A . Typical examples of Centrinone - B treated or control human iPSC - derived NSCs immunostained for Pericentrin and Centrin . Inserts represent centriole ( s ) . Scale bar = 5µm in overview , 2µm in inserts . B . Quantifications of the percentage of cells with 0 , 1 or 2 centrioles per cell after 0 ( control ) , 2 or 5 days Centrinone - B treatment and prior to neuronal induction . n = 48 - 51 cells in 2 independent experiments . C Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Bonferroni correction * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 .

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A. Typical examples of Centrinone-B treated or control human iPSC-derived NSCs immunostained for Pericentrin and Centrin. Inserts represent centriole(s). Scale bar = 5µm in overview, 2µm in inserts. B. Quantifications of the percentage of cells with 0, 1 or 2 centrioles per cell after 0 (control), 2 or 5 days Centrinone-B treatment and prior to neuronal induction. n = 48-51 cells in 2 independent experiments. C Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05


C . Typical examples of Centrinone - B treated or control human iPSC - derived neurons ( 12 - 15 days ) immunostained for AnkG , Trim46 , MAP2 and Centrin . Arrowheads mark AIS structures , arrows mark centrosomes . Inserts represent centrosomes , zooms on the right represent AIS structures . Scale bar = 20µm in overview , 2µm in insert , 10µm in zooms .

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C. Typical examples of Centrinone-B treated or control human iPSC-derived neurons (12-15 days) immunostained for AnkG, Trim46, MAP2 and Centrin. Arrowheads mark AIS structures, arrows mark centrosomes. Inserts represent centrosomes, zooms on the right represent AIS structures. Scale bar = 20µm in overview, 2µm in insert, 10µm in zooms.


M . AP threshold , amplitude and after - hyperpolarization recorded in Centrinone - B treated ( n = 43 cells in 3 independent experiments ) and control human iPSC - derived neuron cultures ( n = 59 cells in 4 independent experiments ) . Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Mann Whitney test after - hyperpolarization Student ' s t test ( M : threshold , AP amplitude ) . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 .

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M. AP threshold, amplitude and after-hyperpolarization recorded in Centrinone-B treated (n = 43 cells in 3 independent experiments) and control human iPSC-derived neuron cultures (n = 59 cells in 4 independent experiments). Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Mann Whitney test after-hyperpolarization Student's t test (M: threshold, AP amplitude). *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05


D . Representative images of fan - like growth cones at day 5 of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions . Growth cone visualized by immunostaining for phalloidin . Scale bar = 5µm . E . Quantifications of the average area ( µm² ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 25 - 83 growth cones in 3 independent experiments . F Data information : Data represent mean ± SEM . One - way ANOVA including Tukey ' s post - hoc analysis * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 0 .

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D. Representative images of fan-like growth cones at day 5 of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions. Growth cone visualized by immunostaining for phalloidin. Scale bar = 5µm. E. Quantifications of the average area (µm²) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 25-83 growth cones in 3 independent experiments. F Data information: Data represent mean ± SEM. One-way ANOVA including Tukey's post-hoc analysis *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05


F . Representative images of different growth cone morphological categories : fan - like , torpedo - like and bulb - like . Scale bar = 5µm . G . Quantifications of the ratios of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . H . Quantifications of the average area ( µm² ) of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 5 - 55 growth cones in 3 independent experiments . Da Data information : Chi - square - test including post - hoc analysis with Bonferroni correction ( G ) , One - way ANOVA including Sidak ' s post - hoc analysis ( H ) . * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 0 .

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F. Representative images of different growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 5-55 growth cones in 3 independent experiments. Da Data information: Chi-square-test including post-hoc analysis with Bonferroni correction (G), One-way ANOVA including Sidak's post-hoc analysis (H). *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05


A . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . The top panel is a still of a neurite in mRFP , showing neurite morphology . The other panels show moving GFP - MT + TIP comets pointing in either an anterograde direction ( green arrowheads ) or retrograde direction ( blue arrowheads ) . P indicates the proximal direction and D the distal direction of the neurite . Timestamp in minutes : seconds given on bottom right . Scale bar = 5 µm . B . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( A ) , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Scale bar = 5 µm . C

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A. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. The top panel is a still of a neurite in mRFP, showing neurite morphology. The other panels show moving GFP-MT+TIP comets pointing in either an anterograde direction (green arrowheads) or retrograde direction (blue arrowheads). P indicates the proximal direction and D the distal direction of the neurite. Timestamp in minutes:seconds given on bottom right. Scale bar = 5 µm. B. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (A), for different time points (day 7; day 13) and conditions (control; Centrinone-B). Scale bar = 5 µm. C


J . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . Red line denotes the location of LS . Scale bar = 5 µm . K . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( J ) following LS , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Red line and red arrowhead denote location and time of LS . Scale bar = 5 µm . L

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J. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. Red line denotes the location of LS. Scale bar = 5 µm. K. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (J) following LS, for different time points (day 7; day 13) and conditions (control; Centrinone-B). Red line and red arrowhead denote location and time of LS. Scale bar = 5 µm. L


( C ) Period length decreases with continual differentiation of PSCs into HIOs . Maturation of HIOs into kcHIEs leads to 24 - hour rhythms with greater reproducibility . ( D ) The amplitude of oscillations are significantly higher in kcHIEs / bHIEs compared to HIOs . Bmal1 - luc traces in B were generated by plotting the standard deviation of n ≥ 3 biological replicates .

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(C) Period length decreases with continual differentiation of PSCs into HIOs. Maturation of HIOs into kcHIEs leads to 24-hour rhythms with greater reproducibility. (D) The amplitude of oscillations are significantly higher in kcHIEs/bHIEs compared to HIOs. Bmal1-luc traces in B were generated by plotting the standard deviation of n≥3 biological replicates.


( A - C ) mRNA expression of Bmal1 ( black ) , Rev - erbα ( grey ) and Per2 ( red ) in HIOs ( A ) , kcHIEs ( B ) and bHIEs ( C ) .

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(A-C) mRNA expression of Bmal1 (black), Rev-erbα (grey) and Per2 (red) in HIOs (A), kcHIEs (B) and bHIEs (C).


( A ) Representative images of necrotic cell death ( SYTOX orange - red fluorescence ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in control Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f / f - EsrCRE enteroids without tamoxifen treatment . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( D ) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids .

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(A) Representative images of necrotic cell death (SYTOX orange - red fluorescence) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in control Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f/f-EsrCRE enteroids without tamoxifen treatment. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (D) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids.


( A ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 1μg / mL TcdB in HIOs . Scale bar = 250μm . ( B ) Quantification of necrotic cell death in HIOs . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in kcHIEs . Scale bar = 250μm . ( D ) Quantification of necrotic cell death in kcHIEs . ( E ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in bHIEs . Scale bar = 250μm . ( F ) Quantification of necrotic cell death in bHIEs .

