TOC protein levels were regulated by autophagy in response to short - term heat stress . Eight - day - old plants of indicated genotypes grown in vitro were subjected to short term heat stress , recovered for the indicated times , and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane ( B , upper ) . Coomassie staining indicated that the treatments did not change the overall protein profile ( B , lower ) . Band intensities were quantified and normalized to equivalent Actin data ( C ) . Time 0 was taken as 100 % . Data are means ± SEM from three biological replicates . Unpaired two - tailed Student ' s t - tests revealed that the Toc159 protein level in nbr1 - 2 ( P < 0 . 05 ) and NBR1 - OX ( P < 0 . 01 ) , and the Toc75 protein level in NBR1 - OX ( P < 0 . 05 ) , were significantly different from levels in the WT at Time 12 h ; and that the Toc159 protein level in atg7 - 2 ( P < 0 . 001 ) , nbr1 - 2 ( P < 0 . 01 ) , and NBR1 - OX ( P < 0 . 001 ) , and the Toc75 protein level in atg7 - 2 ( P < 0 . 01 ) , nbr1 - 2 ( P < 0 . 01 ) , and NBR1 - OX ( P < 0 . 01 ) , were significantly different from levels in the WT at Time 24 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O 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-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

TOC protein levels were regulated by autophagy in response to short-term heat stress. Eight-day-old plants of indicated genotypes grown in vitro were subjected to short term heat stress, recovered for the indicated times, and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane (B, upper). Coomassie staining indicated that the treatments did not change the overall protein profile (B, lower). Band intensities were quantified and normalized to equivalent Actin data (C). Time 0 was taken as 100%. Data are means ± SEM from three biological replicates. Unpaired two-tailed Student's t-tests revealed that the Toc159 protein level in nbr1-2 (P < 0.05) and NBR1-OX (P < 0.01), and the Toc75 protein level in NBR1-OX (P < 0.05), were significantly different from levels in the WT at Time 12 h; and that the Toc159 protein level in atg7-2 (P < 0.001), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.001), and the Toc75 protein level in atg7-2 (P < 0.01), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.01), were significantly different from levels in the WT at Time 24 h.


Analysis of protein import efficiency in vitro . Chloroplasts were isolated from 12 - day - old WT , nbr1 - 2 and NBR1 - OX seedlings grown in vitro under normal condition , and were subjected to in vitro protein import assay by using 35S - labeled Rubisco small subunit ( SSU ) precursor protein as the import substrate , followed by corresponding quantification of the maturation of the radiolabeled precursor protein . A representative phosphor screen image is shown ( upper left ) , and Coomassie staining of Rubisco large subunit ( LSU ) indicated equal loading ( lower left ) ; Times indicate minutes after the start of each import reaction . IVT , in vitro translation product used as a control . Together with similar images from two additional experiments , this was used to conduct the quantitative analysis shown ( right ) ; the amount of imported , mature protein in each sample was expressed as a percentage of that present at the final time point for the WT . Data are means ± SEM from three biological replicates .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of protein import efficiency in vitro. Chloroplasts were isolated from 12-day-old WT, nbr1-2 and NBR1-OX seedlings grown in vitro under normal condition, and were subjected to in vitro protein import assay by using 35S-labeled Rubisco small subunit (SSU) precursor protein as the import substrate, followed by corresponding quantification of the maturation of the radiolabeled precursor protein. A representative phosphor screen image is shown (upper left), and Coomassie staining of Rubisco large subunit (LSU) indicated equal loading (lower left); Times indicate minutes after the start of each import reaction. IVT, in vitro translation product used as a control. Together with similar images from two additional experiments, this was used to conduct the quantitative analysis shown (right); the amount of imported, mature protein in each sample was expressed as a percentage of that present at the final time point for the WT. Data are means ± SEM from three biological replicates.


Chloroplast development in WT , nbr1 - 2 and NBR1 - OX seedlings under UVB stress was studied . Cotyledons of eight - day - old plants grown in vitro under normal condition ( D ) or UVB stress ( E ) were analyzed by transmission electron microscopy , and representative images are shown . UVB stress was conducted in the same procedures as in C . In D and E , the upper images are at the same magnification ( scale bar , 2 μm ) ; and below these are high magnification ( 5 × ) images corresponding to the boxed regions above . These and similar micrographs of chloroplasts were used to determine chloroplast shape ( F ) , the number of thylakoid lamellae per granum ( G ) , and the number of starch granules per chloroplast ( H ) in each genotype , with or without UVB stress . Asterisks indicate significance according to an unpaired two - tailed Student ' s t - test ( * , significant at P < 0 . 05 ; * * * , significant at P < 0 . 001 ; * * * * , significant at P < 0 . 0001 ) . Values are means ± SEM from 30 - 50 chloroplasts .

O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chloroplast development in WT, nbr1-2 and NBR1-OX seedlings under UVB stress was studied. Cotyledons of eight-day-old plants grown in vitro under normal condition (D) or UVB stress (E) were analyzed by transmission electron microscopy, and representative images are shown. UVB stress was conducted in the same procedures as in C. In D and E, the upper images are at the same magnification (scale bar, 2 μm); and below these are high magnification (5×) images corresponding to the boxed regions above. These and similar micrographs of chloroplasts were used to determine chloroplast shape (F), the number of thylakoid lamellae per granum (G), and the number of starch granules per chloroplast (H) in each genotype, with or without UVB stress. Asterisks indicate significance according to an unpaired two-tailed Student's t-test (*, significant at P < 0.05; ***, significant at P < 0.001; ****, significant at P < 0.0001). Values are means ± SEM from 30-50 chloroplasts.


Phenotypes of two - week - old WT , nbr1 - 2 , ppi1 and ppi1 nbr1 - 2 seedlings grown on soil . Representative plant images ( A ) and chlorophyll contents ( B ) are shown . The chlorophyll values for the ppi1 and ppi1 nbr1 - 2 plants were statistically significantly different ( Student ' s t test ; * * * * , significant at P < 0 . 0001 ) . Values are means ± SEM from 20 plants .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Phenotypes of two-week-old WT, nbr1-2, ppi1 and ppi1 nbr1-2 seedlings grown on soil. Representative plant images (A) and chlorophyll contents (B) are shown. The chlorophyll values for the ppi1 and ppi1 nbr1-2 plants were statistically significantly different (Student's t test; ****, significant at P < 0.0001). Values are means ± SEM from 20 plants.


Chloroplast development in ppi1 and ppi1 nbr1 - 2 seedlings was studied . Cotyledons of two - week - old plants grown under normal condition as in A were analyzed by transmission electron microscopy , and representative images are shown ( E ) . In E , the upper images are at the same magnification ( scale bar , 1 μm ) ; and below these are high magnification ( 3 × ) images corresponding to the boxed regions above . These and similar micrographs of chloroplasts were used to determine chloroplast size ( F ) and the number of thylakoid lamellae per granum ( G ) in each genotype . Asterisks indicate significance according to an unpaired two - tailed Student ' s t - test ( * * * * , significant at P < 0 . 0001 ) . Values are means ± SEM from 30 - 40 chloroplasts .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chloroplast development in ppi1 and ppi1 nbr1-2 seedlings was studied. Cotyledons of two-week-old plants grown under normal condition as in A were analyzed by transmission electron microscopy, and representative images are shown (E). In E, the upper images are at the same magnification (scale bar, 1 μm); and below these are high magnification (3×) images corresponding to the boxed regions above. These and similar micrographs of chloroplasts were used to determine chloroplast size (F) and the number of thylakoid lamellae per granum (G) in each genotype. Asterisks indicate significance according to an unpaired two-tailed Student's t-test (****, significant at P < 0.0001). Values are means ± SEM from 30-40 chloroplasts.


