( A ) 293T cells were contransfected with HA - cyclin D3 and Strep - tag - SARS - CoV - 2 E or nsp9 , and control plasmid ( EV ) . Immunoprecipitation was performed using anti - HA antibody . The immunoprecipitates were blotted with anti - Strep , anti - HA , and cyclin D3 antibodies .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O

(A) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E or nsp9, and control plasmid (EV). Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA, and cyclin D3 antibodies.


( B ) 293T cells were contransfected with HA - cyclin D3 and Strep - tag - SARS - CoV - 2 E , M , both E and M or nsp9 . Immunoprecipitation was performed using anti - HA antibody . The immunoprecipitates were blotted with anti - Strep , anti - HA antibodies . WCL , whole cell lysate .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O

(B) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E, M, both E and M or nsp9. Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA antibodies. WCL, whole cell lysate.


( C - G ) 293T GFP11 cells were transfected with Spike , and / or with Envelope , Membrane , and cyclin D3 . 24h post - transfection cells were seeded at a 1 : 1 ratio with Vero - GFP10 cells and percentage of GFP + ve area ( syncytia ) were determined 18h later . S , Spike ; E , Envelope ; M , Membrane ; D3 , cyclin D3 . ( C , D ) Representative images of GFP + syncytia . Scale bars : 40μm . ( E - G ) Quantification of cell - to - cell fusion showing percentage of the GFP + ve area to the acquired total cell area . ( E ) n = 4 biological replicates . ( F , G ) n = 4 biological replicates ; technical triplicates shown ; one - way ANOVA with Dunnett ' s multiple comparisons test : ns , non - significant ; ∗ ∗ ∗ ∗ p < 0 . 0001 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ p < 0 . 01 . Bars indicate mean with SD .

O O O O O B-CELL_LINE B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) 293T GFP11 cells were transfected with Spike, and/or with Envelope, Membrane, and cyclin D3. 24h post-transfection cells were seeded at a 1:1 ratio with Vero-GFP10 cells and percentage of GFP+ve area (syncytia) were determined 18h later. S, Spike; E, Envelope; M, Membrane; D3, cyclin D3. (C,D) Representative images of GFP+ syncytia. Scale bars: 40μm. (E-G) Quantification of cell-to-cell fusion showing percentage of the GFP+ve area to the acquired total cell area. (E) n=4 biological replicates. (F,G) n = 4 biological replicates; technical triplicates shown; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01 . Bars indicate mean with SD.


( I ) Representative example of western blot from purified SARS - CoV - 2 Virus Like Particles ( VLPs ) and whole cell lysates ( WCL ) . MCM2 was used as loading control . ( J ) Quantification of protein incorporation ( into VLPs ) or expression ( in WCL ) . n = 4 biological replicates ; one - way ANOVA with Dunnett ' s multiple comparisons test : ns , non - significant ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD . N , nucleocapsid ; M , membrane ; E , envelope ; S , spike .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O

(I) Representative example of western blot from purified SARS-CoV-2 Virus Like Particles (VLPs) and whole cell lysates (WCL). MCM2 was used as loading control. (J) Quantification of protein incorporation (into VLPs) or expression (in WCL). n = 4 biological replicates; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗p < 0.001. Bars indicate mean with SD. N, nucleocapsid; M, membrane; E, envelope; S, spike.


( B - C ) Western blot analysis ( B ) and densitometric quantification ( C ) of OTUD1 in aortas of control and Ang II - induced mice . β - actin was used as the loading control ( n = 5 biological replicates ) . ( D ) mRNA levels of OTUD1 in aortas of control and Ang II - induced mice were determined by RT - qPCR . The values were normalized to Rn18s ( n = 5 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O

(B-C) Western blot analysis (B) and densitometric quantification (C) of OTUD1 in aortas of control and Ang II-induced mice. β-actin was used as the loading control (n=5 biological replicates). (D) mRNA levels of OTUD1 in aortas of control and Ang II-induced mice were determined by RT-qPCR. The values were normalized to Rn18s (n=5 biological replicates).


( E ) Representative immunofluorescence staining of CD31 ( green ) and OTUD1 ( red ) of aortic sections ( scale bar = 25 μm ) . ( F ) Representative immunofluorescence staining of α - SMA ( green ) and OTUD1 ( red ) of aortic sections ( scale bar = 25 μm ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O

(E) Representative immunofluorescence staining of CD31 (green) and OTUD1 (red) of aortic sections (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and OTUD1 (red) of aortic sections (scale bar=25 μm).


( G - H ) Time - course of OTUD1 induction in response to Ang II in HUVECs . HUVECs were exposed to 1 μM Ang II for indicated time . Western blot analysis ( G ) and densitometric quantification ( H ) of OTUD1 were shown ( n = 3 biological replicates ) . GAPDH was used as the loading control .

O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O

(G-H) Time-course of OTUD1 induction in response to Ang II in HUVECs. HUVECs were exposed to 1 μM Ang II for indicated time. Western blot analysis (G) and densitometric quantification (H) of OTUD1 were shown (n = 3 biological replicates). GAPDH was used as the loading control.


( A ) Representative images of H & E staining of aortic sections of the mice from four groups ( scale bar = 25 μm ) . E , endothelium ; M , media ; A , adventitia . ( B ) The quantification of arterial wall thickness in panel A ; n = 5 biological replicates . ( C ) Representative images of Masson ' s Trichrome staining of aortic sections of the mice ( scale bar = 50 μm ) . ( D ) The quantification of collagen deposition in panel C ; n = 5 biological replicates .

O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

(A) Representative images of H&E staining of aortic sections of the mice from four groups (scale bar=25 μm). E, endothelium; M, media; A, adventitia. (B) The quantification of arterial wall thickness in panel A; n=5 biological replicates. (C) Representative images of Masson's Trichrome staining of aortic sections of the mice (scale bar=50 μm). (D) The quantification of collagen deposition in panel C; n=5 biological replicates.