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(A) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.1μg/mL TcdB in HIOs. Scale bar = 250μm. (B) Quantification of necrotic cell death in HIOs. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in kcHIEs. Scale bar = 250μm. (D) Quantification of necrotic cell death in kcHIEs. (E) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in bHIEs. Scale bar = 250μm. (F) Quantification of necrotic cell death in bHIEs.


( A ) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids .

(A) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids.


( B ) Spearman ' s rho for circadian clock and clock - associated genes .

(B) Spearman's rho for circadian clock and clock-associated genes.


( A ) Validation of anti - phasic Rac1 expression in mouse ( mRac1 , red ) and human enteroids ( hRac1 , black ) by qRT - PCR . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1 - KD bHIEs .

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(A) Validation of anti-phasic Rac1 expression in mouse (mRac1, red) and human enteroids (hRac1, black) by qRT-PCR. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1-KD bHIEs.


( C - D ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f / f ; CAGGCre - ERTM without tamoxifen ( C ) or Rac1 f / f ; CAGGCre - ERTM with tamoxifen - treated ( D ) enteroids .

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(C-D) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f/f;CAGGCre-ERTM without tamoxifen (C) or Rac1 f/f;CAGGCre-ERTM with tamoxifen-treated (D) enteroids.


B . FPKM of the 7 LncRNAs identified in the RNA - seq with high expression in PGCs . The solid lines at each end of the box plot represent the maximum and minimum values , respectively . The three dotted lines in the middle of the violin diagram represent the 75 % percentile , the mean , and the 25 % percentile in turn .

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B. FPKM of the 7 LncRNAs identified in the RNA-seq with high expression in PGCs. The solid lines at each end of the box plot represent the maximum and minimum values, respectively. The three dotted lines in the middle of the violin diagram represent the 75% percentile, the mean, and the 25% percentile in turn.


A . Up : The schematic diagram of PGCs induced from chicken ESCs in vitro . Down : Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference ; oeLncBMP4 indicates the overexpression of LncBMP4 ; shLncBMP4 indicates the interference of LncBMP4 ; BMP4 induction was regarded as the control . Scale bar : 60 µm ( n = 3 independent experiments ) .

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A. Up: The schematic diagram of PGCs induced from chicken ESCs in vitro. Down: Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference; oeLncBMP4 indicates the overexpression of LncBMP4; shLncBMP4 indicates the interference of LncBMP4; BMP4 induction was regarded as the control. Scale bar: 60 µm (n = 3 independent experiments).


B . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR after LncBMP4 overexpression and interference , in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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B. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR after LncBMP4 overexpression and interference, in vitro (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA) .


A . The results of IHC examining using anti - DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge , and interference with LncBMP4 reduces the number of PGCs in the genital ridge . The number of PGCs was counted through the Segmentation module of Image J software . Scale bars : 200 μm ( top row ) , 40 μm ( bottom row ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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A. The results of IHC examining using anti-DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge, and interference with LncBMP4 reduces the number of PGCs in the genital ridge. The number of PGCs was counted through the Segmentation module of Image J software. Scale bars:200 μm (top row),40 μm (bottom row) (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA).


B . The number of PGCs in the genital ridge , following the overexpression or interference of LncBMP4 , as determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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B. The number of PGCs in the genital ridge, following the overexpression or interference of LncBMP4, as determined by flow cytometry (Data are shown as mean ± SEM, n = 3 independent experiments, **** p < 0.0001, one-way ANOVA).


C . The number of PGCs labeled with PkH26 ( red ) that migrated to the genital ridge in a frozen section , the number of PGCs labeled with PkH26 ( red ) was counted using Image J software . Scale bar : 70 µm ( Data are shown as mean ± SEM , n = 6 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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C. The number of PGCs labeled with PkH26 (red) that migrated to the genital ridge in a frozen section, the number of PGCs labeled with PkH26 (red) was counted using Image J software. Scale bar: 70 µm (Data are shown as mean ± SEM, n = 6 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


D . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR in the genital ridge after gga - mir - 12211 overexpression and interference in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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D. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR in the genital ridge after gga-mir-12211 overexpression and interference in vitro (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA).


E . The efficiency of PGC differentiation was detected by IF , 4 d after induction , using C - KIT and CVH antibodies . Scale bar : 60 µm ( n = 3 independent experiments ) .

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E. The efficiency of PGC differentiation was detected by IF, 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments).


A . A luciferase reporting system was used to detect the regulatory effects of gga - mir - 12211 on the activity of BMP4 - 3 ' UTR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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A. A luciferase reporting system was used to detect the regulatory effects of gga-mir-12211 on the activity of BMP4-3'UTR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).


B . qRT - PCR was used to detect the regulatory effects of gga - mir - 12211 on LncBMP4 expression ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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B. qRT-PCR was used to detect the regulatory effects of gga-mir-12211 on LncBMP4 expression (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001, **** p < 0.0001, one-way ANOVA).


Enrichment levels of m6A in LncBMP4 were detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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Enrichment levels of m6A in LncBMP4 were detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


H and I . The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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H and I. The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA.).


A . The influence of m6A on the stability of LncBMP4 was detected by qRT - PCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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A. The influence of m6A on the stability of LncBMP4 was detected by qRT-PCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


D . The enrichment level of m6A , for LncBMP4 variants with the progressive accumulation of RRACH site mutations , was detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA ) .

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D. The enrichment level of m6A, for LncBMP4 variants with the progressive accumulation of RRACH site mutations, was detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, one-way ANOVA).


E . The ability of LncBMP4 , modified with different levels of m6A , to bind gga - mir - 12211 was detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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E. The ability of LncBMP4, modified with different levels of m6A, to bind gga-mir-12211 was detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


B . qRT - PCR was used to detect the expression of related genes in each group , during induction ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one - way ANOVA ) .

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B. qRT-PCR was used to detect the expression of related genes in each group, during induction (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA).


D . The number of CVH - positive cells was determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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D. The number of CVH-positive cells was determined by flow cytometry (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).


C . Effects of m6A on small peptide expression , assessed by western blot ( Up ) , Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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C. Effects of m6A on small peptide expression, assessed by western blot (Up), Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


D and E . Western blot ( Up ) was used to detect gga - mir - 12211 and m6A affect the ability of LncBMP4 to translate EPC5 . Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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D and E. Western blot(Up) was used to detect gga-mir-12211 and m6A affect the ability of LncBMP4 to translate EPC5. Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001,**** p < 0.0001, one-way ANOVA.).


( A ) Cardiac GATA4 protein abundance analyzed by immunoblotting . GAPDH was the loading control . + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus . ( B ) Densitometric quantification of the immunoblot shown under ( A ) ; * * * p = 0 . 0003 for P1 vs . P7 and P1 vs . P21 ; p < 0 . 0001 for P1 vs . P60 . The number within bars indicates the number of mice analyzed in that particular group .

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(A) Cardiac GATA4 protein abundance analyzed by immunoblotting. GAPDH was the loading control. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus.(B) Densitometric quantification of the immunoblot shown under (A); ***p=0.0003 for P1 vs. P7 and P1 vs. P21; p<0.0001 for P1 vs. P60. The number within bars indicates the number of mice analyzed in that particular group.