( A ) Comparative expression during murine pre - implantation embryo development of Dppa2 ( red ) , Dppa4 ( blue ) , Dux ( black ) , and Zscan4c ( ZGA marker , green ) . Each dot represents the average value of single - cell RNA -

O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(A) Comparative expression during murine pre-implantation embryo development of Dppa2 (red), Dppa4 (blue), Dux (black), and Zscan4c (ZGA marker, green). Each dot represents the average value of single-cell RNA-seq


( C ) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes , early 2C , mid 2C and late 2C Arcs depict splicing events , and numbers their relative frequency ( i . e . reads across junctions ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes, early 2C, mid 2C and late 2C Arcs depict splicing events, and numbers their relative frequency (i.e. reads across junctions).


( A ) Average expression of Dppa2 and Dppa4 in a single - cell RNA - seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters , respectively , and the double - negative population .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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-CONTROLLED_VAR O O O O O O O O O O O

(A) Average expression of Dppa2 and Dppa4 in a single-cell RNA-seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.


( B ) Fraction of GFP + cells in mESCs carrying a MERVL - GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control . Bars represent the average , error bars the SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - test .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Fraction of GFP+ cells in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


( C ) Dppa2 , Dppa4 , Dux and Zscan4 gene expression in mESCs carrying a MERVL - GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control . Expression was normalized to Actb . Bars represent the average , error bars the SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - test .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Dppa2, Dppa4, Dux and Zscan4 gene expression in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


RNA - seq analysis of WT and Dppa2 ( D ) KO mESC clones . The dot plot displays the average gene expression of three independent clones from each cell type ; n = 3 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq analysis of WT and Dppa2 (D) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


RNA - seq analysis of WT and Dppa4 ( E ) KO mESC clones . The dot plot displays the average gene expression of three independent clones from each cell type ; n = 3 .

RNA-seq analysis of WT and Dppa4 (E) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


( F ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 and Tdpoz4 ) , and two DUX - independent targets of DPPA2 and DPPA4 ( Mael and Tdrd1 ) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control . Expression was normalized to Actb . Bars represent the average , error bars the SD ; n =

O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(F) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and two DUX-independent targets of DPPA2 and DPPA4 (Mael and Tdrd1) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3


( H ) Comparative expression of DUX - dependent , DPPA2 and DPPA4 - dependent , and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters , respectively , and the double - negative population ( unpaired t - test ) .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(H) Comparative expression of DUX-dependent, DPPA2 and DPPA4-dependent, and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population (unpaired t-test).


( A ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 and Tdpoz4 ) , and three targets of DPPA2 and DPPA4 independent of DUX ( Mael , Tdrd1 , Prex2 ) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2 , DPPA4 or GFP as a control Expression was normalized to Actb . Bars represent the average , error bars the SD ; n = 3 . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - test .

O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and three targets of DPPA2 and DPPA4 independent of DUX (Mael, Tdrd1, Prex2) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2, DPPA4 or GFP as a control Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


( B ) Average coverage normalized for sequencing depth of the ChIP - seq signal of DPPA2 , DPPA4 or DPPA4 truncated of the SAP domain overexpressed in mESCs in a window of 500bp around the Dux gene ( n = 2 ) . Total input ( TI ) is shown in gray . Peaks over the Dux gene were called in DPPA2 , DPPA4 and DPPA4ΔSAP ChIP - seq .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O

(B) Average coverage normalized for sequencing depth of the ChIP-seq signal of DPPA2, DPPA4 or DPPA4 truncated of the SAP domain overexpressed in mESCs in a window of 500bp around the Dux gene (n=2). Total input (TI) is shown in gray. Peaks over the Dux gene were called in DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq.


( C ) Heatmap showing the distribution of DUX , DPPA2 , DPPA4 and DPPA4ΔSAP coverage in a ± 5kb window around the TSS of DUX - dependent , DPPA2 and DPPA4 - dependent and random genes in mESCs .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Heatmap showing the distribution of DUX, DPPA2, DPPA4 and DPPA4ΔSAP coverage in a ± 5kb window around the TSS of DUX-dependent, DPPA2 and DPPA4-dependent and random genes in mESCs.


( D ) Boxplot / Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2 , DPPA4 and DUX ChIP , and in a random shuffle of peaks .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(D) Boxplot/Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2, DPPA4 and DUX ChIP, and in a random shuffle of peaks.


Total expression of transposable element subfamilies in ( A ) Dppa2 KO compared to WT mESC clones .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

Total expression of transposable element subfamilies in (A) Dppa2 KO compared to WT mESC clones.


Total expression of transposable element subfamilies in ( B ) Dppa4 KO compared to WT mESC clones .

Total expression of transposable element subfamilies in (B) Dppa4 KO compared to WT mESC clones.


( C ) RNA - seq analysis of MERVL - int , L1Md _ A and L1Md _ T in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters , respectively , and the double - negative population .

O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O

(C) RNA-seq analysis of MERVL-int, L1Md_A and L1Md_T in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.


( D ) Heatmap displaying the enrichment of DUX , DPPA2 , DPPA4 and DPPA4ΔSAP binding at different transposable element families . Every subfamily enriched ( pval < 0 . 05 ) in at least one DPPA2 , DPPA4 or DPPA4ΔSAP replicate are shown .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O

(D) Heatmap displaying the enrichment of DUX, DPPA2, DPPA4 and DPPA4ΔSAP binding at different transposable element families. Every subfamily enriched (pval < 0.05) in at least one DPPA2, DPPA4 or DPPA4ΔSAP replicate are shown.


( E ) MSA plot of DPPA2 , DPPA4 and DPPA4ΔSAP ChIP - seq in mESCs showing enrichment over L1Md _ A , L1Md _ T and MERVL - int sequences . Gaps are in grey and sequences in white with overlap of color - coded ChIP - seq signals . Upper plot shows average coverage of the signal over the aligned TEs .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) MSA plot of DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq in mESCs showing enrichment over L1Md_A, L1Md_T and MERVL-int sequences. Gaps are in grey and sequences in white with overlap of color-coded ChIP-seq signals. Upper plot shows average coverage of the signal over the aligned TEs.


A . Immunofluorescence of SATB2 ( green ) and the GSC marker SOX2 or OLIG2 ( red ) on frozen tissue sections of human GBM surgical specimens . SATB2 is preferentially expressed by GSCs in human GBMs . Scale bar , 25 μm . B . Quantification of the fraction of SATB2 + cells in SOX2 + or OLIG2 + cells in human GBMs . More than 90 % SOX2 + or OLIG2 + cells showed SATB2 staining . n = 3 GBMs .

O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O

A. Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm. B. Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n=3 GBMs.


C . qPCR analysis of SATB2 mRNA expression in GSCs and matched non - stem tumor cells ( NSTCs ) ( n = 5 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

C. qPCR analysis of SATB2 mRNA expression in GSCs and matched non-stem tumor cells (NSTCs) (n=5).


D . Immunoblot analysis of SATB2 , SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O

D. Immunoblot analysis of SATB2, SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs.


E . Immunofluorescenc of SATB2 ( green ) and SOX2 ( red ) in T3359 GSCs and matched NSTCs . Scale bar , 50 μm .

O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

E. Immunofluorescenc of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm.


F . qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells ( NPCs ) ( n = 3 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

F. qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n=3).


G . Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

G. Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs.


H . Immunoblot analysis of SATB2 , GSC marker SOX2 and differentiation marker GFAP expression during serum - induced GSC differentiation .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O

H. Immunoblot analysis of SATB2, GSC marker SOX2 and differentiation marker GFAP expression during serum-induced GSC differentiation.


A . Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral - mediated non - targeting shRNA ( shNT ) or SATB2 shRNA ( shSATB2 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

A. Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral-mediated non-targeting shRNA (shNT) or SATB2 shRNA (shSATB2).


B . Cell viability of GSCs transduced with shNT or shSATB2 ( n = 5 ) .

B. Cell viability of GSCs transduced with shNT or shSATB2 (n=5).


C . EdU incorporation assay of GSCs transduced with shNT or shSATB2 . Scale bar : 50 μm . D . Quantification of ( C ) showing the percentage of EdU + cells ( n = 5 ) .