( E - F ) Western blot analysis ( E ) of VE - cadherin , Vimentin , Twist , Snail , and OTUD1 and densitometric quantification ( F ) of VE - cadherin , Vimentin , Twist and Snail in aortic tissues of the mice . β - actin was used as the loading control ( n = 5 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

(E-F) Western blot analysis (E) of VE-cadherin, Vimentin, Twist, Snail, and OTUD1 and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues of the mice. β-actin was used as the loading control (n=5 biological replicates).


( G - H ) mRNA levels of Cdh5 , Vim , Twist1 , Snai1 ( G ) , Tgfb1 and Col1a1 ( H ) in aortic tissues of the mice were examined by real - time qPCR assay . The values were normalized to Rn18s ( n = 5 biological replicates ) .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O

(G-H) mRNA levels of Cdh5, Vim, Twist1, Snai1 (G), Tgfb1 and Col1a1 (H) in aortic tissues of the mice were examined by real-time qPCR assay. The values were normalized to Rn18s (n=5 biological replicates).


( B ) HUVECs transfected with si - OTUD1 or NC were challenged with 1 μM Ang II for 24 h . Western blot analysis of VE - cadherin , Vimentin , α - SMA , Twist and Snail were performed . GAPDH was used as the loading control .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O

(B) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( E ) HUVECs transfected with Flag - OTUD1 or EV were challenged with 1 μM Ang II for 24 h . Western blot analysis of VE - cadherin , Vimentin , α - SMA , Twist and Snail were performed . GAPDH was used as the loading control .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O

(E) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( C ) Co - immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag - OTUD1 . Exogenous Flag - OTUD1 was immunoprecipitated by anti - Flag antibody . IgG , immunoglobulin G .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O

(C) Co-immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag-OTUD1. Exogenous Flag-OTUD1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G.


( G ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub and Flag - OTUD1 and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( H ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub , and Flag - OTUD1 or Flag - OTUD1 ( C320S ) , and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( I ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , Flag - OTUD1 , and HA - Ub , HA - Ub - K48 ( K48 only ) , or HA - Ub - K63 ( K63 only ) , respectively , and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody .

O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O

(G) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (H) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and Flag-OTUD1 or Flag-OTUD1(C320S), and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (I) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, Flag-OTUD1, and HA-Ub, HA-Ub-K48 (K48 only), or HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody.


( A - B ) 293T cells were transfected with various amount of Flag - OTUD1 . Western blot analysis ( A ) and densitometric quantification ( B ) of SMAD3 . GAPDH was used as the loading control ( n = 3 biological replicates ) .

O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(A-B) 293T cells were transfected with various amount of Flag-OTUD1. Western blot analysis (A) and densitometric quantification (B) of SMAD3. GAPDH was used as the loading control (n=3 biological replicates).


( E - F ) Western blot analysis ( E ) and densitometric quantification ( F ) of SMAD3 in aortic tissues of the mice from four groups . β - actin was used as the loading control ( n = 5 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-TISSUE O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

(E-F) Western blot analysis (E) and densitometric quantification (F) of SMAD3 in aortic tissues of the mice from four groups. β-actin was used as the loading control (n=5 biological replicates).


( G ) Co - immunoprecipitation of SMAD3 and SMAD4 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub , and HA - Ub - K63 ( K63 only ) , respectively , and then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O

(G) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody.


( H ) Co - immunoprecipitation of SMAD3 and SMAD4 in 293T cells co - transfected with GFP - SMAD3 and Flag - OTUD1 and then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody . IgG , immunoglobulin G .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O

(H) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3 and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. IgG, immunoglobulin G.


( A - B ) Representative images ( A ) of H & E staining of aortic sections ( scale bar = 25 μm ) and quantification of arterial wall thickness ( B ) ; n = 5 biological replicates . E , endothelium ; M , media ; A , adventitia . ( C - D ) Representative images ( C ) of Masson ' s Trichrome staining of aortic sections ( scale bar = 25 μm ) and quantification of collagen deposition ( D ) ; n = 5 biological replicates .

O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

(A-B) Representative images (A) of H&E staining of aortic sections (scale bar=25 μm) and quantification of arterial wall thickness (B); n=5 biological replicates. E, endothelium; M, media; A, adventitia. (C-D) Representative images (C) of Masson's Trichrome staining of aortic sections (scale bar=25 μm) and quantification of collagen deposition (D); n=5 biological replicates.


( E - F ) Western blot analysis ( E ) and densitometric quantification ( F ) of VE - cadherin , Vimentin , Twist and Snail in aortic tissues . β - actin was used as the loading control ( n = 5 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

(E-F) Western blot analysis (E) and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues. β-actin was used as the loading control (n=5 biological replicates).


Immunostaining for CCN1 ( green ) in the adult brain coronal section . Scale bar : 50 μm . The insert refers to the same image with DAPI staining to show cell nuclei ( blue ) . LV , lateral ventricle .

B-EXP_ASSAY O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-TISSUE O B-TISSUE I-TISSUE O

Immunostaining for CCN1 (green) in the adult brain coronal section. Scale bar: 50 μm. The insert refers to the same image with DAPI staining to show cell nuclei (blue). LV, lateral ventricle.


Adult V - SVZ whole - mounts stained for CCN1 ( white ) and S100β ( green ) . Scale bar : 10 μm .

O B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O

Adult V-SVZ whole-mounts stained for CCN1 (white) and S100β (green). Scale bar: 10 μm.


Adult V - SVZ whole - mounts stained for CCN1 ( white ) , GFAP ( green ) and VCAM1 ( magenta ) . Arrowhead points to a VCAM1 + B1 cell surrounded by CCN1 - expressing ependymal cells . Scale bar : 10 μm .

