylation - dependent tau antibodies : PHF1 ( p396 , 404 ) ( J - L ) , AT8 ( pSer 199 , 202 , and pThr 205 ) ( M - O ) . ( P - R ) Representative photomicrographs of GFAP immunostaining of the CA1 region ( P , Q ) and quantitation of GFAP immunoreactivity ( R ) .

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Figure 3Coronal brain sections from 2-4-month-old mice of the indicated genotypes were immunostained with different tau antibodies. (A-I) Sections of hemibrain (A-C), whole hippocampus (D-F), and CA1 region (G-I) immunostained for hTau (HT7). (J-O) Hippocampal sections immunostained with phosphorylation-dependent tau antibodies: PHF1 (p396, 404) (J-L), AT8 (pSer 199, 202, and pThr 205) (M-O).(P-R) Representative photomicrographs of GFAP immunostaining of the CA1 region (P, Q) and quantitation of GFAP immunoreactivity (R).


Figure 4Six - month - old hTau - A152T ( L1 ) mice and NTG controls were or were not treated with DOX for 2 months and analyzed at 8 months of age by immunohistochemistry . ( A - I ) Untreated hTau - A152T ( L1 ) mice had increased neuronal labeling with the hTau antibody HT7 ( A ) , the phosphorylation - dependent tau antibody PHF1 ( D ) , and the conformation - dependent tau antibody MC1 ( G ) . HT7 ( A ) and PHF1 ( D ) staining patterns were similar . MC1 ( G ) specifically stained hippocampal mossy fibers . In hTau - A152T ( L1 ) mice , DOX reduced hippocampal staining with HT7 , PHF1 , and MC1 ( B , E , H ) to the levels in DOX - treated NTG controls ( C , F , I ) . ( J - M ) DOX also normalized CA1 levels of GFAP immunoreactivity ( IR ) in hTau - A152T ( L1 ) mice , as shown by representative photomicrographs ( J - L ) and quantitation of GFAP immunoreactivity ( M ) . Scale bars : 300 µm ( A - I ) , 25 µm ( J - L ) . n = 3 - 4 mice per genotype . * P < 0 . 05 by two - tailed Welch ' s t test without Holm adjustment . Values are means ± SEM . a . u . , arbitrary units .

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

Figure 4Six-month-old hTau-A152T(L1) mice and NTG controls were or were not treated with DOX for 2 months and analyzed at 8 months of age by immunohistochemistry. (A-I) Untreated hTau-A152T(L1) mice had increased neuronal labeling with the hTau antibody HT7 (A), the phosphorylation-dependent tau antibody PHF1 (D), and the conformation-dependent tau antibody MC1 (G). HT7 (A) and PHF1 (D) staining patterns were similar. MC1 (G) specifically stained hippocampal mossy fibers. In hTau-A152T(L1) mice, DOX reduced hippocampal staining with HT7, PHF1, and MC1 (B, E, H) to the levels in DOX-treated NTG controls (C, F, I).(J-M) DOX also normalized CA1 levels of GFAP immunoreactivity (IR) in hTau-A152T(L1) mice, as shown by representative photomicrographs (J-L) and quantitation of GFAP immunoreactivity (M). Scale bars: 300 µm (A-I), 25 µm (J-L). n = 3-4 mice per genotype. *P < 0.05 by two-tailed Welch's t test without Holm adjustment. Values are means ± SEM. a.u., arbitrary units.


Figure 5Nesting behavior of mice at 10 - 14 months of age was scored 0 - 7 by an investigator blinded to genotype . n = 17 NTG , 12 CaMKII - tTA , 13 hTau - WT ( L32 ) , and 10 hTau - A152T ( L1 ) . Nest building was significantly impaired only in hTau - A152T ( L1 ) mice ( P < 0 . 01 at 2 and 6 h ) and CaMKII - tTA mice ( P < 0 . 05 at 2 h , P < 0 . 01 at 6 hours ) by nonparametric Wilcoxon rank - sum test with gatekeeping approach and Holm adjustment . Values are means ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Nesting behavior of mice at 10-14 months of age was scored 0-7 by an investigator blinded to genotype. n = 17 NTG, 12 CaMKII-tTA, 13 hTau-WT(L32), and 10 hTau-A152T(L1). Nest building was significantly impaired only in hTau-A152T(L1) mice (P < 0.01 at 2 and 6 h) and CaMKII-tTA mice (P < 0.05 at 2 h, P < 0.01 at 6 hours) by nonparametric Wilcoxon rank-sum test with gatekeeping approach and Holm adjustment. Values are means ± SEM.