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

C. EdU incorporation assay of GSCs transduced with shNT or shSATB2. Scale bar: 50 μm. D. Quantification of (C) showing the percentage of EdU+ cells (n=5).


E . Tumorsphere images of GSCs transduced with shNTor shSATB2 . Scale bar : 100 μm . Quantification of the diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) .

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

E. Tumorsphere images of GSCs transduced with shNTor shSATB2. Scale bar: 100 μm. Quantification of the diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).


G . Quantification of the number of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) .

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

G. Quantification of the number of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).


H . In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2 . Silencing SATB2 attenuated the self - renewal capacity of GSCs .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

H. In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2. Silencing SATB2 attenuated the self-renewal capacity of GSCs.


A . Bioluminescent images of the GBM xenografts derived from the luciferase - labeled T3359 GSCs expressing NT or SATB2 shRNA . Representative images on day 21 posttransplantation are shown ( n = 5 mice per group ) . Silencing SATB2 significantly delayed GBM growth .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth .


A . Bioluminescent images of the GBM xenografts derived from the luciferase - labeled T3359 GSCs expressing NT or SATB2 shRNA . Representative images on day 21 posttransplantation are shown ( n = 5 mice per group ) . Silencing SATB2 significantly delayed GBM

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth


C . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 ( shNT : n = 7 mice ; shSATB2 - 1 or shSATB2 - 2 : n = 5 mice ) . Median survival : shNT , 28 days ; shSATB2 - 1 , 48 days ; shSATB2 - 2 , 45 Days . Animals bearing GSC - derived xenografts expressing SATB2 shRNA survived longer than the control animals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O

C. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 (shNT: n=7 mice; shSATB2-1 or shSATB2-2: n=5 mice). Median survival: shNT, 28 days; shSATB2-1, 48 days; shSATB2-2, 45 Days. Animals bearing GSC-derived xenografts expressing SATB2 shRNA survived longer than the control animals.


D . Immunofluorescence of SATB2 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . E . Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) .

O O O O B-MEASURED_VAR O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. E. Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).


D . Immunofluorescence of SATB2 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.


F . Immunofluorescence of Ki67 ( Green ) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . G . Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) .

O O O O B-MEASURED_VAR O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. G. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).


F . Immunofluorescence of Ki67 ( Green ) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.


H . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) . Scale bar : 40 μm . I . Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) .

O O O O B-MEASURED_VAR O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm. I. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group).


H . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) . Scale bar : 40 μm .

H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm.


A . Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs ( shSATB2 ) and control H2S GSCs ( shNT ) . Differentially expressed genes had a 1 . 8 - fold or greater expression difference . Among differentially expressed genes , 160 are upregulated and 185 are downregulated .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT). Differentially expressed genes had a 1.8-fold or greater expression difference. Among differentially expressed genes, 160 are upregulated and 185 are downregulated.


C . qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 ( n = 3 ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

C. qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 (n=3).


D . Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

D. Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2.


E . Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts - derived from T3359 GSCs expressing shNT or shSATB2 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

E. Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts-derived from T3359 GSCs expressing shNT or shSATB2.


F . Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs ( shSATB2 ) and control H2S GSCs ( shNT ) from microarray analysis . Differentially expressed genes had a 1 . 5 - fold or greater expression difference .

O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT) from microarray analysis. Differentially expressed genes had a 1.5-fold or greater expression difference.


G . qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 ( n = 3 ) .

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

G. qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 (n=3).


A . Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A. Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2.


B . Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O

B. Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing.


C . Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O

C. Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing.


In vivo bioluminescent images of the tumor xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative images on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

In vivo bioluminescent images of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.


E . In vivo bioluminescent images or quantification of the tumor xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative images on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

E. In vivo bioluminescent images or quantification of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.


F . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 8 mice ; shNT + FOXM1 : n = 7 mice ; shSATB2 : n = 6 mice ; shSATB2 + FOXM1 : n = 8 mice ) . Median survival : shNT , 30 days ; shNT + FOXM1 , 28 days ; shSATB2 , 50 . 5 Days ; shSATB2 + FOXM1 , 34 days . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC - derived GBMs .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

F. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=8 mice; shNT+FOXM1: n=7 mice; shSATB2: n=6 mice; shSATB2+ FOXM1: n=8 mice). Median survival: shNT, 30 days; shNT+FOXM1, 28 days; shSATB2, 50.5 Days; shSATB2+FOXM1, 34 days. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC-derived GBMs.


G . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . H . Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. H. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).


G . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.


I . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . J . Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) .

I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. J. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).


I . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm .

I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.


B . ChIP assays with the SATB2 antibody or IgG using GSCs , NSTCs and NPCs . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Note that abundant SATB2 binds to the MAR of FOXM1 gene locus in GSCs .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O

B. ChIP assays with the SATB2 antibody or IgG using GSCs, NSTCs and NPCs. PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Note that abundant SATB2 binds to the MAR of FOXM1 gene locus in GSCs.


C . qPCR analysis of ChIP assays with the SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Silencing SATB2 decreased its binding amount to the MAR of FOXM1 gene locus .

O O O O O O O O O B-MEASURED_VAR O 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-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O

C. qPCR analysis of ChIP assays with the SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 decreased its binding amount to the MAR of FOXM1 gene locus.


D . CoIP assays of endogenous protein interaction in GSCs . Immunoblots of precipitated proteins or total lysates were performed using indicated antibodies . Note that SATB2 associates with endogenous CBP while not P300 .

O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O

D. CoIP assays of endogenous protein interaction in GSCs. Immunoblots of precipitated proteins or total lysates were performed using indicated antibodies. Note that SATB2 associates with endogenous CBP while not P300.


E . qPCR analysis of ChIP assays with the CBP antibody or IgG using GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Silencing SATB2 reduced the binding of CBP to the MAR of FOXM1 gene locus .

O O O O O O O O O B-MEASURED_VAR O 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-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O

E. qPCR analysis of ChIP assays with the CBP antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced the binding of CBP to the MAR of FOXM1 gene locus.


F . qPCR analysis of ChIP assays with the indicated antibody ( AcH3K18 , AcH3K27 , AcH4 ) using T3359 GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Silencing SATB2 reduced acetylation of H3K18 , H3K27 and H4 levels on the MAR of FOXM1 locus .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR 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-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O

F. qPCR analysis of ChIP assays with the indicated antibody (AcH3K18, AcH3K27, AcH4) using T3359 GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced acetylation of H3K18, H3K27 and H4 levels on the MAR of FOXM1 locus.


G . qPCR analysis of FOXM1 mRNA expression in T3359 GSCs transduced with shSATB2 or shCBP or both ( n = 3 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

G. qPCR analysis of FOXM1 mRNA expression in T3359 GSCs transduced with shSATB2 or shCBP or both (n=3).


H . Immunoblot analysis of FOXM1 expression in T3359 GSCs transduced with shSATB2 or shCBP or both .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O

H. Immunoblot analysis of FOXM1 expression in T3359 GSCs transduced with shSATB2 or shCBP or both.


A . Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control ( n = 5 ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A. Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control (n=5).


B . Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days . Scale bar : 100 μm .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

B. Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days. Scale bar: 100 μm.


C . qPCR analysis of SATB2 , FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours ( n = 3 ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

C. qPCR analysis of SATB2, FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours (n=3).


E . Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase - labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation ( n = 5 mice per group ) . F . Quantification of tumor growth from ( E ) ( n = 5 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase-labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation (n=5 mice per group). F. Quantification of tumor growth from (E) (n=5 mice per group).


G . Kaplan - Meier survival curves of mice bearing T3359 GSC - derived xenografts treated with C646 or the vehicle control ( Vehicle control : n = 9 mice ; C646 : n = 8 mice ) . Median survival : Vehicle control , 27 days ; C646 , 33 . 5 days .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

G. Kaplan-Meier survival curves of mice bearing T3359 GSC-derived xenografts treated with C646 or the vehicle control (Vehicle control: n=9 mice; C646: n= 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days.