O B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O O O O O

Adult V-SVZ whole-mounts stained for CCN1 (white), GFAP (green) and VCAM1 (magenta). Arrowhead points to a VCAM1+ B1 cell surrounded by CCN1-expressing ependymal cells. Scale bar: 10 μm.


Confocal images tracing a VCAM1 + GFAP + cell reveal CCN1 expression is absent in B1 cells . Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit . White arrowheads point to the process of the B1 cell . Scale bar : 10 μm .

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Confocal images tracing a VCAM1+GFAP+ cell reveal CCN1 expression is absent in B1 cells. Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit. White arrowheads point to the process of the B1 cell. Scale bar: 10 μm.


V - SVZ whole - mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment . Scale bar : 10 μm .

B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O

V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) and GFAP ( red ) . Arrowheads point to B1 cells in the pinwheel center . Scale bars : 500 μm ( left ) , 50 μm ( middle ) , 10 μm ( right ) .

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V-SVZ whole-mounts stained for γ-tubulin (cyan) and GFAP (red). Arrowheads point to B1 cells in the pinwheel center. Scale bars: 500 μm (left), 50 μm (middle), 10 μm (right).


Densities ( cells / mm2 ) of B1 cells in the adult V - SVZ . n = 4 , * * * p = 0 . 0005 .

B-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O

Densities (cells/mm2) of B1 cells in the adult V-SVZ. n=4, ***p=0.0005.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) and VCAM1 ( white ) . Arrowheads point to VCAM1 + B1 cells in NSC units . Scale bars : 50 μm ( left ) , 10 μm ( right ) .

B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O

V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).


Densities ( cells / mm2 ) of VCAM1 + B1 cells in the adult V - SVZ . n = 4 , * * p = 0 . 002 . Densities ( units / mm2 ) of NSC units in the adult V - SVZ . n = 4 , * * p = 0 . 0022 . Distribution of NSC units with different numbers of B1 cells . n = 4 , * * * * p < 0 . 0001 ( 1 B1 cell / unit ) , * * p = 0 . 0025 ( 2 B1 cells / unit ) , * * p = 0 . 0063 ( 3 B1 cells / unit ) , * p = 0 . 0385 ( 4 B1 cells / unit ) .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ. n=4, **p=0.002. Densities (units/mm2) of NSC units in the adult V-SVZ. n=4, **p=0.0022. Distribution of NSC units with different numbers of B1 cells. n=4, ****p<0.0001 (1 B1 cell/unit), **p=0.0025 (2 B1 cells/unit), **p=0.0063 (3 B1 cells/unit), *p=0.0385 (4 B1 cells/unit).


Densities ( cells / mm2 ) of B1 cells in the aged V - SVZ . n = 3 , * * p = 0 . 0099 .

B-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O

Densities (cells/mm2) of B1 cells in the aged V-SVZ. n=3, **p=0.0099.


Densities ( cells / mm2 ) of VCAM1 + B1 cells in the aged V - SVZ . n = 3 , * * p = 0 . 0041 .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the aged V-SVZ. n=3, **p=0.0041.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) and VCAM1 ( white ) in aged mice . Arrowheads point to VCAM1 + B1 cells in NSC units . Scale bars : 50 μm ( left ) , 10 μm ( right ) .

B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-ORGANISM O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O

V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white) in aged mice. Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).


Densities ( units / mm2 ) of NSC units in the aged V - SVZ . n = 3 , * * p = 0 . 0093 . Average numbers of B1 cells in a NSC unit . n = 3 , n . s . not significant .

B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O

Densities (units/mm2) of NSC units in the aged V-SVZ. n=3, **p=0.0093. Average numbers of B1 cells in a NSC unit. n=3, n.s. not significant.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 2 days after TAM treatment . n = 4 , * p = 0 . 0141 . V - SVZ whole - mounts stained for GFAP ( red ) and Ki67 ( cyan ) . Arrowheads point to dividing GFAP + cells . Scale bar : 10 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 2 days after TAM treatment. n=4, *p=0.0141. V-SVZ whole-mounts stained for GFAP (red) and Ki67 (cyan). Arrowheads point to dividing GFAP+ cells. Scale bar: 10 μm.


Numbers of ASCL1 + cells in V - SVZ coronal sections 2 days after TAM treatment . n = 3 , * * p = 0 . 0087 . V - SVZ coronal sections stained for ASCL1 ( red ) and nuclei ( blue ) . Scale bar : 50 μm .

B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O

Numbers of ASCL1+ cells in V-SVZ coronal sections 2 days after TAM treatment. n=3, **p= 0.0087. V-SVZ coronal sections stained for ASCL1 (red) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB granule cell layer ( GCL ) 2 days after TAM treatment . n = 4 ( Ctrl ) and 3 ( Ccn1cKO ) , * * p = 0 . 0086 . OB sections stained for BrdU ( yellow ) and nuclei ( blue ) . Scale bar : 50 μm .

B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O

Densities (cells/mm2) of BrdU+ cells in the OB granule cell layer (GCL) 2 days after TAM treatment. n=4 (Ctrl) and 3 (Ccn1cKO), **p=0.0086. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 4 weeks after TAM treatment . Arrowheads point to dividing GFAP + cells . n = 4 , n . s . not significant . Scale bar : 10 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 4 weeks after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Numbers of ASCL1 + cells in V - SVZ coronal sections 4 weeks after TAM treatment . n = 3 , n . s . not significant . Scale bar : 20 μm .

B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

Numbers of ASCL1+ cells in V-SVZ coronal sections 4 weeks after TAM treatment. n=3, n.s. not significant. Scale bar: 20 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL 4 weeks after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of BrdU+ cells in the OB GCL 4 weeks after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 16 months after TAM treatment . Arrowheads point to dividing GFAP + cells . n = 4 , n . s . not significant . Scale bar : 10 μm .

