EMBO/SourceData huggingface dataset:
Fig 4HCT-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 NaBHCT-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.
Fig 5  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.
Fig 6  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.
Fig 7  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.
Fig 8HCT-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
Fig 9  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.
Fig 10  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.
Figure 1(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.(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.(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).(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).(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.
Figure 2Sphinganine 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.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.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.
Figure 3(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.(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.Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMsAssessment 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).
Figure 4(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).(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.(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.(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.(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).
Figure 5(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.
Figure 6Representative 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.(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.(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.
Figure 2A-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.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.
Figure 3C 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.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.
Figure 4A,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.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.F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag- AIM2, -NLRP3, -ASC and HA-CCDC50 or empty control vector. The expression levels of Flag-tagged proteins and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.G Immunoblot analysis of HEK293 cells transfected with FLAG-NLRP3 and an increasing amount of HA-CCDC50 plasmids. Data information: actin was used as a loading control. All the experiments were repeated at least once.H Immunoblot analysis of lysates from CCDC50-KO and CCDC50-WT THP-1 cells induced with LPS plus ATP and then treated with cycloheximide (CHX) for indicated time points. The expression levels of NLRP3 and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.I HEK293 cells were co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h and then treated with MG132, NH4Cl, 3-MA, CQ or DMSO for another 6 h. Data information: actin was used as a loading control. All the experiments were repeated at least once.J Immunoblot analysis of NLRP3 in CCDC50-WT and CCDC50-KO THP-1 cells stimulated with LPS in combination with ATP. Data information: actin was used as a loading control. All the experiments were repeated at least once.K Western blotting analysis of ATG5 wild-type or knockout HEK293T cells co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h. Data information: actin was used as a loading control. All the experiments were repeated at least once.L Immunoblot analyses of NLRP3 in HEK293 cells in the presence of CCDC50 or its mutant CCDC50 with the deletion of MIUs. Data information: actin was used as a loading control. All the experiments were repeated at least once.
Figure 5A Denature-IP (with anti-Flag) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293 cells transfected with Flag-NLRP3, HA-Ub (ubiquitin) as well as HA- CCDC50 or empty vector for 24 h.B Denature-IP analysis (with anti-Flag) of K63-linked polyubiquitination of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h.C The colocalization analysis of K63-Ub (Red), Flag-NLRP3 (Purple) and GFP- CCDC50 in HeLa cells; scale bars, 5 μM.D,E Images of HeLa cells transfected with CCDC50 (red) (D) or K63-Ub (red) (E) and Flag-NLRP3 (Purple) and GFP-LC3B for 24 h and then induced with LPS plus ATP; scale bars, 5 μM.F The colocalization analysis of NLRP3 and LC3B in CCDC50-WT and CCDC50- KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.G The oligomerization analysis of NLRP3 in CCDC50 wild-type and knockout THP-1 cells stimulated with LPS as well as ATP. LPS-primed (1 μg/ml, 3 h) THP-1 cells were stimulated with ATP (5 mM) for another 30 min and then were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.H The oligomerization analysis of NLRP3 in HEK293 cells transfected with plasmids expressing Flag-NLRP3, HA-CCDC50 or empty vector for 24 h, and then treated with DMSO or CQ for 6 h. After treatment, the cells were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.I Co-IP (with anti-HA) and immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h, and treated with or without CQ for 6 h.J Confocal microscopy analysis of colocalization between NLRP3 and ASC in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.K Confocal microscopy analysis of ASC speck formation in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.
Figure 1(A) Expression patterns of Hoxa5 and Hoxc8 in MNs (demarcated by white dashed lines) along the rostrocaudal axis of the spinal cord (cervical C4 to thoracic T1) from mouse embryos from E9.5 to E12.5. Pink scale bars: 12.5 μm for E9.5; 12.5 μm for E10.5; 25 μm for E11.5; and 50 μm for E12.5.
Figure 3Expression patterns of Hoxa5 and Hoxc8 mRNAs, as revealed by in situ hybridization, aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs (demarcated with pink/white dashed lines) along the cervical spinal cord (cervical C5 to C7) in mouse embryos at E12.5. Pink scale bar, 50 μm. White and pink dash lines demarcate the postmitotic MN region.Dot-plots showing expression of genes (rows) that distinguish MN subtypes (columns). The height of each colored shape is proportional to the percentage of cells expressing the marker (≥1 UMI), and its width reflects the average transcript count within expressing cells.
Figure 6Immunostaining (C) of EBs 96 h after ESC differentiation, and quantification (D) of Hoxa5on in MNs (Hb9on).Hoxa5 expression upon miR-27 knockdown, with exposure to RAhi for 72 h, or with exposure to RAhi for 48 h, and then switching to RAlow plus RAR inhibitor (1 µM) for a further 24 h, or with exposure to RAmed for 72 h.
Figure 8(C) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6/7 segments of miR-23~27~24 double KO (DKO) mice. miR-23a~27a~24-2+/-;miR-23b~27b~24-1-/- mice were used as a control (ctrl). (E) Conversely, Hoxc8 is shifted into the MNs of cervical C5 segments of both miR-196a2/b DKO and miR-196a1/a2/b triple knockout (TKO) embryos. Age-matched wild type embryos were used as controls. Scale bar represents 50 μm. White dash lines demarcate the postmitotoic MN domain (D, F) Quantifications of Hoxa5on, Hoxc8on, and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos (mean ± SD, N=3 embryos, *p < 0.01, Student's t-tests ).