H . Immunofluorescence of Ki67 ( Green ) in T3359 GSC - derived xenografts from mice treated with with C646 or the vehicle control ( n = 6 tumors per group ) . Scale bar : 40 μm . I . Quantification of Ki67 positive cells in T3359 GSC - derived xenografts from mice treated with with C646 or the vehicle control ( n = 6 tumors per group ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm. I. Quantification of Ki67 positive cells in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group).


H . Immunofluorescence of Ki67 ( Green ) in T3359 GSC - derived xenografts from mice treated with with C646 or the vehicle control ( n = 6 tumors per group ) . Scale bar : 40 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm.


Immunohistochemistry performed on 12 month - old THY - Tau22 mice showing AT100 - positive neurons ( green ) , CBP - positive nuclei ( red ) and DAPI - stained nuclei ( blue ) in the CA1 region of the dorsal hippocampus . A representative image is shown with a focus . Scale bars : 40µm and 20µm as noted . ( n = 5 mice ) . Arrows depict neurons bearing neurofibrillary tangles and do not display CBP immunostaining . The star depicts a ghost tangle ( AT100 - positive neuron and no nucleus )

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O

Immunohistochemistry performed on 12 month-old THY-Tau22 mice showing AT100-positive neurons (green), CBP-positive nuclei (red) and DAPI-stained nuclei (blue) in the CA1 region of the dorsal hippocampus. A representative image is shown with a focus. Scale bars: 40µm and 20µm as noted. (n=5 mice). Arrows depict neurons bearing neurofibrillary tangles and do not display CBP immunostaining. The star depicts a ghost tangle (AT100-positive neuron and no nucleus)


Western blot analyses perfomed in the dorsal hippocampus of 12 month - old THY - Tau22 mice compared to age - matched controls . NeuN , actin , tubulin and total H2B levels are not changed . Phosphorylated tau on serine 404 ( Tau - Ph404 ) attest for samples from tauopathic mice . Quantification represents the ration of the protein level detected on the total amount of proteins on the membranes , with WT arbitrarily set at 1 ( fold induction ) . Bar graphs are mean ± SEM . n = 5 - 7 / group as noted , multiple t tests , CBP * p = 0 . 003 and GFAP * * p = 0 . 0001 for THY - Tau vs . WT

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O

Western blot analyses perfomed in the dorsal hippocampus of 12 month-old THY-Tau22 mice compared to age-matched controls. NeuN, actin, tubulin and total H2B levels are not changed. Phosphorylated tau on serine 404 (Tau-Ph404) attest for samples from tauopathic mice. Quantification represents the ration of the protein level detected on the total amount of proteins on the membranes, with WT arbitrarily set at 1 (fold induction). Bar graphs are mean ± SEM. n=5-7/group as noted, multiple t tests, CBP * p=0.003 and GFAP **p=0.0001 for THY-Tau vs. WT mice


RNA - sequencing data performed in the hippocampus of 8 month - old mice . Functional analyses ( DAVID GOTERM ) performed on significantly deregulated genes in THY - Tau22 mice compared to WT mice . Representative genes are presented below . | log2 Fold Change | > 0 . 2 ; * indicates FDR < 0 . 05 . IEG , Immediate Early Genes . Predicted promoter motif ( right ) analyses performed with GREAT . Groups : WT mice ( n = 3 ) , THY - Tau22 mice ( n = 2 )

O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O

RNA-sequencing data performed in the hippocampus of 8 month-old mice. Functional analyses (DAVID GOTERM) performed on significantly deregulated genes in THY-Tau22 mice compared to WT mice. Representative genes are presented below. |log2 Fold Change| > 0.2; * indicates FDR<0.05. IEG, Immediate Early Genes. Predicted promoter motif (right) analyses performed with GREAT. Groups: WT mice (n=3), THY-Tau22 mice (n=2)


Long - term spatial memory testing : Mice were injected 3 times ( 1 per week ) with Vehicle ( WT mice saline , WT VEH , n = 17 ) , Vehicle ( THY - Tau22 mice CSP , 500 μg / mouse , TAU VEH , n = 10 ) or Molecule ( THY - Tau22 mice CSP - TTK21 , 500 μg / mice TAU MOL , n = 13 ) before training of spatial memory in the Morris water maze ( MWM ) ; retention ( Probe test ) was tested 10 days after the last training session . Acquisition ( Escape latencies , seconds ) and retention performances ( Time in target quadrant , seconds ) are shown for the 3 groups of mice . All groups of mice displayed significant acquisition of the platform location ( Day effect , F ( 4 , 148 ) = 26 . 45 , p < 0 . 001 ; Group and Group X Day effects , ns ) , but only TAU VEH exhibited impaired retention . CSP - TTK21 treatment fully restored the ability of THY - Tau22 mice to remember the platform location . Bar graphs are mean ± SEM . Student ' s t test to a constant value , # when compared to random ( dotted line , 15sec ) WT VEH , t ( 16 ) = 4 . 6323 , # p = 0 . 0002 ; TAU VEH , t ( 9 ) = 0 . 3606 , p = 0 . 7267 ; TAU MOL , t ( 12 ) = 3 . 945 , # p = 0 . 0019 . One - way ANOVA ; F ( 2 , 37 ) = 3 . 55 ; p = 0 . 03 ; * in the different comparisons : TAU MOL vs . TAU VEH , * p = 0 . 0166 ; WT VEH vs . TAU VEH , * p = 0 . 040 ; TAU MOL vs WT VEH , non significant p = 0 . 437 ) . TQ , Target Quadrant ; O , Other , corresponds to the mean of the 3 other

O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Long-term spatial memory testing: Mice were injected 3 times (1 per week) with Vehicle (WT mice saline, WT VEH, n=17), Vehicle (THY-Tau22 mice CSP, 500 μg /mouse, TAU VEH, n=10) or Molecule (THY-Tau22 mice CSP-TTK21, 500 μg/mice TAU MOL, n=13) before training of spatial memory in the Morris water maze (MWM); retention (Probe test) was tested 10 days after the last training session. Acquisition (Escape latencies, seconds) and retention performances (Time in target quadrant, seconds) are shown for the 3 groups of mice. All groups of mice displayed significant acquisition of the platform location (Day effect, F(4,148)= 26.45, p<0.001; Group and Group X Day effects, ns), but only TAU VEH exhibited impaired retention. CSP-TTK21 treatment fully restored the ability of THY-Tau22 mice to remember the platform location. Bar graphs are mean ± SEM. Student's t test to a constant value, # when compared to random (dotted line, 15sec) WT VEH, t(16)=4.6323, #p=0.0002; TAU VEH, t(9)=0.3606, p=0.7267; TAU MOL, t(12)=3.945, #p=0.0019. One-way ANOVA; F(2,37)= 3.55; p= 0.03; * in the different comparisons: TAU MOL vs. TAU VEH, *p=0.0166; WT VEH vs. TAU VEH, *p= 0.040; TAU MOL vs WT VEH, non significant p=0.437). TQ, Target Quadrant; O, Other, corresponds to the mean of the 3 other quadrants