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 16 months after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL 16 months after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Numbers of GFAP + BrdU + label - retaining NSCs in the V - SVZ . Arrowheads point to label - retaining NSCs . n = 4 , * p = 0 . 039 . V - SVZ coronal sections stained for GFAP ( cyan ) and BrdU ( red ) . Scale bar : 10 μm .

B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O

Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 6 , * p = 0 . 0173 . Scale bar : 10 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm.


Densities ( cells / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts 3 days after TMZ treatment . n = 3 , * p = 0 . 0179 . V - SVZ whole - mounts stained for DCX . Scale bar : 500 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O

Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL labeled 24h after TMZ treatment . n = 5 ( Ctrl ) and 6 ( Ccn1cKO ) , * p = 0 . 0484 . OB sections stained for BrdU ( yellow ) and nuclei ( blue ) . Scale bar : 50 μm .

B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O

Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts of aged mice 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 3 , * p = 0 . 0321 . Scale bar : 10 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm.


Densities ( cells / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts of aged mice 3 days after TMZ treatment . n = 3 , n . s . not significant . Scale bar : 500 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL of aged mice labeled 3 days after TMZ treatment . n = 4 ( Ctrl ) and 3 ( Ccn1cKO ) , n . s . not significant . Scale bar : 50 μm .

B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.


Representative FACS plots showing gating strategy for the analysis of cell types in the adult V - SVZ .

O B-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O

Representative FACS plots showing gating strategy for the analysis of cell types in the adult V-SVZ.


Total number of V - SVZ cells isolated from one mouse . n = 3 , n . s . not significant .

O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O

Total number of V-SVZ cells isolated from one mouse. n=3, n.s. not significant.


Representative FACS plots showing aNSCs ( GFP + EGFR + ) , qNSCs ( GFP + EGFR - ) and C cells ( GFP - EGFR + ) in the adult V - SVZ .

O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE I-TISSUE O

Representative FACS plots showing aNSCs (GFP+EGFR+), qNSCs (GFP+EGFR-) and C cells (GFP-EGFR+) in the adult V-SVZ.


Percentage of each cell population in the adult V - SVZ . n = 3 , * * p = 0 . 0014 ( GFP + EGFR - ) , * * * p = 0 . 0002 ( GFP + EGFR + ) , * p = 0 . 0111 ( GFP - EGFR + ) , * * * p = 0 . 0006 ( GFP + ) , n . s . not significant .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

Percentage of each cell population in the adult V-SVZ. n=3, **p=0.0014 (GFP+EGFR-), ***p=0.0002 (GFP+EGFR+), *p=0.0111 (GFP-EGFR+), ***p=0.0006 (GFP+), n.s. not significant.


Densities ( cells / mm2 ) of VCAM1 + B1 cells in the adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle and Ccn1cKO + vehicle ) and 3 ( Ccn1cKO + erlotinib ) , * * p = 0 . 0021 ( Ccn1cKO + vehicle vs Ccn1cKO + erlotinib ) , * * p = 0 . 0014 ( Ccn1cKO + erlotinib vs Ctrl + vehicle ) , * * * * p < 0 . 0001 .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001.


Densities ( units / mm2 ) of NSC units in the adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle and Ccn1cKO + vehicle ) and 3 ( Ccn1cKO + erlotinib ) , * p = 0 . 017 , * * p = 0 . 0061 , * * * p = 0 . 0002 .

B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) and VCAM1 ( white ) . Scale bar : 50 μm .

B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O

V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Scale bar: 50 μm.


A Neonatal rat ventricular myocytes ( NRVMs ) were infected with the 3xMEF2 - Luc reporter , serum starved for 20 hours and stimulated for 24 hours with different agonists : 100 nM endothelin - 1 ( ET1 ) , 1 μM sphingosine - 1 - phosphate ( S1P ) , 10 μM lisophosphatidic acid ( LPA ) , 1 μM WIN55 , 212 - 2 ( WIN55 , cannabinoid receptor agonist ) , 1 μM isoproterenol ( ISO , β - adrenergic receptor agonist ) , 100 nM angiotensin II ( AngII ) , 10 μM prostaglandin E1 ( PGE1 ) , 10 μM prostaglandin E2 ( PGE2 ) or 100 nM prostanoid F receptor agonist fluprostenol ( Flupro ) , 100 nM treprostinil ( Trepro , prostacyclin receptor agonist ) Data information : values are mean ± s . e . m . In ( A ) , the experiment was performed in triplicates , similar results were obtained in 3 different experiments . Student ' s two - tailed t - test , * , P < 0 . 05 vs . control ( ET1 , P = 0 . 0013 ; S1P , P = 0 . 0144 ; LPA , P = 0 . 026 ; WIN55 , P = 0 . 6205 ; ISO = 0 . 0399 ; AngII , P = 0 . 5812 ; PGE1 , P = 0 . 001 ; PGE2 , P < 0 . 0001 ; Flupro , P = 0 . 0846 ; Trepro , P = 0 . 1958 )

O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist) Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958)


B PGE2 induces the expression of MEF2 target genes . NRVMs were serum - starved for 24 hours , then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information : values are mean ± s . e . m In ( B ) , n = 5 , technical replicates , Student ' s two - tailed t - test , * , P < 0 . 05 vs . control ( Nur77 , P = 0 . 0148 ; Myomaxin , P = 0 . 0417 ; Adamts1 , P = 0 . 0112 ; BNP , P = 0 . 0065 )

O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O

B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information: values are mean±s.e.m In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065)