Figure 6Mice from cohorts 1 - 3 were tested in the MWM at the indicated ages . ( A - D , M , N ) Learning curves of mice in cohorts 1 ( A - D ) and 3 ( M , N ) . Data at day 0 are from the first trial on day 1 . Data from cohort 2 are shown in EV5B - G . Compared with NTG controls , only hTau - A152T ( L1 ) mice showed significant learning impairments at 4 - 6 , 17 - 19 ( cohort 1 , P < 0 . 001 ) and 17 - 21 ( cohort 3 , P < 0 . 01 ) months of age by Cox proportional hazards model analysis with mixed effects and Holm adjustment for the following comparisons for each age range : NTG versus CaMKII - tTA , NTG versus TRE - hTau - A152T ( L1 ) , and NTG versus hTau - A152T ( L1 ) for cohort 1 , and NTG versus CaMKII - tTA , NTG versus hTau - WT ( L32 ) , NTG versus hTau - A152T ( L1 ) , and hTau - WT ( L32 ) versus hTau - A152T ( L1 ) for cohort 3 . ( E - L , O - V ) Performance of mice from cohorts 1 ( E - L ) , 3 ( O - R ) and 1 - 3 combined ( S - V ) in a probe trial ( platform removed ) 24 h after the last training trial . ( E - H , O , P , S , T ) Latency to reach original platform location . ( I - L , Q , R , U , V ) Number of times mice crossed the original platform location ( black bars ) or the average of their crossings of corresponding locations in the three other quadrants ( white bars ) . Cohort 1 : n = 12 ( A - L ) NTG ; 13 ( A , B , E , F , I , J ) , 11 ( C , G , K ) , or 9 ( D , H , L ) TRE - hTau - A152T ( L1 ) ; 11 ( A , B , E , F , I , J ) , 10 ( C , G , K ) , or 9 ( D , H , L ) CaMKII - tTA ; and 14 ( A , B , E , F , I , J ) , 13 ( C , G , K ) , or 12 ( D , H , L ) hTau - A152T ( L1 ) mice . Cohort 3 : n = 17 ( M - R ) NTG ; 12 ( M , O , Q ) or 11 ( N , P , R ) CaMKII - tTA ; 13 ( M , O , Q ) or 12 ( N , P , R ) hTau - WT ( L32 ) ; and 10 ( M - R ) hTau - A152T ( L1 ) mice . Cohorts 1 + 2 + 3 : n = 47 ( S , U ) , or 39 ( T , V ) NTG ; 11 ( S , U ) or 9 ( T , V ) TRE - hTau - A152T ( L1 ) ; 34 ( S , U ) or 31 ( T , V ) CaMKII - tTA ; 13 ( S , U ) or 12 ( T , V ) hTau - WT ( L32 ) ; and 43 ( S , U ) or 36 ( T , V ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Cox proportional hazards / mixed effects model analysis ( A - D , M , N ) , one - way ANOVA with post - hoc Tukey ( E - H , O , P ) or Dunnett ' s ( S , T ) test , or one - tailed paired t test with Holm adjustment ( I - L , Q , R , U , V ) . Values are means ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Mice from cohorts 1-3 were tested in the MWM at the indicated ages. (A-D, M, N) Learning curves of mice in cohorts 1 (A-D) and 3 (M, N). Data at day 0 are from the first trial on day 1. Data from cohort 2 are shown in EV5B-G. Compared with NTG controls, only hTau-A152T(L1) mice showed significant learning impairments at 4-6, 17-19 (cohort 1, P < 0.001) and 17-21 (cohort 3, P < 0.01) months of age by Cox proportional hazards model analysis with mixed effects and Holm adjustment for the following comparisons for each age range: NTG versus CaMKII-tTA, NTG versus TRE-hTau-A152T(L1), and NTG versus hTau-A152T(L1) for cohort 1, and NTG versus CaMKII-tTA, NTG versus hTau-WT(L32), NTG versus hTau-A152T(L1), and hTau-WT(L32) versus hTau-A152T(L1) for cohort 3. (E-L, O-V) Performance of mice from cohorts 1 (E-L), 3 (O-R) and 1-3 combined (S-V) in a probe trial (platform removed) 24 h after the last training trial. (E-H, O, P, S, T) Latency to reach original platform location. (I-L, Q, R, U, V) Number of times mice crossed the original platform location (black bars) or the average of their crossings of corresponding locations in the three other quadrants (white bars). Cohort 1: n = 12 (A-L) NTG; 13 (A, B, E, F, I, J), 11 (C, G, K), or 9 (D, H, L) TRE-hTau-A152T(L1); 11 (A, B, E, F, I, J), 10 (C, G, K), or 9 (D, H, L) CaMKII-tTA; and 14 (A, B, E, F, I, J), 13 (C, G, K), or 12 (D, H, L) hTau-A152T(L1) mice. Cohort 3: n = 17 (M-R) NTG; 12 (M, O, Q) or 11 (N, P, R) CaMKII-tTA; 13 (M, O, Q) or 12 (N, P, R) hTau-WT(L32); and 10 (M-R) hTau-A152T(L1) mice. Cohorts 1+2+3: n = 47 (S, U), or 39 (T, V) NTG; 11 (S, U) or 9 (T, V) TRE-hTau-A152T(L1); 34 (S, U) or 31 (T, V) CaMKII-tTA; 13 (S, U) or 12 (T, V) hTau-WT(L32); and 43 (S, U) or 36 (T, V) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Cox proportional hazards/mixed effects model analysis (A-D, M, N), one-way ANOVA with post-hoc Tukey (E-H, O, P) or Dunnett's (S, T) test, or one-tailed paired t test with Holm adjustment (I-L, Q, R, U, V). Values are means ± SEM.