The timeline of GFP - lentivirus and CSP or CSP - TTK21 ( one injection , 500 μg / mouse ) injection is shown . ( Left ) The total number of spines was significantly decreased in TAU VEH compared to WT VEH mice . TAU MOL hippocampi showed a significant increase in the total number of spines compared to TAU VEH ( One - way ANOVA p = 0 . 0011 , post hoc Holm Sidak ' s multiple comparisons test F ( 2 , 243 ) = 7 . 03 ; WT VEH vs . TAU VEH , p < 0 . 0009 ; TAU MOL vs . TAU VEH , p = 0 . 0455 ) . ( Middle ) Based on spine type , the number of head spines ( Mushrooms , thins ) was significantly lower in both TAU VEH and TAU MOL than in WT VEH controls ( One - way ANOVA p = 0 . 0018 , post hoc Holm Sidak ' s multiple comparisons test F ( 2 , 243 ) = 6 . 467 ; WT VEH vs . TAU VEH , p = 0 . 0013 ; WT VEH vs . TAU MOL , p = 0 . 0272 . Stubby spine density was significantly increased in TAU MOL compared to TAU VEH mice ( One - way ANOVA F ( 2 , 243 ) = 4 . 311 , p = 0 . 0145 , post hoc Holm Sidak ' s multiple comparisons test : TAU MOL vs . TAU VEH , p = 0 . 0109 ) , as the number of filopodia ( One - way ANOVA F ( 2 , 243 ) = 3 . 845 , p = 0 . 0227 , post hoc Holm Sidak ' s multiple comparisons test TAU MOL vs . TAU VEH , p = 0 . 0179 ) . ( Right ) Typical images are presented showing a dendrite fragment for each condition . White arrowhead depicts stubby spines . Scale bar , 2 μm . Number of dendritc segments : WT VEH , n = 67 ; TAU VEH , n = 93 , TAU MOL , n = 87 ; Number of neurons : WT VEH , n = 20 ; TAU VEH , n = 28 , TAU MOL , n = 16 ; Number of mice : WT VEH , n = 2 ; TAU VEH , n = 3 , TAU MOL , n = 3

O O O O O O O O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O

The timeline of GFP-lentivirus and CSP or CSP-TTK21 (one injection, 500 μg/mouse) injection is shown. (Left) The total number of spines was significantly decreased in TAU VEH compared to WT VEH mice. TAU MOL hippocampi showed a significant increase in the total number of spines compared to TAU VEH (One-way ANOVA p=0.0011, post hoc Holm Sidak's multiple comparisons test F(2,243) = 7.03; WT VEH vs. TAU VEH, p < 0.0009; TAU MOL vs. TAU VEH, p=0.0455). (Middle) Based on spine type, the number of head spines (Mushrooms, thins) was significantly lower in both TAU VEH and TAU MOL than in WT VEH controls (One-way ANOVA p=0.0018, post hoc Holm Sidak's multiple comparisons test F(2,243) = 6.467; WT VEH vs. TAU VEH, p=0.0013; WT VEH vs. TAU MOL, p=0.0272. Stubby spine density was significantly increased in TAU MOL compared to TAU VEH mice (One-way ANOVA F(2,243) = 4.311, p = 0.0145, post hoc Holm Sidak's multiple comparisons test: TAU MOL vs. TAU VEH, p=0.0109), as the number of filopodia (One-way ANOVA F(2,243) = 3.845, p = 0.0227, post hoc Holm Sidak's multiple comparisons test TAU MOL vs. TAU VEH, p=0.0179). (Right) Typical images are presented showing a dendrite fragment for each condition. White arrowhead depicts stubby spines. Scale bar, 2 μm. Number of dendritc segments: WT VEH, n=67; TAU VEH, n=93, TAU MOL, n=87; Number of neurons: WT VEH, n=20; TAU VEH, n=28, TAU MOL, n=16; Number of mice: WT VEH, n=2; TAU VEH, n=3, TAU MOL, n=3


CSP - TTK21 injection into THY - Tau22 mice rescues mature dendritic spines formation in response to learning . ( Top left ) The time line of injections is shown : Mice were injected 3 times ( 1 per week ) with Vehicle ( WT mice , WT VEH , NaCl 0 . 9 % ) , Vehicle ( THY - Tau22 mice ( TAU VEH ) , CSP 500 μg / mouse ) or Molecule ( THY - Tau22 mice ( TAU MOL ) , 500 μg / mice ) and either trained over a 4 - day acquisition period in the MWM ( " learning " group ) or left in their home cage ( " basal " group ) . Mushroom - shaped spines were counted in dorsal CA1 , four days post - training . ( Bottom left ) The number of mature spines was significantly increased by learning in WT VEH and TAU MOL mice . Bar graphs are mean ± SEM . ( Two - way ANOVA ; Learning effect , F ( 1 , 139 ) = 54 . 18 ; p < 0 . 0001 ; # # # post hoc Holm Sidak ' s multiple comparisons test : Learning vs . Basal in WT VEH ( p = 0 . 0001 ) and in TAU MOL mice ; ( p = 0 . 0001 ) . After learning , WT VEH and TAU MOL mice displayed significantly higher number of mature spines than TAU VEH mice ( Genotype X Treatment effect , F ( 2 , 139 ) = 9 . 704 ; p = 0 . 0001 ; * * * post hoc Holm Sidak ' s multiple comparisons test : TAU MOL vs . TAU VEH ( p = 0 . 0001 ) , WT VEH vs . TAU VEH ( p = 0 . 0001 ) . ( Right ) Typical examples of a dendritic fragment bearing mushroom spines ( arrows ) are shown for each sub - group in response to learning . Number of dendritc segments : Learning : WT VEH , n = 27 ; TAU VEH , n = 27 , TAU MOL , n = 30 ; WT VEH _ HC , n = 27 ; Number of mice : WT VEH , n = 3 ; TAU VEH , n = 3 , TAU MOL , n = 3 . Basal : WT VEH , n = 27 ; TAU VEH , n = 7 , TAU MOL , n = 15 ; Number of mice : WT VEH , n = 3 ; TAU VEH , n = 3 , TAU MOL , n = 2

O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O

CSP-TTK21 injection into THY-Tau22 mice rescues mature dendritic spines formation in response to learning. (Top left) The time line of injections is shown: Mice were injected 3 times (1 per week) with Vehicle (WT mice, WT VEH, NaCl 0.9%), Vehicle (THY-Tau22 mice (TAU VEH), CSP 500 μg /mouse) or Molecule (THY-Tau22 mice (TAU MOL), 500 μg/mice) and either trained over a 4-day acquisition period in the MWM ("learning" group) or left in their home cage ("basal" group). Mushroom-shaped spines were counted in dorsal CA1, four days post-training. (Bottom left) The number of mature spines was significantly increased by learning in WT VEH and TAU MOL mice. Bar graphs are mean ± SEM. (Two-way ANOVA; Learning effect, F(1, 139)=54.18; p< 0.0001; ### post hoc Holm Sidak's multiple comparisons test: Learning vs. Basal in WT VEH (p=0.0001) and in TAU MOL mice; (p=0.0001). After learning, WT VEH and TAU MOL mice displayed significantly higher number of mature spines than TAU VEH mice (Genotype X Treatment effect, F(2,139)= 9.704; p=0.0001; *** post hoc Holm Sidak's multiple comparisons test: TAU MOL vs. TAU VEH (p=0.0001), WT VEH vs. TAU VEH (p=0.0001). (Right) Typical examples of a dendritic fragment bearing mushroom spines (arrows) are shown for each sub-group in response to learning. Number of dendritc segments: Learning: WT VEH, n=27; TAU VEH, n=27, TAU MOL, n=30; WT VEH_HC, n=27; Number of mice: WT VEH, n=3; TAU VEH, n=3, TAU MOL, n=3. Basal: WT VEH, n=27; TAU VEH, n=7, TAU MOL, n=15; Number of mice: WT VEH, n=3; TAU VEH, n=3, TAU MOL, n=2


Mice were injected 3 times ( 1 per week ) with saline ( WT VEH ) , CSP ( Vehicle , VEH ) or CSP - TTK21 ( Molecule , MOL ) ( 500 μg / mice ; THY - Tau22 mice ( TAU ) before euthanasia . Long Term Depression measurements were performed on hippocampal slices . ( Top left ) Examples of analog traces recorded 10 min before ( a ) and 55 min after LTD induction ( b ; dotted line ) in the three groups of mice . ( Bottom left ) Time course of LTD ; LTD is expressed as a percent change in fEPSP ( field excitatory postsynaptic potentials ) slope over time . After the 20 - min baseline recording , a low - frequency stimulation ( LFS , 2Hz for 10 min ) was applied ( arrow ) . Recording was stopped during the 10 - min conditioning stimulation and resumed after completion of LFS . LFS induced a strong depression of the fEPSP slope , which recovered partially to reach a stable level of depression about 20 min after stimulation . ( Right ) Average depression measured in the last 10 min of LTD . LTD was significantly different in TAU VEH ( 88 . 4 ± 4 . 1 % of the baseline , n = 10 ) compared to controls ( WT VEH , 71 . 1 ± 4 . 4 % , n = 9 ) ( F ( 1 , 17 ) = 8 . 8 , p = 0 . 008 ) . CSP - TTK21 treatment restored LTD to control levels ( 64 . 9 ± 5 . 2 % , n = 10 ) ( WT VEH vs . TAU MOL : F ( 1 , 17 ) = 0 . 83 , p = 0 . 37 , ns ; TAU VEH vs . TAU MOL : F ( 1 , 18 ) = 13 . 2 . p = 0 . 0019 ) . Bar graphs are mean ±