( C ) Immunoblot of cardiac GATA4 abundance in control ( Con ) and cardiomyocyte specific Gata4 knock - out mice ( CM - G4 - KO ) at postnatal day ( P ) 1 . ( D ) Quantification of the immunoblot shown in ( C ) ; * p = 0 . 018 .

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(C) Immunoblot of cardiac GATA4 abundance in control (Con) and cardiomyocyte specific Gata4 knock-out mice (CM-G4-KO) at postnatal day (P) 1.(D) Quantification of the immunoblot shown in (C); *p=0.018.


( E ) Cardiac immunofluorescence staining and its quantification from Con and CM - G4 - KO mice at P1 . Scale bar : 25 µm ; * * * * p < 0 . 0001 .

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(E) Cardiac immunofluorescence staining and its quantification from Con and CM-G4-KO mice at P1. Scale bar: 25 µm; ****p<0.0001.


( F ) Transversal sections of mouse hearts 7 days after cryoinjury as indicated , stained with Sirius - Red . Scale bar : 500 µm . The quantification of the left ventricular ( LV ) scar area ( red , arrows ) 7 days after injury is shown on the right ; * * p = 0 . 0027 . ( G ) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury ; * p = 0 . 0261 . ( H ) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury ; * * * * p < 0 . 0001 .

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(F) Transversal sections of mouse hearts 7 days after cryoinjury as indicated, stained with Sirius-Red . Scale bar: 500 µm. The quantification of the left ventricular (LV) scar area (red, arrows) 7 days after injury is shown on the right; **p=0.0027.(G) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury; *p=0.0261.(H) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury; ****p<0.0001.


( I ) Echocardiography analysis of left ventricular systolic function ( measured as fractional area change ) in the indicated mice 7 days after cryoinjury ; * p = 0 . 033 between Con mice after sham or cryoinjury and p = 0 . 04 between Con and CM - G4 - KO mice after cryoinjury .

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(I) Echocardiography analysis of left ventricular systolic function (measured as fractional area change) in the indicated mice 7 days after cryoinjury; *p=0.033 between Con mice after sham or cryoinjury and p=0.04 between Con and CM-G4-KO mice after cryoinjury.


( A ) Representative pictures of TUNEL , WGA ( wheat germ agglutinin to mark cardiomyocyte membranes ) and DAPI immunofluorescence staining of mouse hearts as indicated . The white dashed line encircles the infarcted area ( arrows ) . Scale bar : 200µm . The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right ( A ´ ) . The number within bars indicates the number of mice analyzed in that particular group .

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(A) Representative pictures of TUNEL, WGA (wheat germ agglutinin to mark cardiomyocyte membranes) and DAPI immunofluorescence staining of mouse hearts as indicated. The white dashed line encircles the infarcted area (arrows). Scale bar: 200µm. The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right (A´). The number within bars indicates the number of mice analyzed in that particular group.


( B ) Representative merged and single channel pictures of cardiac CD31 , DAPI and WGA immunostaining in mouse hearts as indicated . The quantification of the myocardial capillary density as capillaries / cardiomyocyte ( CM ) ratio , 7 days ( d ) after cryoinjury or sham surgery is shown as bar graph ( B ´ ) . Scale bar : 20µm . * * p = 0 . 0039 .

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(B) Representative merged and single channel pictures of cardiac CD31, DAPI and WGA immunostaining in mouse hearts as indicated. The quantification of the myocardial capillary density as capillaries/cardiomyocyte (CM) ratio, 7 days (d) after cryoinjury or sham surgery is shown as bar graph (B´). Scale bar: 20µm. **p=0.0039.


( C ) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho - histone H3 ( pH3 ) , troponin T and DAPI in mice as indicated . Enlarged pH3 positive cardiomyocyte nuclei are shown separately . The arrows indicate pH3 positive cardiomyocyte nuclei . Scale bar : 25µm . The quantification of pH3 positive ( + ) cardiomyocytes per microscopic field is shown as bar graph ( C ´ ) ; * p = 0 . 046 .

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(C) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho-histone H3 (pH3), troponin T and DAPI in mice as indicated. Enlarged pH3 positive cardiomyocyte nuclei are shown separately. The arrows indicate pH3 positive cardiomyocyte nuclei. Scale bar: 25µm. The quantification of pH3 positive (+) cardiomyocytes per microscopic field is shown as bar graph (C´); *p=0.046.


( D ) Representative myocardial immunostaining for aurora B , DAPI and troponin T ( TropT ) . The arrow points towards a cardiomyocyte cell division , in which aurora B localizes at the midbody in cytokinesis . Scale bar : 25µm . The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph ( D ´ ) ; * p = 0 . 0104 .

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(D) Representative myocardial immunostaining for aurora B, DAPI and troponin T (TropT). The arrow points towards a cardiomyocyte cell division, in which aurora B localizes at the midbody in cytokinesis. Scale bar: 25µm. The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph (D´); *p=0.0104.


( E ) Determination of cardiac gene - expression by qPCR 7 days after sham or cryoinjury for the indicated genes ; * p = 0 . 039 for Il13 and p = 0 . 049 for Vegfa ; * * p = 0 . 0043 for Ccne1 , p = 0 . 0027 for Cdk4 , p = 0 . 0096 for Cenpa , p = 0 . 0015 for Zfp191 ; * * * p = 0 . 0009 for Ccna2 , p = 0 . 0002 for Cenpa .

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(E) Determination of cardiac gene-expression by qPCR 7 days after sham or cryoinjury for the indicated genes; *p=0.039 for Il13 and p=0.049 for Vegfa; **p=0.0043 for Ccne1, p=0.0027 for Cdk4, p=0.0096 for Cenpa, p=0.0015 for Zfp191; ***p=0.0009 for Ccna2, p=0.0002 for Cenpa.


( A ) Representative immunoblots for GATA4 and GAPDH ( as loading control ) of fetal cardiomyocytes treated with adenoviruses ( Ad ) expressing control shRNA ( shCon ) or shRNA against Gata4 ( shGATA4 ) for 48 hours .

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(A) Representative immunoblots for GATA4 and GAPDH (as loading control) of fetal cardiomyocytes treated with adenoviruses (Ad) expressing control shRNA (shCon) or shRNA against Gata4 (shGATA4) for 48 hours.


( B ) Number of cardiomyocytes ( CM ) in a defined low magnification ( 50x ) microscopic field before the adenoviral infection and 48h later . The number in the bars indicates the number of cell culture plates analyzed . * * * * p < 0 . 0001 .

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(B) Number of cardiomyocytes (CM) in a defined low magnification (50x) microscopic field before the adenoviral infection and 48h later. The number in the bars indicates the number of cell culture plates analyzed. ****p<0.0001.


( C ) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU , troponin I ( Trop I ) and DAPI . Scale bar : 50µm ; p = 0 . 0169 .

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(C) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU, troponin I (Trop I) and DAPI. Scale bar: 50µm; p=0.0169.


