EMBO/SourceData huggingface dataset:
Figure 4(A) Suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons treated with the NMDA receptor antagonist D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence or absence of D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 24 min (5-29 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence of D-AP5 and Aβo (assigned a value of 1.0). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test (means ± SE; n = 4), and significant p-values (p < 0.05, p < 0.01) are indicated on the graph.(D) Non-synergistic suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons by p3-Alcβ and D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence (+) or absence (-) of p3-Alcβ9-19 (50 μM) and D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 23 min (5-28 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in experiment No.1 (assigned a value of 1.0). Statistical significance was determined with a one-way ANOVA with Tukey's multiple comparison test (means ± SEM; n = 12), and significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graph.
Figure 5(E-G) The SUVRs of groups administered p3-Alcβ9-19 versus the vehicle-treated group. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni correction for multiple comparisons (means ± SEM; n = 6), and significant p-values (p < 0.05, p < 0.01) are indicated on the graphs.(I, J) The SUVRs of [18F]BCPP-EF in the cortex (I) and hippocampus (J) are compared across the three groups. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni correction for multiple comparisons (means ± SEM; n = 6-10), and significant p-values (p < 0.05, p < 0.01, p < 0.001) are indicated on the graphs.(K) Improvement of the inverse SUVR correlation between [18F]BCPP-EF and [11C]DPA713 in AD mice following administration of p3-Alcβ9-19. AD mice with (closed circle) or without (open circle) subcutaneous injection of p3-Alcβ9-19 peptide (1 mg/kg body weight) are also analyzed with [11C]DPA713. A solid linear line shows a significant inverse correlation (Pearson's correlation, r = 0.594, p < 0.05) in AD mice, whereas a dotted linear line shows a positive correlation tendency in AD mice in response to p3-Alcβ9-19 administration.
Figure 1C Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. Data are representative of three biological replicates.D Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. Data are representative of three biological replicates.G Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. Data are representative of three biological replicates.H Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Data are representative of three biological replicates. Scale bar: 100 µm.
Figure 2A RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant (two-tailed unpaired Student's t-test).F IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. The red dashed contour represents the group of K48-linked ubiquitination we focused on studying here. Data are representative of three biological replicates.H IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.
Figure 3C IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. Data are representative of three biological replicates.G Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. Data are representative of three biological replicates.K Fluorescence microscopy of the VSV virus with a SARS-CoV-2-S gene and a GFP gene (VSV-Spike) in Usp50+/+ and Usp50-/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with the VSV-Spike virus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. The average intensity each unit area was shown as mean and s.d. of three-unit areas (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).
Figure 4D IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. The red dashed contour represents the group of K48-linked ubiquitination we focused on studying here. Data are representative of three biological replicates.E IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). Data are representative of three biological replicates.
Figure 5B IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. Data are representative of three biological replicates.C Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. Data are representative of three biological replicates.D IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. Data are representative of three biological replicates.G ACE2-KO Caco-2 cells transfected with ACE2 (WT or K788R) were treated with VitC (5 mM) for 12 hrs, and then were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).
Figure 6A IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.D Flag-USP50 proteins were obtained as (C). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na+ (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three biological replicates.
Figure 7D Western blot analysis of hACE2 levels in lung tissues of hACE2 mice (n = 6) administrated with VitC as (B). Data are representative of three biological replicates.G RT-qPCR analysis of the SARS-CoV-2-Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and the VSV-Spike viruses as (F). All graphs show the mean ± SEM for five individual mice (n = 5). ***p < 0.001 (two-tailed unpaired Student's t-test).
Figure 1(A-C) qPCR and western blot analysis indicated an upregulation of hepatic ZBTB22 in diabetic db/db mice, obese ob/ob mice and high-fat diet for 12 weeks mice (n=6 mice). Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.(D) qPCR and western blot analysis showed an increase of hepatic ZBTB22 in normal C57 mice after overnight fasting, and a recovery of ZBTB22 expression after refeeding (n=6 mice). Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.(E) qPCR and immunofluorescence analysis displayed an enhanced expression of ZBTB22 in MPHs after FSK treatment for 24h and a reduction of ZBTB22 in MPHs after insulin treatment for 15min (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.