Figure 7 ( A - C ) One cohort of mice was assessed with the social approach test at young ( A ) and old ( B ) age and another cohort of mice at middle age ( C ) . Mice were placed individually into the empty center chamber of a three - chamber apparatus . One side - chamber contained an empty cup , and the other an identical cup with an unfamiliar live mouse in it . Close contact with each cup was monitored for 10 min . n = 12 ( A , B ) or 17 ( C ) NTG ; 13 ( A ) or 10 ( B ) TRE - hTau - A152T ( L1 ) ; 12 ( A ) , 9 ( B ) , or 12 ( C ) CamKII - tTA ; 13 ( C ) hTau - WT ( L32 ) ; and 14 ( A ) , 12 ( B ) , or 10 ( C ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - tailed paired t test with Holm adjustment . Values are means ± SEM .

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

Figure 7(A-C) One cohort of mice was assessed with the social approach test at young (A) and old (B) age and another cohort of mice at middle age (C). Mice were placed individually into the empty center chamber of a three-chamber apparatus. One side-chamber contained an empty cup, and the other an identical cup with an unfamiliar live mouse in it. Close contact with each cup was monitored for 10 min. n = 12 (A, B) or 17 (C) NTG; 13 (A) or 10 (B) TRE-hTau-A152T(L1); 12 (A), 9 (B), or 12 (C) CamKII-tTA; 13 (C) hTau-WT(L32); and 14 (A), 12 (B), or 10 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-tailed paired t test with Holm adjustment. Values are means ± SEM.