( D ) Representative pictures and quantification of cardiomyocytes positive for phospho - histone H3 ( pH3 ) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3 , troponin I and DAPI . Scale bar : 50µm ; * p = 0 . 0145 .

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(D) Representative pictures and quantification of cardiomyocytes positive for phospho-histone H3 (pH3) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3, troponin I and DAPI. Scale bar: 50µm; *p=0.0145.


( E ) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B ( Auro B ) , troponin I and DAPI . Scale bar : 50µm .

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(E) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B (Auro B), troponin I and DAPI. Scale bar: 50µm.


( F ) Representative pictures , single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3 , troponin I and DAPI . Scale bar : 100µm .

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(F) Representative pictures, single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3, troponin I and DAPI. Scale bar: 100µm.


( G ) Representative pictures of cardiac explants of the indicated mice embedded in Matrigel shortly after cryoinjury and immunostained for troponin I 7 days later . Quantification of outgrowing cardiomyocytes is shown on the right . The number within bars indicates the number of mice analyzed in that particular group . Scale bar : 50µm . * * p = 0 . 0037 .

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(G) Representative pictures of cardiac explants of the indicated mice embedded in Matrigel shortly after cryoinjury and immunostained for troponin I 7 days later. Quantification of outgrowing cardiomyocytes is shown on the right. The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 50µm. **p=0.0037.


( H ) Representative pictures of cardiac explants like in ( G ) , stained for pH3 in addition . The quantification is shown on the right . Scale bar : 50µm . * p = 0 . 0215 .

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(H) Representative pictures of cardiac explants like in (G), stained for pH3 in addition. The quantification is shown on the right. Scale bar: 50µm. *p=0.0215.


( A ) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control ( Ad . Con ) or GATA4 expressing adenovirus ( Ad . GATA4 ) .

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(A) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control (Ad.Con) or GATA4 expressing adenovirus (Ad.GATA4).


( B ) Quantification of BrdU incorporation , pH3 labelling and cytokinesis in neonatal cardiomyocytes treated as shown . * p = 0 . 0272 for BrdU incorporation , p = 0 . 0329 for pH3 labelling , p = 0 . 0217 for cytokinesis .

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(B) Quantification of BrdU incorporation, pH3 labelling and cytokinesis in neonatal cardiomyocytes treated as shown. *p=0.0272 for BrdU incorporation, p=0.0329 for pH3 labelling, p=0.0217 for cytokinesis.


( C ) Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental time scale above . ( D ) A quantification of the immunoblot from ( C ) is shown ; * * p = 0 . 0062 .

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(C) Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental time scale above.(D) A quantification of the immunoblot from (C) is shown; **p=0.0062.


( E ) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad . Con or Ad . GATA4 . The quantification of cardiomyocytes ( CM ) staining positive for GATA4 vs . total cardiomyocytes is shown as bar graph ( E ´ ) . The number within bars indicates the number of mice analyzed in that particular group . Scale bar : 20µm . * * * * p < 0 . 0001 .

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(E) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E´). The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 20µm. ****p<0.0001.


( F ) Representative Sirius - Red stained heart sections of mice 7 days after cryoinjury ( performed at P7 ) and treated with adenoviruses as indicated . The scar is displayed in red ( arrows ) . Scale bar : 500 µm . The quantification of left ventricular ( LV ) scar area of mice 7 days after cryoinjury is shown as bar graph ( F ´ ) . * p = 0 . 0135 .

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(F) Representative Sirius-Red stained heart sections of mice 7 days after cryoinjury (performed at P7) and treated with adenoviruses as indicated. The scar is displayed in red (arrows). Scale bar: 500 µm. The quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury is shown as bar graph (F´). *p=0.0135.


( G ) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury . Scale bar : 10 µm . Quantification of Ki67 positive cardiomyocytes in these mice ( G ´ ) . * * p = 0 . 0023 .

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(G) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury. Scale bar: 10 µm. Quantification of Ki67 positive cardiomyocytes in these mice (G´). **p=0.0023.


( H ) Quantification of pH3 positive cardiomyocytes and cardiomyocyte ( CM ) cytokinesis in mice 7 days after cryoinjury ; * p = 0 . 0264 , * * * p = 0 . 0006 .

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(H) Quantification of pH3 positive cardiomyocytes and cardiomyocyte (CM) cytokinesis in mice 7 days after cryoinjury; *p=0.0264, ***p=0.0006.


( I ) Quantification of the capillary / cardiomyocyte ratio in mice 7 days after cryoinjury . * * * p = 0 .

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(I) Quantification of the capillary/cardiomyocyte ratio in mice 7 days after cryoinjury. ***p=0.0002


( B ) Quantification of left ventricular ( LV ) scar area of mice 7 days after cryoinjury and treated as indicated . The number within bars indicates the number of mice analyzed in that particular group . * * p = 0 . 0017 between Con and CM - G4 - KO after PBS treatment and p = 0 . 0078 between CM - G4 - KO mice with PBS or IL - 13 treatment .

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(B) Quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury and treated as indicated. The number within bars indicates the number of mice analyzed in that particular group. **p=0.0017 between Con and CM-G4-KO after PBS treatment and p=0.0078 between CM-G4-KO mice with PBS or IL-13 treatment.


( C ) Echocardiography analysis of left ventricular systolic function in the indicated mice 7 days after cryoinjury ; * p = 0 .

(C) Echocardiography analysis of left ventricular systolic function in the indicated mice 7 days after cryoinjury; *p=0.0218


( D ) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in ( A ) ; * p = 0 . 0106 and * * p = 0 . 0052 between Con and CM - G4 - KO after PBS treatment and * p = 0 . 0439 or * * p = 0 . 0023 between CM - G4 - KO mice with PBS or IL - 13 treatment .

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(D) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in (A); *p=0.0106 and **p=0.0052 between Con and CM-G4-KO after PBS treatment and *p=0.0439 or **p=0.0023 between CM-G4-KO mice with PBS or IL-13 treatment.


( E ) Quantification of capillary density as capillary / cardiomyocyte ( CM ) ratio in mice treated as described in ( A ) . * p = 0 . 0365 , * * p = 0 . 0031 .

(E) Quantification of capillary density as capillary/cardiomyocyte (CM) ratio in mice treated as described in (A). *p=0.0365, **p=0.0031.


( F ) Quantification of α - SMA positive small conductance vessel ( ~ 20 - 50µM in diameter ) in myocardial sections of the mice treated as indicated .

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(F) Quantification of α-SMA positive small conductance vessel (~20-50µM in diameter) in myocardial sections of the mice treated as indicated.


( G ) Representative immunoblots for STAT6 and GAPDH from neonatal mouse hearts ( KO = CM - G4 - KO ) treated as indicated . The quantification of the blots is shown on the right . * p = 0 . 0114 .

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(G) Representative immunoblots for STAT6 and GAPDH from neonatal mouse hearts (KO = CM-G4-KO) treated as indicated. The quantification of the blots is shown on the right. *p=0.0114.