C Correlation between MEF2 activation and NRVM hypertrophy . MEF2 activity was measured as in ( A ) upon increasing concentration of PGE2 . Cell size of NRVMs was determined after 48 - hour stimulation with the indicated concentrations of PGE2 . Left : representative images of α - actinin stained NRVMs stimulated with DMSO , 300 nM PGE2 , or 1 nM ET1 . Scale bar is 10 µm Data information : values are mean ± s . e . m In ( C ) , NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2 , MEF2 - activity was parallel assessed from 6 technical replicates . Correlation was statistically analyzed by calculating Pearson correlation coefficient ( Pearson r = 0 . 9626 , P = 0 . 0021 )

O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-hour stimulation with the indicated concentrations of PGE2. Left: representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 µm Data information: values are mean±s.e.m In (C), NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2, MEF2-activity was parallel assessed from 6 technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r=0.9626, P=0.0021)


D , PGE2 activates MEF2 via EP3 receptor . NRVMs were infected with the 3xMEF2 - Luc reporter and serum - starved for 20 hours . The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists : AH6809 ( 10 μM , EP1 - and EP2 - antagonist ) or 798106 ( 200 nM , EP3 - antagonist ) or L161 , 982 ( 2 μM , EP4 - antagonist ) Data information : values are mean ± s . e . m In ( D ) , n = 3 , independent experiments , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , AH6809 , P = 0 . 1092 ; L798106 , P = 0 . 0002 ; L161 , 982 , P = 0 . 5579 ) . The exact n and P values can be also found in the Source Data excel file for Figure

O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist) Data information: values are mean±s.e.m In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1


B - D ( B , D ) NRVMs were infected with the 3xMEF2 - Luciferase reporter and with recombinant adenoviruses encoding EGFP ( AdGFP ) , RGS16 , RGS2 , p63ΔN , RGS - LSCII , or Gαt , as indicated . The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours . ( C ) NRVMs were infected with the 3xMEF2 - Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 ( 100 μM ) for 20 minutes or the Gi / o - protein inhibitor pertussis toxin ( PTX , 100 ng / ml ) for 20 hours in serum - starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours . Data information : In ( B - D ) , n = 3 , independent experiments , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , RGS16 , P < 0 . 0001 ; RGS2 , P = 0 . 1725 ; p63ΔN , P = 0 . 0666 ; RGS - LSCII , P = 0 . 0818 , SQ22536 , P = 0 , 385 ; PTX , P < 0 . 0001 ; Gαt , P < 0 , 0001 ) , values are mean ± s . e . m . The exact n and P values can be also found in the Source Data excel file for Figure 2

O O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours. Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2


A , B NRVMs were infected with recombinant adenovirus encoding Flag - HDAC5 ( A ) or not infected ( B ) and serum starved for 20 hours . The cells were pretreated with the PKC - inhibitor bisindolylmaleimide I ( 2 μM ) or the PKD - inhibitor BPKDi ( 3 μM ) for 20 minutes and stimulated with 100 nM endothelin - 1 ( ET1 ) or 100 μM phenylephrine ( PE ) or 1 μM PGE2 for 4 hours , as indicated . HDAC phosphorylation was detected by immunoblotting with the anti - HDAC5 phospho - Ser - 498 antibody , phosphorylation of PKD was assessed using anti - phospho - PKD antibodies ( Ser - 744 / Ser - 748 and Ser - 916 ) , representative images are shown from 3 independent experiments

O O O B-CELL_TYPE O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

A,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments


C NRVMs were infected with the 3xMEF2 - Luc reporter and serum - starved for 20 hours . The cells were pretreated with the PKD - inhibitor BPKDi ( 3 μM ) or the CamKII - inhibitor AIP ( 1 μM ) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours . N = 3 , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , BPKDi , P < 0 . 0001 ; AIP , P = 0 . 2197 ) , values are mean ± s . e . m . The exact n and P values can be also found in the Source Data excel file for Figure

O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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 NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3


A NRVMs were serum - starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2 . Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information : In ( A - C ) representative images from at least 3 independent experiments are shown

O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

A NRVMs were serum-starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2. Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information: In (A-C) representative images from at least 3 independent experiments are shown


B NRVMs were infected with recombinant adenoviruses encoding EGFP ( AdGFP ) or RGS16 24 hours prior to stimulation , then Rac1 activity was measure Data information : In ( A - C ) representative images from at least 3 independent experiments are shown . In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS16 , P = 0 . 0415 ( B ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure

O B-CELL_TYPE O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O 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 NRVMs were infected with recombinant adenoviruses encoding EGFP (AdGFP) or RGS16 24 hours prior to stimulation, then Rac1 activity was measure Data information: In (A-C) representative images from at least 3 independent experiments are shown. In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS16, P=0.0415 (B ), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4


C Cells were pretreated with 100 ng / ml pertussis toxin ( PTX , Gi / o - inhibitor ) for 24 hours or with 50 μM NSC23766 ( NSC , Tiam1 - Rac1 interaction inhibitor ) for 2 hours . Thereafter Rac1 activation was determined by effector pulldown assay Data information : In ( A - C ) representative images from at least 3 independent experiments are shown In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS1 PTX , P = 0 . 039 ( C ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure

O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O 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 Cells were pretreated with 100 ng/ml pertussis toxin (PTX, Gi/o-inhibitor) for 24 hours or with 50 μM NSC23766 (NSC, Tiam1-Rac1 interaction inhibitor) for 2 hours. Thereafter Rac1 activation was determined by effector pulldown assay Data information: In (A-C) representative images from at least 3 independent experiments are shown In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS1 PTX, P=0.039 (C), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4


D NRVMs were transduced with the 3xMEF2 - Luc reporter and were serum - starved for 20 hours . After 2 - hour of 50 μM NSC23766 ( A ) pretreatments , the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours , then MEF2 activity was determined . NSC abolished the MEF2 activation by PGE2 Data information In ( B - D ) values are mean ± s . e . m . ( D ) , n = 3 ; * , P < 0 . 05 ( Two - Way ANOVA ,