Figure 8 ( A - F ) Coronal brain sections obtained from mice at 4 - 6 ( A - C ) or 20 - 23 ( D - F ) months of age were immunostained for NeuN . The number of NeuN - positive cells with neuronal morphology was determined in dentate gyrus ( A , D ) , CA3 ( B , E ) , and CA1 ( C , F ) . n = 11 ( A - C ) or 18 ( D - F ) NTG ; 11 ( A - C ) or 5 ( D - F ) CamKII - tTA ; 7 ( A - C ) or 9 ( D - F ) hTau - WT ( L32 ) ; and 14 ( A - C ) or 10 ( D - F ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - way ANOVA and Tukey test . Values are means ± SEM .

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

Figure 8(A-F) Coronal brain sections obtained from mice at 4-6 (A-C) or 20-23 (D-F) months of age were immunostained for NeuN. The number of NeuN-positive cells with neuronal morphology was determined in dentate gyrus (A, D), CA3 (B, E), and CA1 (C, F). n = 11 (A-C) or 18 (D-F) NTG; 11 (A-C) or 5 (D-F) CamKII-tTA; 7 (A-C) or 9 (D-F) hTau-WT(L32); and 14 (A-C) or 10 (D-F) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey test. Values are means ± SEM.


Figure 9fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy fibers in acute hippocampal slices . ( A ) Representative fEPSP traces from slices of 4 - 8 - month - old mice . ( B , C ) Input / output curves reflecting the change in the fEPSP slope as a function of the fiber volley amplitude from slices obtained at 4 - 8 ( B ) or 20 ( C ) months of age . ( D , E ) Short - term plasticity assessed by measuring the PPR ( second pulse / first pulse ) with 50 - msec interpulse interval at 4 - 8 ( D ) or 20 ( E ) months of age . ( F ) Mossy fiber LTP at 20 months of age . n ( mice / slices ) = 5 - 9 / 8 - 21 ( NTG ) , 2 - 5 / 5 - 12 ( CaMKII - tTA ) , 3 - 6 / 8 - 14 ( TRE - hTau ) , 3 - 6 / 8 - 14 ( hTau - WT ) , and 5 - 6 / 8 - 14 ( hTau - A152T ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus NTG by linear regression analysis with Holm adjustment ( B , C ) or by one - way ANOVA and Dunnett ' s test ( D , E ) . No significant difference was detected by two - way ANOVA in ( F ) . Values are means ± SEM .

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

Figure 9fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy fibers in acute hippocampal slices. (A) Representative fEPSP traces from slices of 4-8-month-old mice. (B, C) Input/output curves reflecting the change in the fEPSP slope as a function of the fiber volley amplitude from slices obtained at 4-8 (B) or 20 (C) months of age. (D, E) Short-term plasticity assessed by measuring the PPR (second pulse/first pulse) with 50-msec interpulse interval at 4-8 (D) or 20 (E) months of age.(F) Mossy fiber LTP at 20 months of age. n (mice/slices) = 5-9/8-21 (NTG), 2-5/5-12 (CaMKII-tTA), 3-6/8-14 (TRE-hTau), 3-6/8-14 (hTau-WT), and 5-6/8-14 (hTau-A152T). *P < 0.05, **P < 0.01, ***P < 0.001 versus NTG by linear regression analysis with Holm adjustment (B, C) or by one-way ANOVA and Dunnett's test (D, E). No significant difference was detected by two-way ANOVA in (F). Values are means ± SEM.


Figure 10 ( A - C ) Subdural EEG recordings from freely behaving mice at 4 - 9 months of age before ( A top trace , B ) and after ( A bottom trace , C ) injection of PTZ at a dose ( 30 mg / kg ) that did not produce convulsions . ( A ) Representative EEG traces from an hTau - A152T ( L1 ) mouse . Arrows indicate epileptic discharges . ( B , C ) Quantitation of spikes per hour at baseline ( B ) and of spikes per 20 - min intervals after PTZ injection ( C ) . n = 31 ( B ) or 23 ( C ) NTG ; 13 ( B ) or 11 ( C ) CaMKII - tTA ; 12 ( B ) or 10 ( C ) hTau - WT ( L32 ) ; and 22 ( B ) or 21 ( C ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Welch ' s one - way ANOVA and two - tailed Welch ' s t test with Holm adjustment ( B ) or by two - way repeated - measures ANOVA and Tukey test ( C ) . Values are means ± SEM .