Figure 2(A-B) qPCR analysis the change of genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in primary hepatocytes after ZBTB22 overexpression (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D-E) ZBTB22 overexpression increase lipid deposition in primary hepatocytes, as shown by the enhanced lipid TOX probes and TG content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(F-G) qPCR analysis the change of genes involved in gluconeogenesis, lipogenesis, and fatty acid oxidation in primary hepatocytes after ZBTB22 knockdown (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(I-J) ZBTB22 knockdown suppress lipid deposition in primary hepatocytes, as shown by the weakened lipid TOX probes and TG content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(K) qPCR analysis of mRNA levels of Pgc1α, PCK1 in primary hepatocytes infected with Ad-shCtrl or Ad-shZBTB22 with or without forskolin treatment (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.
Figure 3ZBTB22 deletion effectively decrease the fat mass of mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) ZBTB22 deletion enhanced the expression of genes involved in thermogenesis and fatty acid oxidation in brown adipose tissue from mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(E) ZBTB22 deletion increased the heat production, CO2 generation and O2 consumption in mice fed on HFD (n=3 mice).
Figure 4ZBTB22 deletion effectively decrease the fasting blood glucose of mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) ZBTB22 deletion significantly changed hepatic expression of genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(E) ZBTB22 deletion significantly improved the phosphorylation of AKT and GSK3β in the liver of mice fed on HFD.(I) Hepatic ZBTB22 overexpression induces glucose intolerance and insulin resistance in ZBTB22 deficiency mice fed on HFD (n=6 mice).(J) Hepatic ZBTB22 overexpression decreased the insulin-induced phosphorylation of AKT and GSK3β in the liver of ZBTB22 deficiency mice fed on HFD.(K) qPCR analysis of hepatic genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in ZBTB22 deficiency mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.
Figure 5(A-B) Hepatic ZBTB22 silencing decreases the expression of gene involved in gluconeogenesis, lipogenesis, and fatty acid oxidation in db/db mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) Hepatic ZBTB22 silencing improved the glucose tolerance, insulin sensitivity and pyruvate tolerance in db/db mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(J-K) Hepatic ZBTB22 silencing decreases serum inflammatory cytokines levels and hepatic inflammatory genes expression (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.
Figure 6Hepatic ZBTB22 overexpression increases the expression of genes involved in gluconeogenesis in normal C57 mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(C) Hepatic ZBTB22 overexpression significantly induced glucose intolerance and insulin resistance in normal C57 mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) Hepatic ZBTB22 overexpression significantly decreased insulin-induced phosphorylation of hepatic AKT and GSK3β in normal C57 mice.Hepatic ZBTB22 overexpression increase serum inflammatory cytokines levels Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.Hepatic ZBTB22 overexpression increase hepatic expression of inflammatory genes (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.
Figure 7Western Blot analysis showed that ZBTB22-mediated induced the up-regulated expression of PEPCK1 in Ad-ZBTB22 infected mice primary hepatocytes and livers (C)Chip and Chip-qPCR displayed increase occupancy of ZBTB22 on the promoter region of PCK1 (n=3 biological replicates).PCK1 silencing , as well as the stimulatory effects of ZBTB22 on SREBP1C expression (J) (n=4 biological replicates) and cellular glucose output (K) (n=4 biological replicates) Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.
Figure 8Hepatic ZBTB22 overexpression failed to change glucose tolerance, insulin sensitivity in PCK1-silencing C57 mice (n=6 mice). Data information: Data are means ± SEM. *p < 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(G) Hepatic ZBTB22 overexpression failed to change serum and hepatic TG and TC levels in PCK1-silencing C57 mice (n=6 mice). Data information: Data are means ± SEM. *p < 0.05, **p< 0.01, ***p< 0.001 by Student's t test.