HCT - 116 ( A ) and HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 ( grey bars ) , 48 ( pink bars ) , or 72 ( yellow bars ) hours and cell proliferation was assessed . HCT -

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HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 (grey bars), 48 (pink bars), or 72 (yellow bars) hours and cell proliferation was assessed. HCT-116


( C , E ) and HT - 29 ( D , F ) cells were treated with the indicated concentrations of NaB for 24 h . C . D . The percentage of apoptotic cells in HCT - 116 ( C ) and HT - 29 ( D ) cells was quantified in three independent experiments using an annexinV / PI assay ( annexinV + / PI - ) by flow cytometry and expressed as mean ± SD . One - way ANOVA was used to compare between the control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 compared to control . E . F . Representative Western blots of full - length and cleaved PARP products in HCT - 116 ( E ) and HT - 29 ( F ) cells .

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

(C, E) and HT-29 (D, F) cells were treated with the indicated concentrations of NaB for 24 h. C.D. The percentage of apoptotic cells in HCT-116 (C) and HT-29 (D) cells was quantified in three independent experiments using an annexinV/PI assay (annexinV+/PI-) by flow cytometry and expressed as mean ± SD. One-way ANOVA was used to compare between the control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. E.F. Representative Western blots of full-length and cleaved PARP products in HCT-116 (E) and HT-29 (F) cells.


HCT - 116 ( A , C ) and HT - 29 ( B , D ) cells were treated with 2mM sodium butyrate ( NaB ) for 24 h and then stained with acridine orange ( AO ; A - B ) or monodansylcadaverine ( MDC ; C - D ) .

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

HCT-116 (A, C) and HT-29 (B, D) cells were treated with 2mM sodium butyrate (NaB) for 24 h and then stained with acridine orange (AO; A-B) or monodansylcadaverine (MDC; C-D).


Flow cytometry measured the percentage of AO + cells and the results of three independent AO fluorescence experiments for HTC - 116 ( E ) and HT - 29 ( F ) cells were expressed as mean ± SD . One - way ANOVA was used to compare control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 when compared to control .

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

Flow cytometry measured the percentage of AO+ cells and the results of three independent AO fluorescence experiments for HTC-116 (E) and HT-29 (F) cells were expressed as mean ± SD. One-way ANOVA was used to compare control cells and NaB treatments. *p<0.05, ** p<0.01 when compared to control.


HCT - 116 ( A ) or HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 h . Representative blots showed expression of Beclin1 , ATG3 , and LC3 as well as GAPDH , the loading control .

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

HCT-116 (A) or HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative blots showed expression of Beclin1, ATG3, and LC3 as well as GAPDH, the loading control.


C . Electron microscopy images of control and NaB ( 2mM ) treated HCT - 116 cells . ER , endoplasmic reticulum ; Mt , mitochondrion ; AL .

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C. Electron microscopy images of control and NaB (2mM) treated HCT-116 cells. ER, endoplasmic reticulum; Mt, mitochondrion; AL.


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of LC3 - I and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( A ) HCT - 116 cells treated with 3 - MA and NaB ;

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

HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (A) HCT-116 cells treated with 3-MA and NaB;


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of LC3 - I and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( B ) HT - 29 cells treated with 3 - MA and

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

HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (B) HT-29 cells treated with 3-MA and NaB


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of null and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( C ) HCT - 116 cells treated with CQ and NaB ;

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

HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of null and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (C) HCT-116 cells treated with CQ and NaB;


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of LC3 - I and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( D ) HT - 20 cells treated with CQ and NaB .

HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group.(D) HT-20 cells treated with CQ and NaB.


HCT - 116 ( A ) and HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 h . Representative Western blots show the expression of IRE - 1a , BIP , PDI , and CHOP . GAPDH was used as the loading control .

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

HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative Western blots show the expression of IRE-1a, BIP, PDI, and CHOP. GAPDH was used as the loading control.


HCT - 116 or HT - 29 cells were treated with 10 μg / mL cycloheximide for 30 min followed by sodium butyrate ( NaB ) for 24 h . Representative Western blots showed the expression of IRE - 1a , BIP , PDI , and GADPH ( loading control ) in HCT - 116 ( A ) or HT - 29 ( C ) cells . Protein expression was quantified by densitometry and normalized to GAPDH ( ratio of protein : GAPDH ) . The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT - 116 ( B ) and HT - 29 ( D ) cells . are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the respective control group .

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

HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showed the expression of IRE-1a, BIP, PDI, and GADPH (loading control) in HCT-116 (A) or HT-29 (C) cells. Protein expression was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.


HCT - 116 ( A , B ) or HT - 29 ( C , D ) cells were treated with 0 . 1μM mithramycin for 30 min followed by sodium butyrate ( NaB ) for 24 h . Representative Western blots showing the expression of IRE - 1a , BIP , and PDI in HCT - 116 ( A ) or HT - 29 ( C ) cells are shown . GAPDH was used as loading control . Protein expression ( IRE - 1a , BIP , and PDI ) was quantified by densitometry and normalized to GAPDH ( ratio of protein : GAPDH ) . The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT - 116 ( B ) and HT - 29 ( D ) cells . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the respective control group .

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

HCT-116 (A, B) or HT-29 (C,D) cells were treated with 0.1μM mithramycin for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showing the expression of IRE-1a, BIP, and PDI in HCT-116 (A) or HT-29 (C) cells are shown. GAPDH was used as loading control. Protein expression (IRE-1a, BIP, and PDI) was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.


HCT - 116 or HT - 29 cells were treated with 10 μg / mL cycloheximide or 0 . 1μM mithramycin for 30 min and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of the expression of LC3 - II are shown . The level of LC3 - II expression was quantified by densitometry and normalized to GAPDH ( ratio of LC3 - II : GAPDH ) . The fold change from control cells is shown . Means and standard deviation ( SD ) of three independent experiments are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the control group . ( A - B ) HCT - 116 cells treated with cyclohexamide ; ( E - F ) HT - 29 cells treated with cyclohexamide ;

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

HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (A-B) HCT-116 cells treated with cyclohexamide; (E-F) HT-29 cells treated with cyclohexamide;


HCT - 116 or HT - 29 cells were treated with 10 μg / mL cycloheximide or 0 . 1μM mithramycin for 30 min and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of the expression of LC3 - II are shown . The level of LC3 - II expression was quantified by densitometry and normalized to GAPDH ( ratio of LC3 - II : GAPDH ) . The fold change from control cells is shown . Means and standard deviation ( SD ) of three independent experiments are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the control group . ( C - D ) HCT - 116 cells treated with mithramycin ; ( G - H ) HT - 29 cells treated with mithramycin . cancel

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

HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (C-D) HCT-116 cells treated with mithramycin;  (G-H) HT-29 cells treated with mithramycin. cancel


HCT - 116 cells were transfected with BIP or CHOP specific siRNAs for 48 h and then treated with or without 2mM sodium butyrate ( NaB ) for 24 h . Negative control ( NC ) scramble siRNA was used the negative control for the transfection . Representative Western blots are shown for BIP siRNA ( A ) and CHOP siRNA ( C ) . The expression level of each protein was determined by densitometry and normalized to GAPDH ( ratio of protein : GAPDH ) . ( B ) Normalized expression levels of BIP and LC3 - II in HCT - 116 cells treated with BIP specific siRNA . ( C ) Normalized expression levels of CHOP and LC3 - II in HCT - 116 cells treated with CHOP specific siRNA . Means and standard deviation ( SD ) of three independent experiments are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the respective control group .