O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were injected 3 times (1 per week) with saline (WT VEH), CSP (Vehicle, VEH) or CSP-TTK21 (Molecule, MOL) (500 μg/mice; THY-Tau22 mice (TAU) before euthanasia. Long Term Depression measurements were performed on hippocampal slices. (Top left) Examples of analog traces recorded 10 min before (a) and 55 min after LTD induction (b; dotted line) in the three groups of mice. (Bottom left) Time course of LTD; LTD is expressed as a percent change in fEPSP (field excitatory postsynaptic potentials) slope over time. After the 20-min baseline recording, a low-frequency stimulation (LFS, 2Hz for 10 min) was applied (arrow). Recording was stopped during the 10-min conditioning stimulation and resumed after completion of LFS. LFS induced a strong depression of the fEPSP slope, which recovered partially to reach a stable level of depression about 20 min after stimulation. (Right) Average depression measured in the last 10 min of LTD. LTD was significantly different in TAU VEH (88.4 ± 4.1% of the baseline, n=10) compared to controls (WT VEH, 71.1 ± 4.4%, n=9) (F(1,17)=8.8, p=0.008). CSP-TTK21 treatment restored LTD to control levels (64.9 ± 5.2%, n=10) (WT VEH vs. TAU MOL: F(1,17)=0.83, p=0.37, ns; TAU VEH vs. TAU MOL: F(1,18)=13.2. p=0.0019). Bar graphs are mean ± SEM


Volcano plot showing that 2756 genes are differentially regulated between THY - Tau22 and WT mice during learning . The log2 ( Fold - Change ) was estimated by DESeq2 . FDR < 0 . 05 and | log2 Fold Change | > 0 . 2 . Red dots correspond to genes with adjusted p - value < 0 . 05 . Numbers in the corners represent the number of down - regulated ( left ) and up - regulated ( right ) genes . C Heatmap representing expression of z - score of the 2756 deregulated genes in all experimental conditions . Color - coding was performed according to the z - score of the normalized reads counts divided by gene length . Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson " s distance . Groups : WT VEH _ HC ( n = 3 ) , WT VEH ( n = 2 ) ; TAU VEH ( n = 2 ) ; TAU

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR

Volcano plot showing that 2756 genes are differentially regulated between THY-Tau22 and WT mice during learning. The log2(Fold-Change) was estimated by DESeq2. FDR<0.05 and |log2 Fold Change| > 0.2. Red dots correspond to genes with adjusted p-value <0.05. Numbers in the corners represent the number of down-regulated (left) and up-regulated (right) genes. C Heatmap representing expression of z-score of the 2756 deregulated genes in all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s distance. Groups: WT VEH_HC (n=3), WT VEH (n=2); TAU VEH (n=2); TAU MOL


Volcano plot showing that 1756 genes are differentially regulated between treated - THY - Tau22 and WT mice during learning . Same parameters as in

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

Volcano plot showing that 1756 genes are differentially regulated between treated-THY-Tau22 and WT mice during learning. Same parameters as in B


F , G Volcano plots showing differentially regulated genes by the molecule in THY - Tau22 mice ( F ) and by learning in WT mice ( G ) . Same parameters as in

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

F, G Volcano plots showing differentially regulated genes by the molecule in THY-Tau22 mice (F) and by learning in WT mice (G). Same parameters as in B


Heatmap representing expression z - score of the 98 significantly over - expressed genes in the TAU MOL vs . TAU VEH comparison , for all experimental conditions . Color - coding was performed according to the z - score of the normalized reads counts divided by gene length . Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson " s

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heatmap representing expression z-score of the 98 significantly over-expressed genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s dis


RNA - seq data showing expression of several immediate early genes ( IEGs ) : Nr4a1 , Arc , Egr1 , Dusp1 , Fos and Junb . Most of the down - regulated IEGs in tauopathic compared to WT mice present a significant induction in THY - Tau22 mice after CSP - TTK21 injection . Adjusted p - values corresponding to p < 0 . 05 are indicated by * , for WT VEH vs . WT VEH _ HC , $ for TAU VEH vs . WT VEH and # , for TAU MOL vs . TAU

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR

RNA-seq data showing expression of several immediate early genes (IEGs) : Nr4a1, Arc, Egr1, Dusp1, Fos and Junb. Most of the down-regulated IEGs in tauopathic compared to WT mice present a significant induction in THY-Tau22 mice after CSP-TTK21 injection. Adjusted p-values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH


RNA - seq data showing that CSP - TTK21 treatment of tauopathic mice restored the expression of plasticity / memory - relevant target genes : Klotho ( Kl ) , and Neurotensin ( Nts ) . Adjusted p values corresponding to p < 0 . 05 are indicated by * , for WT VEH vs . WT VEH _ HC , $ for TAU VEH vs . WT VEH and # , for TAU MOL vs . TAU VEH . F RT - qPCR validations performed in a different cohort of mice from the RNA - seq study ( n = 4 - 5 / group ) . One - way ANOVA with uncorrected Fisher " s test . Klotho , Kl : F ( 3 , 16 ) = 2 . 949 , p = 0 . 0643 ; * learning p = 0 . 0145 for WT VEH vs . WT VEH _ HC , $ pathology p = 0 . 0395 for WT VEH vs . TAU VEH . Nts : F ( 3 , 14 ) = 4 . 290 , p = 0 . 0241 ; $ pathology p = 0 . 0036 for WT VEH vs . TAU VEH , # molecule p = 0 . 0081 for TAU MOL vs .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O

RNA-seq data showing that CSP-TTK21 treatment of tauopathic mice restored the expression of plasticity/memory-relevant target genes : Klotho (Kl), and Neurotensin (Nts). Adjusted p values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH. F RT-qPCR validations performed in a different cohort of mice from the RNA-seq study (n=4-5/group). One-way ANOVA with uncorrected Fisher"s test. Klotho, Kl: F(3,16)=2.949, p=0.0643; * learning p=0.0145 for WT VEH vs. WT VEH_HC, $ pathology p=0.0395 for WT VEH vs. TAU VEH. Nts: F(3,14)=4.290, p=0.0241; $ pathology p=0.0036 for WT VEH vs. TAU VEH, # molecule p=0.0081 for TAU MOL vs. TA


Western blot for Klotho and Neurotensin expression in the different experimental conditions . Immunoblot results are shown ( n = 5 / group ) . Bar graphs represent the quantification of the protein levels as percentage of the control group WT VEH _ HC , arbitrarily set at 100 % . Each detected protein was normalized to the corresponding amount of total proteins in the gels or the nitrocellulose membrane . Klotho and Neurotensin were further normalized to the level of actin . One - way ANOVA with uncorrected Fisher " s test . Klotho : F ( 3 , 16 ) = 5 . 192 , p = 0 . 0107 , * learning p = 0 . 0117 for WT VEH vs . WT VEH _ HC , $ pathology p = 0 . 0027 for WT VEH vs . TAU VEH , # molecule p = 0 . 0282 for TAU MOL vs . TAU VEH . Nts : F ( 3 , 16 ) = 3 . 748 , p = 0 . 0326 , $ pathology p = 0 . 0200 for WT VEH vs . TAU VEH , # molecule p = 0 . 0079 for TAU MOL vs .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O