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

Figure 10(A-C) Subdural EEG recordings from freely behaving mice at 4-9 months of age before (A top trace, B) and after (A bottom trace, C) injection of PTZ at a dose (30 mg/kg) that did not produce convulsions. (A) Representative EEG traces from an hTau-A152T(L1) mouse. Arrows indicate epileptic discharges. (B, C) Quantitation of spikes per hour at baseline (B) and of spikes per 20-min intervals after PTZ injection (C). n = 31 (B) or 23 (C) NTG; 13 (B) or 11 (C) CaMKII-tTA; 12 (B) or 10 (C) hTau-WT(L32); and 22 (B) or 21 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch's one-way ANOVA and two-tailed Welch's t test with Holm adjustment (B) or by two-way repeated-measures ANOVA and Tukey test (C). Values are means ± SEM.


Figure 11 ( A - D ) Numbers of offspring from crosses of hAPP - J20 mice with hTau - A152T ( L1 ) ( A , B ) or hTau - WT ( L32 ) ( C , D ) mice that were ( A , C ) or were not ( B , D ) treated with DOX . ( E ) Numbers of offspring from crosses of lower expresser hAPP - J9 mice with hTau - A152T ( L1 ) mice that were not treated with DOX . n = 141 ( A ) , 112 ( B ) , 94 ( C ) , 83 ( D ) , and 282 ( E ) mice per cohort . Yields from two crosses indicated significant deviations from Mendelian inheritance : hAPP - J20 x TRE - hTau - A152T ( L1 ) x CaMKII - tTA without DOX ( Holm - adjusted P = 0 . 003 , unadjusted P = 0 . 0008 ) and hAPP - J9 x TRE - hTau - WT ( L32 ) x CaMKII - tTA without DOX ( adjusted and unadjusted P < 0 . 0001 ) by chi - square goodness - of - fit test . ( F ) Subdural EEG recordings from untreated 3 - 10 - month - old mice were analyzed to compare their number of spikes at baseline . n = 8 NTG , 6 hAPP - J9 , 6 hTau - A152T ( L1 ) , and 8 hAPP - J9 / hTau - A152T ( L1 ) . * P < 0 . 05 , * * P < 0 . 01 by logistic regression analysis for three - way interaction of the three transgenes with Holm adjustment ( A - E ) or by two - tailed Welch ' s t test with Holm adjustment ( F ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 11(A-D) Numbers of offspring from crosses of hAPP-J20 mice with hTau-A152T(L1) (A, B) or hTau-WT(L32) (C, D) mice that were (A, C) or were not (B, D) treated with DOX. (E) Numbers of offspring from crosses of lower expresser hAPP-J9 mice with hTau-A152T(L1) mice that were not treated with DOX. n = 141 (A), 112 (B), 94 (C), 83 (D), and 282 (E) mice per cohort. Yields from two crosses indicated significant deviations from Mendelian inheritance: hAPP-J20 x TRE-hTau-A152T(L1) x CaMKII-tTA without DOX (Holm-adjusted P = 0.003, unadjusted P = 0.0008) and hAPP-J9 x TRE-hTau-WT(L32) x CaMKII-tTA without DOX (adjusted and unadjusted P < 0.0001) by chi-square goodness-of-fit test.(F) Subdural EEG recordings from untreated 3-10-month-old mice were analyzed to compare their number of spikes at baseline. n = 8 NTG, 6 hAPP-J9, 6 hTau-A152T(L1), and 8 hAPP-J9/hTau-A152T(L1). *P < 0.05, **P < 0.01 by logistic regression analysis for three-way interaction of the three transgenes with Holm adjustment (A-E) or by two-tailed Welch's t test with Holm adjustment (F).


Figure 2E . Naïve MCF7 cells and MCF7aTT cells were exposed to the indicated stress conditions for 3 h , and cell viability was assessed by staining dead cells with trypan blue . The bar graphs represent the percentage of trypan blue excluding cells . ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) .

