Journal: Nature cell biology

Article Title: Diurnal oscillations of endogenous H 2 O 2 sustained by p66 Shc regulate circadian clocks

doi: 10.1038/s41556-019-0420-4

Figure Lengend Snippet: a, Environment of the C195 thiol in the CLOCK:BMAL1 crystal structure. b, Representative western blot showing the interactions between recombinant BMAL1 (amino acids 49–434) and recombinant WT- or C195S-CLOCK after treatment with or without H2O2 (10−5 mM). c,d, Scatchard analysis of the equilibrium binding of recombinant BMAL1 to recombinant WT-CLOCK (c) and C195S-CLOCK (d) treated with or without H2O2 (10−6 mM). e, Scatchard analysis of the equilibrium binding of the heterodimer of recombinant BMAL1:WT-CLOCK treated with or without H2O2 (10−6 mM) to a G-box probe. f, Representative EMSA of the heterodimer of BMAL1 and WT- or C195S-CLOCK proteins treated with or without H2O2 (10−6 mM) binding to the G-box probe. g, Relative mRNA levels of Per2, Per1, Cry2, Rev-erbα, Dbp, and Tef in WT MAFs and ClockC195S MAFs treated with or without H2O2 (50 μM) for 10 h (n = 3 independent biological samples). h-m, Relative mRNA levels of Per2 (h), Per1 (i), Cry2 (j), Rev-erbα (k), Dbp (l), and Tef (m) in WT and ClockC195S MAFs over the circadian cycle (n = 3 independent biological samples per time point). P values are shown for the comparisons of ClockC195S with WT. n, Representative baseline detrended bioluminescence recordings from synchronized Clock KO MAFs rescued by WT or C195S mutant CLOCK. P values were calculated using one-way ANOVA with a Bonferroni’s post hoc test (g) and an unpaired two-tailed Student’s t test (h-m). Data are presented as the means ± SEM. n = 3 independent experiments for b,f and n = 2 independent experiments for c-e and n = 6 independent experiments for n with similar results. Source data are provided in Statistics Source Data Figure 4. Unprocessed blots are shown in Source Data Figure 4.

Article Snippet: One microgram of recombinant CLOCK protein (Creative Biomart) was incubated with 1 mM hydrogen peroxide for 10 min at 37°C, at which point dimedone was added to a final concentration of 5 mM and the samples were incubated for an additional 1 h. After the dimedone treatment, 10 mM DTT was added, and the samples were incubated for an additional 30 min at 37°C.

Techniques: Western Blot, Recombinant, Binding Assay, Mutagenesis, Two Tailed Test

Journal: Nature cell biology

Article Title: Diurnal oscillations of endogenous H 2 O 2 sustained by p66 Shc regulate circadian clocks

doi: 10.1038/s41556-019-0420-4

Figure Lengend Snippet: a, Analysis of evolutionary conservation of cysteines in CLOCK. b, Relative luciferase activities of Per1:Luc in HEK293T cells transfected with WT or a series of cysteine mutant CLOCK plasmids in the presence of BMAL1 overexpression (n = 3 independent biological samples). Data are presented as the means ± SEM. c, Relative mRNA levels of Dbp in N2a cells transfected with WT or corresponding mutant CLOCK in the presence of BMAL1 overexpression (n = 3 independent biological samples). P values were calculated using an unpaired two-tailed Student’s t test. Data are presented as the means ± SEM. d,e, Representative western blot of free thiols in WT and corresponding mutant CLOCK (d) or followed by treatment with 200 μM H2O2 (e). f, Representative western blot showing the levels of sulfenylated thiols in WT- and C195S-CLOCK at 28 h and 40 h post serum shock. g, Representative mass spectrum (MS) ion chromatograms for the peptides corresponding to the iodoacetamide-alkyne probe (IPM)-labelled cysteines from H2O2 (150 μM)-treated recombinant CLOCK (amino acids 1–351) or control CLOCK. H2O2 treatment significantly decreased the levels of reduced C195 and C267 labelled by IPM in recombinant CLOCK (0.5-fold as the cut-off value). h, Representative MS of dimedone-labelled recombinant CLOCK peptide at C195. C#, dimedone-labelled C195. i, Sequencing of PCR products from wild-type mice (top) and ClockC195S mice (bottom). Black arrow indicates the location of mutation introduced by CRISPR/Cas9. The replaced nucleotide is shown in red. j, Representative western blot showing levels of CLOCK-SH labelled by BIAM in livers from WT and ClockC195S mice at CT28. Each genotype consists of a mixture of liver extracts from three mice. k,l, Representative western blot (k) and quantification (l) of CLOCK-SH labelled by BIAM in MAFs from WT and ClockC195S mice for one circadian cycle. n = 2 independent experiments for f-l and n = 3 independent experiments for d,e with similar results. Source data are provided in Statistics Source Data Figure 3. Unprocessed blots are shown in Source Data Figure 3.

Article Snippet: One microgram of recombinant CLOCK protein (Creative Biomart) was incubated with 1 mM hydrogen peroxide for 10 min at 37°C, at which point dimedone was added to a final concentration of 5 mM and the samples were incubated for an additional 1 h. After the dimedone treatment, 10 mM DTT was added, and the samples were incubated for an additional 30 min at 37°C.

Techniques: Luciferase, Transfection, Mutagenesis, Over Expression, Two Tailed Test, Western Blot, Recombinant, Sequencing, CRISPR