Western blot for Klotho and Neurotensin expression in the different experimental conditions. Immunoblot results are shown (n=5/group). Bar graphs represent the quantification of the protein levels as percentage of the control group WT VEH_HC, arbitrarily set at 100%. Each detected protein was normalized to the corresponding amount of total proteins in the gels or the nitrocellulose membrane. Klotho and Neurotensin were further normalized to the level of actin. One-way ANOVA with uncorrected Fisher"s test. Klotho: F(3,16)=5.192, p=0.0107, * learning p=0.0117 for WT VEH vs. WT VEH_HC, $ pathology p=0.0027 for WT VEH vs. TAU VEH, # molecule p=0.0282 for TAU MOL vs. TAU VEH. Nts: F(3,16)=3.748, p=0.0326, $ pathology p=0.0200 for WT VEH vs. TAU VEH, # molecule p=0.0079 for TAU MOL vs. TA


A Functional annotation charts ( Benjamini , p < 0 . 05 ) using using DAVID ( GOTERM _ Cellular Component ) perfomed on the 82 significantly down - regulated genes by CSP - TTK21 treatment in tauopathic mice ( TAU MOL vs . TAU VEH ) , showing a significant association with neuronal terms . Significance is indicated as - log10 ( Benjamini p _ value ) . B Heatmap representing expression z - score of the 82 significantly down - regulated genes in the TAU MOL vs . TAU VEH comparison , for all experimental conditions . Color - coding was performed according to the z - score of the normalized reads counts divided by gene length . Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson " s

O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Functional annotation charts (Benjamini, p<0.05) using using DAVID (GOTERM_Cellular Component) perfomed on the 82 significantly down-regulated genes by CSP-TTK21 treatment in tauopathic mice (TAU MOL vs. TAU VEH), showing a significant association with neuronal terms. Significance is indicated as -log10(Benjamini p_value). B Heatmap representing expression z-score of the 82 significantly down-regulated genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s dis


C RNA - seq data showing expression of several genes belonging to « synapse » annotation ( DAVID ) : Grik4 , Homer3 and Sv2b , which are significantly down - regulated by CSPTTK21 treatment in THY - Tau22 mice . Adjusted p values corresponding to p < 0 . 05 are indicated by * for WT VEH vs . WT VEH _ HC , $ for TAU VEH vs . WT VEH and # , for TAU MOL vs . TAU

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR

C RNA-seq data showing expression of several genes belonging to « synapse » annotation (DAVID): Grik4, Homer3 and Sv2b, which are significantly down-regulated by CSPTTK21 treatment in THY-Tau22 mice. Adjusted p values corresponding to p<0.05 are indicated by * for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH


Mean H2Bac profiles at TSS relative to gene expression obtained from RNA - seq data . Expressed genes were separated into 4 groups ( Q1 to Q4 , 6483 genes / group ) , Q1 representing the less expressed genes ( 0 - 25 % ) and Q4 , the most expressed genes ( 75 - 100 % ) . Profiles were established with SeqMiner centered on TSS ± 2Kb . C Mean H2Bac profiles established with SeqMiner for all genes at TSS ( top ) and gene bodies ( bottom ) for the 4th quartile ( most expressed genes ) in the two replicates ( rep1 and rep2 ) . Significance ( Benjamini , p < 0 . 05 ) of the annotations associated with both TSS ( green ) or Gene profiles ( red ) are shown ( DAVID , KEGG Pathways )

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean H2Bac profiles at TSS relative to gene expression obtained from RNA-seq data. Expressed genes were separated into 4 groups (Q1 to Q4, 6483 genes/group), Q1 representing the less expressed genes (0-25%) and Q4, the most expressed genes (75-100%). Profiles were established with SeqMiner centered on TSS ±2Kb. C Mean H2Bac profiles established with SeqMiner for all genes at TSS (top) and gene bodies (bottom) for the 4th quartile (most expressed genes) in the two replicates (rep1 and rep2). Significance (Benjamini, p<0.05) of the annotations associated with both TSS (green) or Gene profiles (red) are shown (DAVID, KEGG Pathways)


Differentially regulated H2Bac peaks were analyzed with SICER and significant Fold Changes ( FC > 1 ; FDR < 0 . 001 ) between comparisons are shown for the pathology ( TAU VEH vs . WT VEH , blue ) and the effect of the molecule ( TAU MOL vs . TAU VEH , red ) . Fold Change is shown as log2 ( Fold Change ) ( X - axis ) and significance , as - log10 ( FDR ) ( Y - axis )

O O B-MEASURED_VAR O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Differentially regulated H2Bac peaks were analyzed with SICER and significant Fold Changes (FC>1; FDR<0.001) between comparisons are shown for the pathology (TAU VEH vs. WT VEH, blue) and the effect of the molecule (TAU MOL vs. TAU VEH, red). Fold Change is shown as log2 (Fold Change) (X-axis) and significance, as -log10(FDR) (Y-axis)


Mean H2Bac and H3K27ac profiles established with SeqMiner for the 1624 decreased peaks obtained in the comparison TAU VEH vs . WT VEH . H2Bac but not H3K27ac peaks show differential

O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O

Mean H2Bac and H3K27ac profiles established with SeqMiner for the 1624 decreased peaks obtained in the comparison TAU VEH vs. WT VEH. H2Bac but not H3K27ac peaks show differential regulation


Time series Like plot . For every significant H2Bac decreased regions ( 1624 ) in WT VEH vs TAU VEH , normalized read counts were computed for each of the different conditions ( WT VEH , TAU VEH , TAU MOL ) . Fold changes are all referring to WT VEH . Y - axis : Fold change . X - axis :

O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Time series Like plot. For every significant H2Bac decreased regions (1624) in WT VEH vs TAU VEH, normalized read counts were computed for each of the different conditions (WT VEH, TAU VEH, TAU MOL). Fold changes are all referring to WT VEH. Y-axis: Fold change. X-axis: Conditions


A Mean enrichment profiles performed with SeqMiner presenting the genomic distribution of H2Bac and H3K27ac reads obtained in ChIP - seq experiments in the different experimental groups , established along putative neuronal CBP enhancers ( ± 4Kb ) ( Kim et al . , 2010 ) . In the dorsal hippocampus , H2Bac levels are enriched at CBP enhancers in WT WEH ( blue ) , decreased in tauopathic mice ( TAU VEH , pink ) and induced by CSP - TTK21 treatment ( TAU MOL , green ) . B Mean profiles were separated in two sets of loci , including 17817 peaks highly enriched in H2Bac and showing a differential regulation in the experimental groups and 23331 peaks that were poorly enriched in H2Bac and not

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

A Mean enrichment profiles performed with SeqMiner presenting the genomic distribution of H2Bac and H3K27ac reads obtained in ChIP-seq experiments in the different experimental groups, established along putative neuronal CBP enhancers (± 4Kb) (Kim et al., 2010). In the dorsal hippocampus, H2Bac levels are enriched at CBP enhancers in WT WEH (blue), decreased in tauopathic mice (TAU VEH, pink) and induced by CSP-TTK21 treatment (TAU MOL, green). B Mean profiles were separated in two sets of loci, including 17817 peaks highly enriched in H2Bac and showing a differential regulation in the experimental groups and 23331 peaks that were poorly enriched in H2Bac and not regulated


Mean profiles established with SeqMiner for all genes at TSS for H2Bac ( Left ) and H3K27ac ( Right ) . H2Bac but not H3K24ac presents a differential regulation at

O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O

Mean profiles established with SeqMiner for all genes at TSS for H2Bac (Left) and H3K27ac (Right). H2Bac but not H3K24ac presents a differential regulation at TSS


Mean H2Bac profiles established with SeqMiner on the differentially regulated genes obtained in transcriptomic experiments ( RNA - seq ) , either decreased ( left ) or increased ( right ) . The ' basal ' H2Bac TSS enrichment state of these genes is differentially regulated whether the genes are decreased or increased in response to