O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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 NRVMs were transduced with the 3xMEF2-Luc reporter and were serum-starved for 20 hours. After 2-hour of 50 μM NSC23766 (A) pretreatments, the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours, then MEF2 activity was determined. NSC abolished the MEF2 activation by PGE2 Data information In (B-D) values are mean±s.e.m. (D), n=3; *,P<0.05 (Two-Way ANOVA, RGS1


A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour . Total and phosphorylated PKD levels were analyzed by immunoblot . PKD was activated by PGE2 , but it was not affected by NSC Data information : In ( A - C ) representative images from 3 independent experiments are shown

O O B-TISSUE O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC Data information: In (A-C) representative images from 3 independent experiments are shown


B After infection with Flag - HDAC5 and serum starvation , NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation . The cells were stimulated with 1 μM PGE2 for 1 hour . HDAC5 phosphorylation at Ser - 498 was detected by immunoblot . The phosphorylation of HDAC5 at this site was not prevented by NSC Data information : In ( A - C ) representative images from 3 independent experiments are shown

O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC Data information: In (A-C) representative images from 3 independent experiments are shown


C NRVMs and neonatal cardiac fibroblasts ( CFs ) were serum - starved , pretreated with vehicle or 200 nM L798106 ( EP3 receptor antagonist ) for 20 minutes , and were stimulated with 1 μM PGE2 for 10 minutes . PAK2 and PKD phosphorylation was assessed by immunoblot . Calsequestrin was used as a myocyte - marker Data information : In ( A - C ) representative images from 3 independent experiments are shown

O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD 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 NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker Data information: In (A-C) representative images from 3 independent experiments are shown


D MEF2 activity was determined in cells infected with the 3xMEF2 - Luc reporter after serum starvation . Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1 - hour of 30 μM IPA - 3 pretreatment . IPA - 3 inhibited the MEF2 activation by PGE2 Data information In ( D ) values are mean ± s . e . m . , n = 3 , biological replicates , * , P < 0 . 05 ( two - way ANOVA , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure

O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2 Data information In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5


Top : GFP - HDAC5 infected NRVMs were serum - starved for a day , pretreated for two hours with the indicated compounds ( BPKDi , 3 µM , IPA - 3 , 30 µM ) alone or in combination , then were stimulated for 4 hours . Representative images . Scale bar is 10 µm . Bottom : Statistical analysis of cellular localization of HDAC5 . GFP - HDAC5 localization ( nuclear or cytosolic ) was assessed in > 100 cells of each sample ( n = 3 , technical replicates ) . * , P < 0 , 05 ( one - way ANOVA with Bonferroni post - hoc test , vehicle vs . ET1 or vs . PGE2 , P < 0 . 0001 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0004 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0114 ; PGE2 vs . PGE2 + BPKDi + IPA - 3 , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure 6

O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours A mRNA - levels of different inflammatory cytokines , as indicated . The graphs show relative mRNA - levels , fold increase compared to saline - treated controls , normalized to 18s - content Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values : IL6 , P = 0 . 0007 ( A ) ; TNFα , P < 0 . 0001 ( A )

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours A mRNA-levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA-levels, fold increase compared to saline-treated controls, normalized to 18s-content Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values: IL6, P=0.0007 (A); TNFα, P<0.0001 (A)


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano - liquid chromatography tandem mass spectrometry Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values PGE2 , P = 0 . 0016 (

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano-liquid chromatography tandem mass spectrometry Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values PGE2, P=0.0016 (B


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β - galactosidase - activity in MEF2 - lacZ reporter mice show MEF2 - activation ( blue cells and precipitates , respectively ) in the myocardium upon LPS - treatment . Saline - treated littermates served as control . Scale bar of histological stainings is 100 µm . Quantification of whole - heart stainings is shown in the right panel ( pixel intensity in blue channel normalized to total intensity ) Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant . In ( C ) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments . P values blue pixel intensity , P = 0 . 0083 ( C )

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β-galactosidase-activity in MEF2-lacZ reporter mice show MEF2-activation (blue cells and precipitates, respectively) in the myocardium upon LPS-treatment. Saline-treated littermates served as control. Scale bar of histological stainings is 100 µm. Quantification of whole-heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity) Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant. In (C) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments. P values blue pixel intensity, P=0.0083 (C)


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD , HDAC5 and PAK2 in myocardial inflammations . Immunoblots were performed on extracts of hearts of saline - and LPS - treated mice . Representative blots are shown in the left panel , the quantification is in the right panel Data information : values are mean ± s . e . m The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values ) ; phospho - PKD - Ser - 744 / Ser - 748 , P = 0 . 0049 ( D ) ; phospho - PKD - Ser - 916 , P = 0 . 2684 ( D ) ; phospho - HDAC5 - Ser - 498 ( D ) , P = 0 . 0019 ; phospho - HDAC5 - Ser - 259 , P = 0 . 0003 ( D ) ; phospho - PAK2 - Ser - 192 / Ser - 197 , P = 0 . 1962 ( D ) ; phospho - PAK2 - Thr - 402 , P = 0 . 0446 ( D ) . The exact n and P values can be also found in the Source Data excel file for Figure

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD, HDAC5 and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, the quantification is in the right panel Data information: values are mean±s.e.m The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values ); phospho-PKD-Ser-744/Ser-748, P=0.0049 (D); phospho-PKD-Ser-916, P=0.2684 (D); phospho-HDAC5-Ser-498 (D), P=0.0019; phospho-HDAC5-Ser-259, P=0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P=0.1962 (D); phospho-PAK2-Thr-402, P=0.0446 (D). The exact n and P values can be also found in the Source Data excel file for Figure 7


A Isothermal calorimetry ( ITC ) assays using ADP - ribose ligand and purified macrodomains of human macroH2A1 . 1 and macroH2A2 .