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

HCT-116 cells were transfected with BIP or CHOP specific siRNAs for 48 h and then treated with or without 2mM sodium butyrate (NaB) for 24 h. Negative control (NC) scramble siRNA was used the negative control for the transfection. Representative Western blots are shown for BIP siRNA (A) and CHOP siRNA (C). The expression level of each protein was determined by densitometry and normalized to GAPDH (ratio of protein:GAPDH). (B) Normalized expression levels of BIP and LC3-II in HCT-116 cells treated with BIP specific siRNA. (C) Normalized expression levels of CHOP and LC3-II in HCT-116 cells treated with CHOP specific siRNA. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.


HCT - 116 ( A ) and HT - 29 ( B ) cells were exposed to 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min and then treated with 2mM sodium butyrate ( NaB ) for 24 h . The percentage of apoptotic cells ( annexinV + / PI - ) was quantified by flow cytometry using an annexinV / PI assay and expressed as mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis to compare control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 compared to control . ( C . D . ) Cells were treated as described above and the proteins were harvested for Western blot analysis . Expression of full - length and cleaved PARP in HCT - 116 ( C ) or HT - 29 ( D ) cells was compared to GAPDH , the loading control . The ratio of cleaved PARP to total PARP was calculated and the fold change in normalized PARP expression relative to cells treated with 2mM NaB is shown as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the 2mM NaB group .

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

HCT-116 (A) and HT-29 (B) cells were exposed to 5mM 3-MA or 5μM chloroquine (CQ) for 30 min and then treated with 2mM sodium butyrate (NaB) for 24 h. The percentage of apoptotic cells (annexinV+/PI-) was quantified by flow cytometry using an annexinV/PI assay and expressed as mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis to compare control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. (C.D.) Cells were treated as described above and the proteins were harvested for Western blot analysis. Expression of full-length and cleaved PARP in HCT-116 (C) or HT-29 (D) cells was compared to GAPDH, the loading control. The ratio of cleaved PARP to total PARP was calculated and the fold change in normalized PARP expression relative to cells treated with 2mM NaB is shown as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. *P<0.05, ** p<0.01, compared to the 2mM NaB group.


( A ) Heatmap of differentially enriched metabolites in heart , liver , skeletal muscle and serum of young ( Y ) and aged ( A ) killifish ( n = 4 ) .

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(A) Heatmap of differentially enriched metabolites in heart, liver, skeletal muscle and serum of young (Y) and aged (A) killifish (n = 4).


( B - C ) Ageing leads to significant cardiac - specific accumulation of sphinganine .

(B-C) Ageing leads to significant cardiac-specific accumulation of sphinganine.


( D ) Targeted lipidomics confirm age - dependent accumulation of sphinganine in killifish ( n = 4 ; per condition ) , zebrafish ( n = 4 , per condition ) , mouse ( n = 4 for young and n = 6 for aged samples ) and human hearts ( n = 1 for young and 2 for aged ) . In the case of human heart tissue , statistical significance was computed from technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Targeted lipidomics confirm age-dependent accumulation of sphinganine in killifish (n = 4; per condition), zebrafish (n = 4, per condition), mouse (n = 4 for young and n=6 for aged samples) and human hearts (n = 1 for young and 2 for aged). In the case of human heart tissue, statistical significance was computed from technical replicates.


( E ) Heatmap of differentially expressed transcripts ( Log2 fold change + / - 1 , adjusted p - value cut off < 0 . 05 ) between young and aged killifish hearts ( n = 3 ) . Significantly enriched relevant GO categories depicted as bar graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap of differentially expressed transcripts (Log2 fold change +/- 1, adjusted p-value cut off <0.05) between young and aged killifish hearts (n = 3). Significantly enriched relevant GO categories depicted as bar graph.


( F ) Transcriptional drift variance of genes involved in genome integrity , epigenetic regulation and sphingolipid metabolism .

(F) Transcriptional drift variance of genes involved in genome integrity, epigenetic regulation and sphingolipid metabolism.


( G ) γH2A . X staining of young , mid - aged and aged killifish hearts show signs of DNA damage , increasing with age ( n = 4 , # of sections per animal = 10 alternate sections , 10 µm thickness ) .

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(G) γH2A.X staining of young, mid-aged and aged killifish hearts show signs of DNA damage, increasing with age (n = 4, # of sections per animal = 10 alternate sections, 10 µm thickness).


( H ) Quantification of γH2A . X + cardiomyocyte nuclei .

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(H) Quantification of γH2A.X+ cardiomyocyte nuclei.


( I ) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays . Representative images of single nuclei from young and old hearts along with comet length on the right ( n = 4 ) .

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(I) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays. Representative images of single nuclei from young and old hearts along with comet length on the right (n = 4).


( J ) γH2A . X staining of young and aged human ventricles show signs of DNA damage accumulating with age ( n = 2 , # of sections per sample = 5 alternate sections , 10 µm thickness ) .

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(J) γH2A.X staining of young and aged human ventricles show signs of DNA damage accumulating with age (n = 2, # of sections per sample = 5 alternate sections, 10 µm thickness).


( K ) Quantification of γH2A . X + cardiomyocyte nuclei is on the right . Scale bar = 50 µm .

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(K) Quantification of γH2A.X+ cardiomyocyte nuclei is on the right. Scale bar = 50 µm.


Sphinganine induces DNA damage in human cardiomyocytes depicted here ( A - C ) by γH2A . X + nuclei in cardiac - specific ACTN2 + cells ( n = 3 biological replicates ; # of cells quantified = 147 per condition )

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Sphinganine induces DNA damage in human cardiomyocytes depicted here (A-C) by γH2A.X+ nuclei in cardiac-specific ACTN2+ cells (n = 3 biological replicates; # of cells quantified = 147 per condition)


Sphinganine induces DNA damage in human cardiomyocytes depicted here ( D ) by neutral comet assay of DHS treated hCMs . ( E ) Quantification of comet length in the indicated conditions .

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Sphinganine induces DNA damage in human cardiomyocytes depicted here (D) by neutral comet assay of DHS treated hCMs. (E) Quantification of comet length in the indicated conditions.


Elevated DHS levels lead to ageing signatures in hCMs shown here by ( F - G ) SA - β - GAL activity , Respective graphs depict the quantification of each of the ageing signatures are on the right .

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Elevated DHS levels lead to ageing signatures in hCMs shown here by (F-G) SA-β-GAL activity, Respective graphs depict the quantification of each of the ageing signatures are on the right.