Figure 1SMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment. B, Representative images from wild type prometaphase cells labelled on one or two sister chromatids as indicated. C, Representative images from wild type G2 cells labelled on one or two sister chromatids as indicated. D, Quantification of sister chromatid separation in one-sister labelled prometaphase cells for experimental conditions as indicated. Dots represent individual cells; red bars indicate the mean. Wild type (n = 83 cells), ΔSMC4 (n = 75 cells) and ΔNIPBL (n = 48 cells) were analysed. Significance was tested using a two-tailed Mann Whitney U test; P = 2.33 x 10-27. Two-sister labelled wild type prometaphase (n = 4), one-sister labelled wild type prometaphase (n = 6), ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3), wild type G2 (n = 4), ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates Two-sister labelled wild type prometaphase (n = 9), one sister-labelled wild type prometaphase (n = 14), ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6), wild type G2 (n = 8), ΔSMC4 G2 (n = 5), ΔNIPBL G2 (n = 8). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 μm, insets: 1 μSMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment. F, Representative images from ΔSMC4 prometaphase cells labelled on one sister chromatid. G, Representative images from ΔSMC4 G2 cells labelled on one sister chromatid. H, Representative images from ΔNIPBL prometaphase cells labelled on one sister chromatid. I, Representative images from ΔNIPBL G2 cells labelled on one sister chromatid. Two-sister labelled , ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3) , ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates: Two-sister labelled , ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6) , ΔSMC4 G2 (n = 5), ΔNIPBL G2 (n = 8).
Figure 2WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. One sister chromatid was labelled per chromosome as in Fig. 1 SCC1 (WAPL-dTAG cells) was visualised by immunofluorescence. A, Representative images of control WAPL-dTAG cells synchronised to G2 by RO-3306. B, Representative images of ΔWAPL cells synchronised to G2 by RO-3306. C, Representative images of ΔSororin cells synchronised to G2 by RO-3306. D, Quantification of sister chromatid separation as in Fig. 1D Control WAPL-dTAG G2 (n = 36 cells), ΔWAPL G2 (n = 53 cells) and ΔSororin G2 (n = 45 cells) cells were analysed. Dots represent individual cells; red bars indicate the mean. Significance was tested using a two-tailed Mann Whitney U test; P = 3.89 x 10-11 (Control G2, ΔWAPL G2), P = 5.90 x 10-7 (Control G2, ΔSororin G2), P = 2.75 x 10-3 (ΔWAPL G2, ΔSororin G2). Biological replicates: Control WAPL-dTAG G2 (n = 3), ΔWAPL G2 (n = 3), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3), wild type late prophase (n = 4). Technical replicates Control WAPL-dTAG G2 (n = 6), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 6), ΔWAPL ΔSororin G2 (n = 6), wild type late prophase (n = 7). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. To aid visualisation, the SCC1 channel is not contrast matched. Scale bars large panels: 5 μm, insets: 2 μWAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. One sister chromatid was labelled per chromosome as in Fig. 1 and SMC4 (wild type late prophase cells) or SCC1 (WAPL-dTAG cells) was visualised by immunofluorescence. E, Representative images of ΔWAPL ΔSororin cells synchronised to G2 by RO-3306. F, Representative images of wild type cells synchronised to late prophase by release from a RO-3306-mediated G2 arrest. G, Quantification of sister chromatid separation as in Fig. 1D, E. ΔWAPL ΔSororin G2 cells (n = 51 cells) and wild type late prophase (n = 55 cells) cells were analysed. Dots represent individual cells; red bars indicate the mean. Biological replicates: Control WAPL-dTAG G2 (n = 3), ΔWAPL G2 (n = 3), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3), wild type late prophase (n = 4). Technical replicates Control WAPL-dTAG G2 (n = 6), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 6), ΔWAPL ΔSororin G2 (n = 6), wild type late prophase (n = 7). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. To aid visualisation, the SCC1 channel is not contrast matched. Scale bars large panels: 5 μm, insets: 2 μm.
Figure 3A-C, Analysis of sister DNA and SMC protein complex distribution in cross sections perpendicular to the long chromosome axis, as shown in Fig. 2B, E, F. Density of Hoechst minus F-ara-EdU was calculated by subtracting F-ara-EdU fluorescence from Hoechst fluorescence. Line profiles indicate the mean curves of individual line profile measurements aligned to the midpoint between two SMC4 peaks (wild type late prophase), the SCC1 peak (ΔWAPL G2 cells), or the midpoint between two SCC1 peaks (ΔWAPL ΔSororin G2 cells). A, Lines from wild type late prophase (n = 157 lines from 31 cells). B, ΔWAPL G2 cells (n = 110 lines from 18 cells). C, ΔWAPL ΔSororin G2 cells (n = 138 lines from 18 cells) were analysed. Wild type late prophase (n = 4), ΔWAPL G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3). Technical replicates: Wild type late prophase (n = 7), ΔWAPL G2 (n = 6), ΔWAPL ΔSororin G2 (n = 5). Scale bars: 2μm.