A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.


B In vitro PARP1 activity assay in the presence of increasing concentrations ( 10 , 25 and 50 μM ) of purified macrodomains of human macroH2A1 . 1 , macroH2A1 . 1 G224E mutant and macroH2A2 . PARP1 activity is assessed by its auto - PARylation detected by an anti - PAR immunoblot . The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction . A representative blot of one of three independent experiments is shown .

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B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.


A The diameter ( d ) of the fluorescence signaling of H2B tagged with photo - activatable GFP ( PAGFP - H2B ) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser .

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A The diameter (d) of the fluorescence signaling of H2B tagged with photo-activatable GFP (PAGFP-H2B) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.


B Chromatin expansion in absence and presence of intercalating DNA dye ( - and + DNA damage ) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor . Boxplots represent single cell measurement of chromatin expansion at 120 sec . post DNA damage from 3 biological replicates with n > 30 cells each . The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values ( * , p < 0 . 05 using unpaired , two - tailed Student ' s T - test ) .

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B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).


C Quantified chromatin expansion in cells not transfected ( control ) and cells transfected with each mCherry - tagged histone ( as indicated ) . Data is represented as in B ( n > 30 cells , 3 bio . repl . ; * , p < 0 . 05 compared to mCherry - H2A - expressing cells using unpaired , two - tailed Student ' s T - test ) .

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C Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry-tagged histone (as indicated). Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 compared to mCherry-H2A-expressing cells using unpaired, two-tailed Student's T-test).


D Chromatin expansion in Hela cells containing a doxocyclin ( doxo . ) - inducible macroH2A1 . 1 transgene . Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti - macroH2A1 . 1 - immunoblotting . Data is represented as in B ( n > 30 cells , 3 bio . repl . ; * , p < 0 . 05 using unpaired , two - tailed Student ' s T - test ) .

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D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).


Quantified chromatin expansion in cells transfected with indicated histone constructs . Boxplots represent single cell measurement of chromatin expansion at 120 sec . post DNA damage ( n > 30 cells , 3 bio . repl . ) . The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test comparing to H2A ; n . s . , not significant ) .

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Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).


A Western blot showing the expression of macroH2A proteins in control HepG2 cells , knock - down for macroH2A1 and macroH2A2 ( DKD ) and DKD with ectopic expression of the indicated YFP - tagged constructs . H3 is used as a loading control .

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A Western blot showing the expression of macroH2A proteins in control HepG2 cells, knock-down for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP-tagged constructs. H3 is used as a loading control.


B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI . The indicated areas are zoomed - in for a better observation of the H3K9me3 signal profile . All images are maximum intensity Z - projections of confocal stacks .

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B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.


Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test ; n . s . , not significant ) .

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Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


Quantification of H3K9me3 foci per nucleus , The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test ; n . s . , not significant ) .

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Quantification of H3K9me3 foci per nucleus, The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


Wild - type , apg12 - 1 mutant and Δapg12 cells were cultured in nitrogen - starvation medium containing 1 mM PMSF . After incubation for 6 h , cells were observed under a phase - contrast microscope . Arrows indicate autophagic bodies .

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Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.


c , Wild - type ( squares ) and Δapg12 ( circles ) were cultured in nitrogen - starvation medium and their viability was determined by phloxine B staining .

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c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.


d , Quantification of autophagic activity of wild - type and Δapg12 cells by alkaline phosphatase ( ALP ) assay before ( black bars ) and after ( white bars ) nitrogen starvation for 4 h . Error bars indicate s . d . of three independent experiments .

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d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.


a , b , Lysates from Δapg12 cells carrying only vector or 3 × HA - APG12 ( a ) , and Δapg5 , apg7 - 1 , apg10 - 1 and Δapg1 cells carrying 3 × HA - Apg12 plasmid ( b ) were immunoblotted using anti - HA antibody . The positions of 3 × HA - Apg12 and the larger product ( asterisks ) are indicated .

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a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated.


c , Immunoblot analysis of wild - type and Δapg12 cells harbouring HA - APG5 plasmid .

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c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid.


d , Δapg5 Δapg12 cells were co - transformed with Myc - APG12 and HA - APG5 . Their lysates were immunoprecipitated with anti - Myc or anti - HA antibodies and detected by immunoblotting using anti - Myc antibody . The position of the crossreacting IgG heavy chain is

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d, Δapg5 Δapg12 cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicated


b , Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti - HA antibody .

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b, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody.


c , Autophagic activity was measured as described for Fig . 1d .

c, Autophagic activity was measured as described for Fig. 1d.


d , Transport of pro - API to the vacuole was examined by immunoblotting with anti - API antiserum . The positions of pro - API and mature API are indicated .

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d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of pro-API and mature API are indicated.


Δapg5 cells were transformed with vector alone , wild - type APG5 or APG5K149R , and then immunoblotted with anti - HA ( b )

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted with anti-HA (b)


Autophagic activity was determined by alkaline phosphatase assay ( c ) .

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Autophagic activity was determined by alkaline phosphatase assay (c).


Δapg5 cells were transformed with vector alone , wild - type APG5 or APG5K149R , and then immunoblotted anti - API ( d ) .

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted anti-API (d).


c , In vitro conjugation of Apg5 and Apg12 . A cell lysate of Δapg12 carrying HA - APG5 ( lane 1 ) was incubated with an equal amount of lysate from Δapg5 carrying HA - APG12 ( 2 µ plasmid ) ( lane 2 ) at 30 ° C with ( lane 4 - 6 ) or without ( lane 7 ) 5 mM ATP . Samples were mixed with SDS - sample buffer at the times indicated .