O B-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

Mean H2Bac profiles established with SeqMiner on the differentially regulated genes obtained in transcriptomic experiments (RNA-seq), either decreased (left) or increased (right). The 'basal' H2Bac TSS enrichment state of these genes is differentially regulated whether the genes are decreased or increased in response to learning


UCSC genome browser view of the c - fos genomic locus with H3K27ac ChIP - seq ( blue ) , H2Bac ChIP - seq ( green ) and Input ( black ) signals . Blue shading indicates locations of the c - fos enhancers ( Joo et al . , 2016 ) ; gray shading indicates location of the promoter . Differential peaks were analyzed with SICER . This set of data shows that H2Bac levels were significantly decreased at e1 ( FDR = 1 . 95 . 10 - 08 ) , e2 ( FDR = 0 . 0005 ) and e5 ( FDR = 2 . 44 . 10 - 05 ) enhancers in THY - Tau22 compared to WT mice , and significantly enriched at e1 ( FDR = 4 . 01 . 10 - 08 ) the regions encompassing e3 , e4 and TSS ( FDR = 7 . 53 . 10 - 07 ) and e5 ( FDR = 0 . 001 ) after CSP - TTK21 treatment . Expanded View Figure

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O

UCSC genome browser view of the c-fos genomic locus with H3K27ac ChIP-seq (blue), H2Bac ChIP-seq (green) and Input (black) signals. Blue shading indicates locations of the c-fos enhancers (Joo et al., 2016); gray shading indicates location of the promoter. Differential peaks were analyzed with SICER. This set of data shows that H2Bac levels were significantly decreased at e1 (FDR=1.95.10-08), e2 (FDR=0.0005) and e5 (FDR= 2.44.10-05) enhancers in THY-Tau22 compared to WT mice, and significantly enriched at e1 (FDR= 4.01.10-08) the regions encompassing e3, e4 and TSS (FDR=7.53.10-07) and e5 (FDR= 0.001) after CSP-TTK21 treatment. Expanded View Figure Legend


B Quantification of the expression of miR - 19a and miR - 19b in bones of mice treated with PTH ( n = 15 ) or Scl - Ab ( n = 14 ) compared to veh ( n = 13 ) control . C Quantification of the expression of miR - 19a and miR - 19b in calvarial osteoblasts upon in vitro stimulation with PTH or Wnt3a compared to veh control ( n = 3 ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

B Quantification of the expression of miR-19a and miR-19b in bones of mice treated with PTH (n=15) or Scl-Ab (n=14) compared to veh (n=13) control. C Quantification of the expression of miR-19a and miR-19b in calvarial osteoblasts upon in vitro stimulation with PTH or Wnt3a compared to veh control (n=3).


D Alkaline phosphatase staining of differentiating calvarial osteoblasts transfected with a scrambled ( scr ) control miRNA , anti - sense microRNA ( anti - miR ) inhibiting miR - 19a or miR - 19b or a combination thereof ( anti - miR - 19a / b ) ( representative image of 4 independent experiments ) . E Quantification of alkaline phosphatase staining of calvarial osteoblasts transfected with scr , anti - miR - 19a , anti - miR - 19b or anti - miR - 19a / b ( n = 4 ) .

O B-MEASURED_VAR I-MEASURED_VAR O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

D Alkaline phosphatase staining of differentiating calvarial osteoblasts transfected with a scrambled (scr) control miRNA, anti-sense microRNA (anti-miR) inhibiting miR-19a or miR-19b or a combination thereof (anti-miR-19a/b) (representative image of 4 independent experiments). E Quantification of alkaline phosphatase staining of calvarial osteoblasts transfected with scr, anti-miR-19a, anti-miR-19b or anti-miR-19a/b (n=4).


F , G Expression analysis of osteoblast marker genes ( F ) Runx2 and ( G ) Type I Collagen ( Col1a1 ) in calvarial osteoblasts transfected with scr , anti - miR - 19a , anti - miR - 19b or anti - miR - 19a / b ( n = 4 ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

F, G Expression analysis of osteoblast marker genes (F) Runx2 and (G) Type I Collagen (Col1a1) in calvarial osteoblasts transfected with scr, anti-miR-19a, anti-miR-19b or anti-miR-19a/b (n=4).


H Images of von Kossa - stained histological sections of the proximal tibiae of 12 - week - old male C57Bl / 6J mice and fluorescence double labeling to visualize bone formation ( insets ) after 4 - weeks of treatment with anti - miR - 19a / b ( n = 12 ) , scr control oligonucleotides ( n = 11 ) or veh ( n = 8 ) . Scale bars indicate 1 mm ( black ) and 50 μm ( white ) . I Bone histomorphometric analysis of the proximal tibiae of the same animals as in ( H ) Abbreviations : BV / TV , bone volume / tissue volume ; MS / BS , mineralizing surface / bone surface ; BFR / BS , bone formation rate / bone surface ; MAR , mineral apposition rate ; OS / BS , osteoid surface / bone surface ; Ob . S / BS , osteoblast surface / bone surface ; N . Ob / BS , number of osteoblasts / bone surface ; ES / BS , eroded surface / bone surface ; Oc . S / BS , osteoclast surface / bone surface ; N . Oc / BS , number of osteoclasts / bone surface .

O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male C57Bl/6J mice and fluorescence double labeling to visualize bone formation (insets) after 4-weeks of treatment with anti-miR-19a/b (n=12), scr control oligonucleotides (n=11) or veh (n=8). Scale bars indicate 1 mm (black) and 50 μm (white). I Bone histomorphometric analysis of the proximal tibiae of the same animals as in (H) Abbreviations: BV/TV, bone volume/tissue volume; MS/BS, mineralizing surface/bone surface; BFR/BS, bone formation rate/bone surface; MAR, mineral apposition rate; OS/BS, osteoid surface/bone surface; Ob.S/BS, osteoblast surface/bone surface; N.Ob/BS, number of osteoblasts/bone surface; ES/BS, eroded surface/bone surface; Oc.S/BS, osteoclast surface/bone surface; N.Oc/BS, number of osteoclasts/bone surface.


J Images of von Kossa - stained histological sections of the fourth lumbar vertebral body of 12 - week - old mice after 4 - weeks of treatment with anti - miR - 19a / b , scr or veh . Scale bar indicates 1 mm . K Quantification of bone mass ( BV / TV ) of the fourth lumbar vertebral body of 12 - week - old mice after 4 - weeks of treatment with anti - miR - 19a / b ( n = 12 ) , scr ( n = 12 ) or veh ( n = 8 ) .

O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

J Images of von Kossa-stained histological sections of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b, scr or veh. Scale bar indicates 1 mm. K Quantification of bone mass (BV/TV) of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b (n=12), scr (n=12) or veh (n=8).


A Images of von Kossa - stained histological sections of the bone and bone marrow ( upper panel ) and of hematoxylin - eosin - stained liver sections ( lower panel ) of 12 - week - old mice after treatment with anti - miR - 19a / b ( n = 12 ) , scrambled ( scr ) control oligonucleotides ( n = 11 ) or vehicle ( veh ) ( n = 8 ) . Scale bars indicate 100 μm ( upper panel ) and 50 μm ( lower panel ) . B , C Quantification of the femur length ( B ) and width ( C ) after treatment with anti - miR - 19a / b ( n = 8 ) , scr ( n = 7 ) or veh ( n = 8 ) .

O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

A Images of von Kossa-stained histological sections of the bone and bone marrow (upper panel) and of hematoxylin-eosin-stained liver sections (lower panel) of 12-week-old mice after treatment with anti-miR-19a/b (n=12), scrambled (scr) control oligonucleotides (n=11) or vehicle (veh) (n=8). Scale bars indicate 100 μm (upper panel) and 50 μm (lower panel). B, C Quantification of the femur length (B) and width (C) after treatment with anti-miR-19a/b (n=8), scr (n=7) or veh (n=8).