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Figure 2E. Naïve MCF7 cells and MCF7aTT cells were exposed to the indicated stress conditions for 3 h, and cell viability was assessed by staining dead cells with trypan blue. The bar graphs represent the percentage of trypan blue excluding cells. (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).


Figure 3A . Whole lysates of MCF7 and MCF7aTT cells exposed to heat shock and / or proteasome ( MG132 , 20 μM , 1h ) and HSP70 ( VER - 155008 , 50 μM , 1h ) inhibitors were prepared as described in Materials and Methods . Total lysate , soluble fractions and insoluble fractions were stained by SYPRO Ruby ( left ) and quantified ( right ) . ( means + / - SD , n = 3 , numbers above the bars indicate p values calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) . C . The soluble and insoluble fractions shown in ( A ) were assayed for the level of K48 - linked polyubiquitin , and polyubiquitylated proteins were quantified ( blue line graph , means + / - SEM , n = 4 , numbers above the bars indicate p values ) . The data for bulk protein obtained in ( A ) were overlaid onto the graph for comparison ( red line , means + / - SEM , n = 3 ) .

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Figure 3A. Whole lysates of MCF7 and MCF7aTT cells exposed to heat shock and/or proteasome (MG132, 20 μM, 1h) and HSP70 (VER-155008, 50 μM, 1h) inhibitors were prepared as described in Materials and Methods. Total lysate, soluble fractions and insoluble fractions were stained by SYPRO Ruby (left) and quantified (right). (means +/- SD, n = 3, numbers above the bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).C. The soluble and insoluble fractions shown in (A) were assayed for the level of K48-linked polyubiquitin, and polyubiquitylated proteins were quantified (blue line graph, means +/- SEM, n = 4, numbers above the bars indicate p values). The data for bulk protein obtained in (A) were overlaid onto the graph for comparison (red line, means +/- SEM, n = 3).


Figure 4B . The same experiment as described in ( A ) but with MCF7 cells exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) . Means + / - SEM , n = 2 . C . Lysate from MCF7 cells exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) as indicated were subjected to immunoprecipitation with anti - puromycin antibodies , followed by assaying for co - precipitation of K48 - ubiquitylated proteins . Actin is shown as a reference .

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Figure 4B. The same experiment as described in (A) but with MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min). Means +/- SEM, n = 2. C. Lysate from MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min) as indicated were subjected to immunoprecipitation with anti-puromycin antibodies, followed by assaying for co-precipitation of K48-ubiquitylated proteins. Actin is shown as a reference.


Figure 5B . Same experiment as in ( A ) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER - 155008 ( 50 μM , 1h ) or MG132 ( 20 μM , 1h ) . The signal obtained for ribosomal protein RPS19 and RPL7 are shown for reference , documenting residual polysomes despite the flat UV trace . Right panels are quantifications of K48 - ub ( means + / - SEM , n = 2 ) , black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity . The bar graphs represent the P / M ratios ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) . C . Sucrose density gradient fractions described in ( B ) were assayed for partitioning of HSPA1 and HSPH1 by immunoblotting . HSPA1 data were quantified in a line graph ( means + / - SEM , n = 4 ) . D D . Cell lysate was spun through a 30 % sucrose cushion to pellet polysomes . Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA ( see Appendix Figures S3 ) . Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48 - linked polyubiquitin . The levels of K48 - linked polyubiquitin were quantified ( means + / - SD , n = 3 ) . Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines .

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Figure 5B. Same experiment as in (A) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER-155008 (50 μM, 1h) or MG132 (20 μM, 1h). The signal obtained for ribosomal protein RPS19 and RPL7 are shown for reference, documenting residual polysomes despite the flat UV trace. Right panels are quantifications of K48-ub (means +/- SEM, n = 2), black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The bar graphs represent the P/M ratios (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). C. Sucrose density gradient fractions described in (B) were assayed for partitioning of HSPA1 and HSPH1 by immunoblotting. HSPA1 data were quantified in a line graph (means +/- SEM, n = 4). D D. Cell lysate was spun through a 30% sucrose cushion to pellet polysomes. Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA (see Appendix Figures S3). Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48-linked polyubiquitin. The levels of K48-linked polyubiquitin were quantified (means +/- SD, n = 3). Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines.