Figure 4A, Representative images of one-sister labelled sister chromatids from ΔWAPL, ΔSMC4 ΔWAPL, or ΔSMC4 ΔWAPL ΔSororin prometaphase cells, as indicated. SMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. B, Quantification of sister chromatid separation as in Fig. 1D, E. ΔWAPL (n = 21 cells), ΔWAPL ΔSMC4 (n = 25 cells), and ΔWAPL ΔSMC4 ΔSororin (n = 28 cells) prometaphase cells were analysed. Dots represent individual cells; red bars indicate the mean. Significance was tested using a two-tailed Mann Whitney U test; P = 6.57 x 10-8 (ΔWAPL); P = 3.64 x 10-7 (ΔWAPL ΔSMC4 ΔSororin). Biological replicates , ΔWAPL prometaphase (n = 3), ΔWAPL ΔSMC4 prometaphase (n = 3), ΔWAPL ΔSMC4 ΔSororin prometaphase (n = 3).C, Representative images of control prometaphase cells labelled on one sister chromatid and depleted of SMC4 for different amounts of time as indicated. SMC4 was visualised by immunofluorescence using an anti-SMC4 antibody. Cells were synchronised to G2 phase by RO-3306, before being arrested in prometaphase by STLC for 60 min. SMC4 was then depleted through the addition of 1 μM 5-Ph-IAA for 120 or 240 minutes, as indicated. Control (n = 2), ΔSMC4 120 min (n = 2), ΔSMC4 240 min (n = 2). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 μm, insets: 1 μm.
Figure 5C, Genomic resolution analysis for G2 cells. Wild type, ΔNIPBL, ΔSMC4, ΔWAPL, ΔSororin, and ΔWAPL ΔSororin cells were analysed. Points indicate the values calculated for each replicate, bars indicate the mean and error bars indicate the standard deviation. Significance was tested using a two-tailed Mann Whitney U test; P = 1.24 x 10-4 (ΔNIPBL G2); P = 1.62 x 10-4 (ΔWAPL G2); P = 5.49 x 10-3 (ΔSororin G2), P = 1.47 x 10-3 (ΔWAPL ΔSororin G2). C, Wild type G2 (n = 11), ΔNIPBL G2 (n = 10), ΔSMC4 G2 (n = 4), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 4).
Figure 1(C) The quantification of labeled-m6A in mRNA and labeled-6mdA in genomic DNA.F) the quantitative results (F) of mRNA [D3]-m6A and genomic [D3]-6mdA of mES, HEK293T, C2C12, and NIH3T3 cells. The cells were treated with [D3]-L-methionine for 7 days, and [15N5]-6mdA nucleoside (50 amol) was spiked in digested DNA samples as external isotopic standard.
Figure 2(D) The determination of kinetic constants for recombinant ADAL catabolizing m6rAMP (red) and 6mdAMP (blue). The kinetic data were fitted with the Michaelis-Menten equation.(E) The catalytic efficiency (Kcat/ Km) of ADAL to 6mdAMP and m6rAMP. Error bars = s.d., three technical replicates.
Figure 3B) quantitative results (B) of DNA 6mdA in [15N5]-rA-treated mES cells after Adal knockdown for 7 days. Two independent biological replicates.D) quantification (D) of genomic 6mdA in Adal-/- mES cells treated with [15N5]-rA for 3 days. Error bars = s.d., three independent biological replicates.(E) The relative levels of Adal in mES, C2C12, and NIH3T3 cells against reference gene TUBB.(F The western blot analysis (F) ADAL expression in mES, C2C12, and NIH3T3 cells. Unpaired t-test, error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope. ND, not detected.G) quantitative results (G) of ADAL expression in mES, C2C12, and NIH3T3 cells. Unpaired t-test, error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope. ND, not detected.