Elevated DHS levels lead to ageing signatures in hCMs shown here by ( H ) p21 activation ( # cells quantified per replicate = 150 - 200 ) , ( I ) HP1 - α + nuclear foci ( # of cells quantified per replicate = 120 - 200 ) and ( J ) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining ( # of cells quantified per replicate = 100 - 150 per condition ) . Respective graphs depict the quantification of each of the ageing signatures are on the right .

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Elevated DHS levels lead to ageing signatures in hCMs shown here by (H) p21 activation (# cells quantified per replicate = 150-200), (I) HP1-α+ nuclear foci (# of cells quantified per replicate = 120-200) and (J) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining (# of cells quantified per replicate = 100-150 per condition). Respective graphs depict the quantification of each of the ageing signatures are on the right.


High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs ( L ) and ( M ) in aged human hearts . ( N ) Violin plot depicting the distributions of the grey - scale nuclear intensity of the indicated markers . ( When not specified , the experiments were conducted in at least 3 biological replicates ) . Scale bar = 10 µm .

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High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs (L) and (M) in aged human hearts. (N) Violin plot depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (When not specified, the experiments were conducted in at least 3 biological replicates). Scale bar = 10 µm.


( A ) Pharmacological inhibition of HDAC1 using romidepsin , MS - 275 and pyroxamide in human cardiomyocytes cause extensive DNA damage , shown here by γH2A . X staining .

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(A) Pharmacological inhibition of HDAC1 using romidepsin, MS-275 and pyroxamide in human cardiomyocytes cause extensive DNA damage, shown here by γH2A.X staining.


( B ) Bar - graphs depict quantification of γH2A . X + hCM nuclei in the indicated conditions .

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(B) Bar-graphs depict quantification of γH2A.X+ hCM nuclei in the indicated conditions.


( E ) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC - activity assay , shown here as bar graph .

(E) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC-activity assay, shown here as bar graph.


( F ) In vitro HDAC activity assay revealed inhibition of nuclear HDAC ' s and purified HDAC1 activity by DHS1P and S1P , shown here as bar graphs .

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(F) In vitro HDAC activity assay revealed inhibition of nuclear HDAC's and purified HDAC1 activity by DHS1P and S1P, shown here as bar graphs.


Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs


Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications .

Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications.


( I ) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co - incubation with RNA Pol II inhibitor , triptolide and ( J ) the respective quantifications of γH2A . X + CM nuclei represented as bar graph . Scale bars for A , I = 10 µm and G = 50 µm . ( When not specified , the experiments were conducted in at least 3 biological replicates ) .

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(I) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co-incubation with RNA Pol II inhibitor, triptolide and (J) the respective quantifications of γH2A.X+ CM nuclei represented as bar graph. Scale bars for A,I=10 µm and G = 50 µm. (When not specified, the experiments were conducted in at least 3 biological replicates).


( A - B ) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS - induced DNA damage as shown here by staining for γH2A . X ( green ) in hCMs .

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(A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) in hCMs.


( C ) Bar graphs representing the percentage of γH2A . X + hCMs in the indicated conditions ( # cells quantified per replicate ~ 200 ) .

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(C) Bar graphs representing the percentage of γH2A.X+ hCMs in the indicated conditions (# cells quantified per replicate ~200).


( D - E ) Treatment of hCMs with ABC294640 , a specific SPHK2 inhibitor prevents DHS - induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining ( ~ 100 cells quantified / condition ) .

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(D-E) Treatment of hCMs with ABC294640, a specific SPHK2 inhibitor prevents DHS-induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining (~100 cells quantified/ condition).


( F ) Violin plots depicting the distributions of the grey - scale nuclear intensity of the indicated markers .

(F) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers.


( G ) HAT inhibition by curcumin prevents the aberrant increase in H3K56ac and H3K27ac levels .

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(G) HAT inhibition by curcumin prevents the aberrant increase in H3K56ac and H3K27ac levels.


( H ) Violin plots depicting the distributions of the grey - scale nuclear intensity of the indicated markers .

(H) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers.


( I ) HAT inhibition prevents DHS - induced DNA damage as shown here by staining for γH2A . X ( green ) on human cardiomyocytes .

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(I) HAT inhibition prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) on human cardiomyocytes.


( J ) Quantification of γH2A . X + hCMs nuclei represented as percentage bar graph .

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(J) Quantification of γH2A.X+ hCMs nuclei represented as percentage bar graph.


( K ) Curcumin - mediated HAT inhibition lead to inactivation of p21 in hCMs .

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(K) Curcumin-mediated HAT inhibition lead to inactivation of p21 in hCMs.


( L ) Violin plots depicting the distributions of the grey - scale nuclear intensity of p21 stained nuclei . ( When not specified , the experiments were conducted in at least 3 biological replicates ) .

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(L) Violin plots depicting the distributions of the grey-scale nuclear intensity of p21 stained nuclei. (When not specified, the experiments were conducted in at least 3 biological replicates).


( D ) Heatmap depicting the expression profile of the markers of DNA damage response in the indicated conditions .

(D) Heatmap depicting the expression profile of the markers of DNA damage response in the indicated conditions.


( E ) Co - treatment of curcumin activates DNA repair pathways as depicted here by heatmaps .

(E) Co-treatment of curcumin activates DNA repair pathways as depicted here by heatmaps.


Representative micrographs depicting ( B ) γH2A . X staining on the ventricle sections for the indicated cohorts .

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Representative micrographs depicting (B) γH2A.X staining on the ventricle sections for the indicated cohorts.


Representative micrographs depicting ( C ) H3K27ac staining on the ventricle sections for the indicated cohorts .

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Representative micrographs depicting (C) H3K27ac staining on the ventricle sections for the indicated cohorts.


( D ) Quantification of γH2A . X + CMs nuclei represented as percentage bar graph .

(D) Quantification of γH2A.X+ CMs nuclei represented as percentage bar graph.


( E ) Box - plot depicting the distributions of the corrected total nuclear fluorescence of the indicated cohorts .

(E) Box-plot depicting the distributions of the corrected total nuclear fluorescence of the indicated cohorts.


( F ) Representative micrographs depicting ventricular view from the echocardiography of the indicated cohorts .

(F) Representative micrographs depicting ventricular view from the echocardiography of the indicated cohorts.


( G - H ) Quantification of fractional shortening and ejection fraction of the indicated groups represented as bean plot and percentage bar graph respectively ( n = 6 animals per condition ) . Scale bars for B , C = 50 µm .

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(G-H) Quantification of fractional shortening and ejection fraction of the indicated groups represented as bean plot and percentage bar graph respectively (n=6 animals per condition). Scale bars for B,C = 50 µm.