Figure 6D . Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells , and puromycin labeled peptides were pull down as illustrated in the scheme . Precipitates were assayed for the presence of K48 - linked polyubiquitin , chaperones , and proteasome subunits by immunoblotting .

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Figure 6D. Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells, and puromycin labeled peptides were pull down as illustrated in the scheme. Precipitates were assayed for the presence of K48-linked polyubiquitin, chaperones, and proteasome subunits by immunoblotting.


Figure 7E . Whole lysates of cells KYSE150 and KYSE150HSPH1 - / - cells exposed to the indicated temperature conditions ( aTT scheme and heat shock ) were fractionated into soluble and insoluble fractions as described in Materials and Methods . Insoluble fractions were assayed for the level of K48 - linked polyubiquitin ( left panel ) . Total levels of HSPA1 and HSPH1 are shown for reference . K48 ubiquitin signals were quantified and displayed in a bar graph ( means + / - SD , n = 4 ) . F . Lysates of cells KYSE150 and KYSE150HSPH1 - / - cells exposed to the indicated temperature conditions ( aTT scheme and heat shock ) were obtained and polysomes were isolated by centrifugation through a 30 % sucrose cushion . Whole cell lysates ( left panel ) and the polysomal pellets ( right panel ) were assayed by immunoblotting for the levels of HSPH1 , HSPA1 and proteasome subunits .

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Figure 7E. Whole lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were fractionated into soluble and insoluble fractions as described in Materials and Methods. Insoluble fractions were assayed for the level of K48-linked polyubiquitin (left panel). Total levels of HSPA1 and HSPH1 are shown for reference. K48 ubiquitin signals were quantified and displayed in a bar graph (means +/- SD, n = 4).F. Lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were obtained and polysomes were isolated by centrifugation through a 30% sucrose cushion. Whole cell lysates (left panel) and the polysomal pellets (right panel) were assayed by immunoblotting for the levels of HSPH1, HSPA1 and proteasome subunits.


Figure 8A . Representative micrographs of tissue sections derived from oral squamous cell carcinomas ( OSCC ) and esophageal squamous cell carcinomas ( ESCC ) stained with the indicated antibodies by immunohistochemistry ( IHC ) . Top panels show routine H & E staining . Black squares point to regions magnified in the adjacent panels to the right . C . Kaplan - Meier plots showing the correlation between HSPH1 mRNA expression level and the survival of patients with ESCC . The expression data was obtained from and visualized with KM Plotter ( www . kmplot . com , ( Nagy et al , 2018 ) using the Pan Cancer algorithm at default settings . D . KYSE150 and KYSE150HSPH1 - / - cells were injected into nude mice , and xenograft tumor growth was monitored over time ( left panel ; mean tumor volume + / - SEM , n = 4 with 5 animals per n ) . Mean tumor weights at the end of the experiments were determined ( means + / - SD , n = 4 with 5 animals per n , number above the bar graph represent p values , * p < 0 . 05 , * * p < 0 . 01 ( calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) ) .

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Figure 8A. Representative micrographs of tissue sections derived from oral squamous cell carcinomas (OSCC) and esophageal squamous cell carcinomas (ESCC) stained with the indicated antibodies by immunohistochemistry (IHC). Top panels show routine H&E staining. Black squares point to regions magnified in the adjacent panels to the right.C. Kaplan-Meier plots showing the correlation between HSPH1 mRNA expression level and the survival of patients with ESCC. The expression data was obtained from and visualized with KM Plotter (www.kmplot.com, (Nagy et al, 2018) using the Pan Cancer algorithm at default settings.D. KYSE150 and KYSE150HSPH1-/- cells were injected into nude mice, and xenograft tumor growth was monitored over time (left panel; mean tumor volume +/- SEM, n = 4 with 5 animals per n). Mean tumor weights at the end of the experiments were determined (means +/- SD, n = 4 with 5 animals per n, number above the bar graph represent p values, * p<0.05, **p<0.01 (calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli)).