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c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.


Spheroplasts were generated from cells expressing either HA - Apg12 or HA - Apg5 . Their lysates were mixed and layered on top of a 10 - step ( 18 - 54 % w / w ) sucrose gradient , and centrifuged at 174 , 000g for 2 . 5 h ( ref . 29 ) . Fifteen fractions were collected and the positions of free Apg5 , free Apg12 and Apg5 / Apg12 conjugate were examined by western blotting . The peak fractions of alcohol dehydrogenase ( ADH ) ( cytosol ) , ALP ( vacuole ) , Kex2 ( Golgi ) and Sec12 ( endoplasmic reticulum ) are indicated by arrows .

B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.


A ) We identified previously unknown telomere regulators in fission yeast using the haploid S . pombe whole - genome gene deletion library including ssu72 + ( SPAC3G9 . 04 ) . B ) Telomere length of wt , ssu72Δ and ssu72 - C13S ( point mutation at the phosphatase active site ) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe .

O O O O O O B-SUBCELLULAR O O B-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR O O

A) We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04). B) Telomere length of wt, ssu72Δ and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe.


C ) Ssu72 recruitment to telomeres is cell cycle regulated . Ssu72 was myc - tagged at the 3 ' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains . Septa formation was used proxy for S - phase . n ≥ 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to ssu72 + control samples . Error bars represent standard error of the mean ( SEM ) .

O O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O

C) Ssu72 recruitment to telomeres is cell cycle regulated. Ssu72 was myc-tagged at the 3' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains. Septa formation was used proxy for S-phase. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).


D ) Telomere length of ssu72Δ is dependent on telomerase . Diploid strains with the appropriate genotypes were sporulated and trt1 ∆ ssu72 ∆ double mutants were streaked for multiple passages ( triangle indicates increased number of generations ) .

O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

D) Telomere length of ssu72Δ is dependent on telomerase. Diploid strains with the appropriate genotypes were sporulated and trt1∆ ssu72∆ double mutants were streaked for multiple passages (triangle indicates increased number of generations).


E ) Telomerase is recruited to telomeres in the absence of Ssu72 . ChIP analysis for Trt1 - myc in wt and ssu72 ∆ was performed as described in material and methods using a non - tagged strain as a control . n ≥ 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) .

O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Telomerase is recruited to telomeres in the absence of Ssu72. ChIP analysis for Trt1-myc in wt and ssu72∆ was performed as described in material and methods using a non-tagged strain as a control. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


F ) Telomerase activator Ccq1 is hyperphosphorylated in ssu72 ∆ cells . rap1 ∆ cells were used as positive control for Ccq1 phosphorylation status . Western blots were performed using Ccq1 - flag tagged strains .

O O B-SUBCELLULAR O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O

F) Telomerase activator Ccq1 is hyperphosphorylated in ssu72∆ cells. rap1∆ cells were used as positive control for Ccq1 phosphorylation status. Western blots were performed using Ccq1-flag tagged strains.


A ) ssu72Δ telomeres present longer G - rich overhangs than wt and rif1 ∆ . In - gel hybridization in native and denaturing conditions was labelled with a radiolabelled C - rich telomere probe and quantified for ssDNA at the telomeres . n = 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) .

O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O 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) ssu72Δ telomeres present longer G-rich overhangs than wt and rif1∆. In-gel hybridization in native and denaturing conditions was labelled with a radiolabelled C-rich telomere probe and quantified for ssDNA at the telomeres. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


B ) RPA ( Rad11 - GFP ) is enriched at ssu72 ∆ telomeres . Colocalization of Rad11 - GFP with Taz1 - mCherry , used as a telomere marker was performed in wt and ssu72 ∆ cells ; n = 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) . More than 1000 cells were analyzed for each

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B) RPA (Rad11-GFP) is enriched at ssu72∆ telomeres. Colocalization of Rad11-GFP with Taz1-mCherry, used as a telomere marker was performed in wt and ssu72∆ cells; n =3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). More than 1000 cells were analyzed for each genotype


C ) ssu72 + controls telomere length independently of rif1 + . Epistasis analysis of telomere length of ssu72 ∆ and ssu72 - C13S ( catalytically inactive mutant ) with rif1 ∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe . D ) ssu72 + and stn1 + regulate telomere length in the same genetic pathway . Epistasis analysis of ssu72 ∆ and stn1 - 75 performed by Southern blotting of ApaI digested DNA using a telomeric probe . Two independently generated ssu72 ∆ stn1 - 75 double mutants are shown .

O O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

C) ssu72+ controls telomere length independently of rif1+. Epistasis analysis of telomere length of ssu72∆ and ssu72-C13S (catalytically inactive mutant) with rif1∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe. D) ssu72+ and stn1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and stn1-75 performed by Southern blotting of ApaI digested DNA using a telomeric probe. Two independently generated ssu72∆ stn1-75 double mutants are shown.


A ) Ssu72 is required for telomere recruitment of Stn1 in late S phase . ChIP analysis of stn1 - myc in wt and ssu72 ∆ cells was performed in synchronized cdc25ts cells . n ≥ 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to ssu72 + control samples . Error bars represent standard error of the mean ( SEM ) .

O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O 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-GENEPROD O O O O O O O O O O O O O O O O

A) Ssu72 is required for telomere recruitment of Stn1 in late S phase. ChIP analysis of stn1-myc in wt and ssu72∆ cells was performed in synchronized cdc25ts cells. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).


B ) 2D - gel analysis of NsiI telomeric fragments of wt and ssu72 ∆ strains . Smart ladder from Eurogentec was used for DNA size measurement .

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B) 2D-gel analysis of NsiI telomeric fragments of wt and ssu72∆ strains. Smart ladder from Eurogentec was used for DNA size measurement.