A - C qRT - PCR detection of mRNA levels of IL - 1β , TNFα and IL - 6 mRNA levels in CCDC50 - WT and CCDC50 - KO THP - 1 cells primed with LPS and then treated with ATP and Nigericin ( A ) , or infected with Listeria monocytogenes at a multiplicity of infection ( MOI ) of 10 and Salmonella typhimurium ( MOI of 10 ) for 6 h ( B ) or MSU ( 40 μg / ml ) and SiO2 ( 40 μg / ml ) for 6 h ( C ) ( n = 3 biological replicates ) . Data information : L . m , Listeria monocytogene ; S . t , Salmonella typhimurium ; MSU , Monosodium urate ; mRNA results are normalized to GAPDH and relative to untreated wild - type cells Data are representative of three biological replicates and shown as mean with SEM ( A - C ) ; * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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A-C qRT-PCR detection of mRNA levels of IL-1β, TNFα and IL-6 mRNA levels in CCDC50-WT and CCDC50-KO THP-1 cells primed with LPS and then treated with ATP and Nigericin (A), or infected with Listeria monocytogenes at a multiplicity of infection (MOI) of 10 and Salmonella typhimurium (MOI of 10) for 6 h (B) or MSU (40 μg/ml ) and SiO2 (40 μg/ml) for 6 h (C) (n = 3 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; mRNA results are normalized to GAPDH and relative to untreated wild-type cells Data are representative of three biological replicates and shown as mean with SEM (A-C); **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


D Enzyme - linked immunosorbent assays ( ELISA ) of IL - 1β production in supernatants of THP - 1 cells treated as in ( A - C ) ( n = 2 biological replicates ) . Data information : L . m , Listeria monocytogene ; S . t , Salmonella typhimurium ; MSU , Monosodium urate ; ; data are representative of two biological replicates ( D ) . * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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D Enzyme-linked immunosorbent assays (ELISA) of IL-1β production in supernatants of THP-1 cells treated as in (A-C) (n = 2 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; ; data are representative of two biological replicates (D). **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


E - G CCDC50 - WT and CCDC50 - KO THP - 1 cells were stimulated as in ( A - C ) . The cells were collected for immunoblot analysis of pro - caspase - 1 , and the supernatants were subjected to cleaved caspase - 1 and IL - 1β analysis . The expression levels of cleaved - caspase - 1 and cleaved - IL - 1β were quantitated using ImageJ software . Data information : L . m , Listeria monocytogene ; S . t , Salmonella typhimurium ; MSU , Monosodium urate ; Casp1 , caspase - 1 ; actin was used as a loading control .

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E-G CCDC50-WT and CCDC50-KO THP-1 cells were stimulated as in (A-C). The cells were collected for immunoblot analysis of pro-caspase-1, and the supernatants were subjected to cleaved caspase-1 and IL-1β analysis. The expression levels of cleaved-caspase-1 and cleaved-IL-1β were quantitated using ImageJ software. Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; Casp1, caspase-1; actin was used as a loading control.


C qRT - PCR detection of Il6 , IL - 1β and Tnfα mRNA levels in Ccdc50 + / + and Ccdc50 - / - BMDMs primed with LPS for 3 h and then induced with ATP and Nigericin ( A ) , L . m and S . t ( B ) , or MSU and SiO2 ( C ) for indicated time points ( n = 3 biological replicates ) . D - F Ccdc50 + / + and Ccdc50 - / - BMDCs treated as BMDMs were collected and subjected to qRT - PCR analysis ( n = 3 biological replicates ) . Data information : mRNA data are normalized to GAPDH and relative to untreated wild - type cells ; Data are representative of three biological replicates and are shown as the mean with SEM * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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C qRT-PCR detection of Il6, IL-1β and Tnfα mRNA levels in Ccdc50+/+ and Ccdc50-/- BMDMs primed with LPS for 3 h and then induced with ATP and Nigericin (A), L.m and S.t (B), or MSU and SiO2 (C) for indicated time points (n = 3 biological replicates). D-F Ccdc50+/+ and Ccdc50-/- BMDCs treated as BMDMs were collected and subjected to qRT-PCR analysis (n = 3 biological replicates). Data information: mRNA data are normalized to GAPDH and relative to untreated wild-type cells; Data are representative of three biological replicates and are shown as the mean with SEM *P <0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


G ELISA analysis of the protein level of secreted IL - 1β in supernatants of Ccdc50 + / + and Ccdc50 - / - BMDMs ( top panel ) and BMDCs ( bottom panel ) treated as in ( A - C ) ( n = 3 biological replicates ) . Data information : Data are representative of three biological replicates and are shown as the mean with SEM * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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G ELISA analysis of the protein level of secreted IL-1β in supernatants of Ccdc50+/+ and Ccdc50-/- BMDMs (top panel) and BMDCs (bottom panel) treated as in (A-C) (n = 3 biological replicates). Data information: Data are representative of three biological replicates and are shown as the mean with SEM *P <0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


H , I Ccdc50 + / + and Ccdc50 - / - BMDMs ( H ) and BMDCs ( I ) were treated as in ( A - C ) . The cells were lysed for western blotting analysis of pro - caspase - 1 and the supernatant was collected for immunoblots of cleaved - caspase - 1 . The expression levels of cleaved - caspase - 1 were quantitated using ImageJ software .

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H,I Ccdc50+/+ and Ccdc50-/- BMDMs (H) and BMDCs (I) were treated as in (A-C). The cells were lysed for western blotting analysis of pro-caspase-1 and the supernatant was collected for immunoblots of cleaved-caspase-1. The expression levels of cleaved-caspase-1 were quantitated using ImageJ software.


A , B Co - Immunoprecipitation ( Co - IP ) with anti - HA ( A ) or anti - Flag ( B ) was performed for the analysis of the association between HA - CCDC50 and Flag - AIM2 , - NLRP3 , and - ASC in cotransfected HEK293 cells . Data information : L . C , light - chain ; actin was used as a loading control . All the experiments were repeated at least once .

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A,B Co-Immunoprecipitation (Co-IP) with anti-HA (A) or anti-Flag (B) was performed for the analysis of the association between HA-CCDC50 and Flag-AIM2, -NLRP3, and -ASC in cotransfected HEK293 cells. Data information: L.C, light-chain; actin was used as a loading control. All the experiments were repeated at least once.


C Immunoprecipitation ( IP ) analysis of the interaction of NLRP3 with endogenous CCDC50 and ASC in THP - 1 cells activated by LPS plus ATP . IgG was used as a negative control . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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C Immunoprecipitation (IP) analysis of the interaction of NLRP3 with endogenous CCDC50 and ASC in THP-1 cells activated by LPS plus ATP. IgG was used as a negative control. Data information: actin was used as a loading control. All the experiments were repeated at least once.


D Representative endogenous colocalization analysis of CCDC50 and NLRP3 in PMA - induced THP - 1 cells treated with LPS and ATP . Scale bar , 10 μm .

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D Representative endogenous colocalization analysis of CCDC50 and NLRP3 in PMA-induced THP-1 cells treated with LPS and ATP. Scale bar, 10 μm.


E Representative confocal microscopy image of HCT116 cells overexpressing GFP - CCDC50 and stimulated with LPS as well as ATP . Scale bar , 10 μm .

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E Representative confocal microscopy image of HCT116 cells overexpressing GFP-CCDC50 and stimulated with LPS as well as ATP. Scale bar, 10 μm.