Journal: Cell

Article Title: BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis

doi: 10.1016/j.cell.2019.05.009

Figure Lengend Snippet: Epidermal BMAL1 Is Required to Prevent Increased Differentiation

Article Snippet: The following antibodies were used: anti-BMAL1 (Abcam, ab93806), antiphophorylated BMAL1 (Ser42) (Cell Signaling, 13936), anti-acetylated BMAL1 (Lys538) (Merck Millipore, AB15396), anti-TUBULIN (Sigma-Aldrich, T7816) and anti-ACTIN (Sigma-Aldrich, A5228).

Techniques:

Journal: Cell

Article Title: BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis

doi: 10.1016/j.cell.2019.05.009

Figure Lengend Snippet: Key Resources Table

Article Snippet: The following antibodies were used: anti-BMAL1 (Abcam, ab93806), antiphophorylated BMAL1 (Ser42) (Cell Signaling, 13936), anti-acetylated BMAL1 (Lys538) (Merck Millipore, AB15396), anti-TUBULIN (Sigma-Aldrich, T7816) and anti-ACTIN (Sigma-Aldrich, A5228).

Techniques: Recombinant, SYBR Green Assay, Expressing, Knock-Out, Software

Journal: bioRxiv

Article Title: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids

doi: 10.1101/2020.10.30.362830

Figure Lengend Snippet: ( A ) Human genomic locus of circadian clock genes, PER1 , and the essential segmentation clock gene, HES7 , are highly conserved in higher vertebrates. The PER1 homolog PER2 is also located adjacent to the HES7 homolog HES6 in the genome. ( B ) ESCs were differentiated into iPSM for 96 hr in vitro , and then the iPSM colonies were treated with or without dox. ( C ) qPCR of Clock and Bmal1 mRNA in the indicated ESCs. 500 ng/mL dox treatment for 6 hr (red) or not (blue). Each number indicates clone number. Mean ± SD (n = 3 biological replicates). ( D ) qPCR of Clock and Bmal1 mRNA in the indicated iPSM colonies. 1000 ng/mL dox treatment for 2 hr (red) or not (blue). Mean ± SD (n = 2 technical replicates). The average expression level of pHes7-luc ESCs or iPSM colonies without dox was set to 1. ( E ) Representative maximum intensity projection of the immunostaining of iPSM colonies treated with 1000 ng/mL dox for 2 hr or not. n = 2–3 biological replicates. Scale = 250 µm.

Article Snippet: The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) , anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody , or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C.

Techniques: In Vitro, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids

doi: 10.1101/2020.10.30.362830

Figure Lengend Snippet: (A) The expression levels of pluripotent marker genes, Nanog, Sox2 , and Oct3/4 , were measured using RNA-seq in ESCs and pHes7-luc iPSM. The data were shown with in vitro 0, 7-, and 28-day differentiated WT ESCs and Per2 Luc ESCs (GSE61184). No circadian clock oscillates in the in vitro 7-day differentiated ESCs and the circadian clock oscillation starts to emerge after 14 days of differentiation culture ( , ). (B) Bioluminescence traces of the iPSM differentiated from Per2 Luc ESCs. The iPSM was stimulated with 10 µM forskolin (Fsk, middle) or 100 nM dexamethasone (Dex, bottom). n = 3 biological replicates. (C) Representative immunostaining of CLOCK, BMAL1, and PER1 in the iPSM. The immunostaining represented the suppression of CLOCK proteins in contrast to the expression of BMAL1. Furthermore, although the nuclei indicated the quite faint signals, PER1 protein in the iPSM is still predominantly accumulated in the cytoplasm.

Article Snippet: The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) , anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody , or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C.

Techniques: Expressing, Marker, RNA Sequencing Assay, In Vitro, Immunostaining

Journal: bioRxiv

Article Title: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids

doi: 10.1101/2020.10.30.362830

Figure Lengend Snippet: Although the expression of BMAL1 protein was observed even in ESCs , dox-dependent sole expression of Clock was insufficient for the E-box-driven expression of clock genes such as Per1/2 and Cry1/2 in undifferentiated ESCs, raising the possibility that the endogenously expressed BMAL1 might be post-translationally modified to not function. Colored boxes indicate 500 ng/mL dox treatment for 6 hr (red) or no treatment (blue). Each number indicates clone number. Mean ± SD (n = 3 biological replicates). The averaged expression level of Per2 Luc ESCs without dox was set to 1. Two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) , anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody , or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C.

Techniques: Expressing, Modification, Two Tailed Test

Journal: bioRxiv

Article Title: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids

doi: 10.1101/2020.10.30.362830

Figure Lengend Snippet: ( A, B ) qPCR of core circadian clock gene ( A ) and Hes 7 or Hes6 gene ( B ) expression in ESCs harboring dox-inducible Clock and Bmal1 or Clock and Bmal1DN . Colored boxes indicate the presence (red) or absence (blue) of 500 ng/mL dox treatment for 6 hr. Each number indicates clone number. Mean ± SD (n = 2–3 biological replicates). The average expression level of ESCs without dox was set to 1.

Article Snippet: The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) , anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody , or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C.

Techniques: Expressing

Journal: bioRxiv

Article Title: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids

doi: 10.1101/2020.10.30.362830

Figure Lengend Snippet: ( A ) Bioluminescence of each dox-inducible Clock/Bmal1 or Clock/Bmal1DN pHes7-luc iPSM colony was observed using PMT or an EM-CCD camera without or with dox. ( B, C ) Representative bioluminescence traces ( B , n = 25 biological replicates) and live imaging ( C , n = 3 biological replicates) of single pHes7-luc iPSM colony with or without dox. The kymograph of the imaging along the arrow is shown. ( D ) Representative bioluminescence traces of the single indicated iPSM colony with and without 1000 ng/mL dox. n = 10-54 biological replicates. ( E–H ) Live imaging of the single tetO-Clock/Bmal1 pHes7-luc iPSM colony with and without dox. Dox-containing medium or only medium was added at the indicated time points at the final dox concentration of 1000 ng/mL ( G, H ). Each kymograph along the arrow is shown. Scales = 250 µm. n = 2-4 biological replicates.

Article Snippet: The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) , anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody , or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C.

Techniques: Imaging, Concentration Assay

Journal: bioRxiv

Article Title: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids

doi: 10.1101/2020.10.30.362830

Figure Lengend Snippet: ( A ) Dox-inducible Clock/Bmal1 pHes7-luc ESCs were differentiated into gastruloids for 96 hr in vitro , and then the gastruloids embedded in 10% Matrigel were treated with or without dox. pHes7-luc bioluminescence was observed using an EM-CCD camera without or with dox. ( B–M ) Time-lapse bioluminescence (red) and bright field imaging of the single pHes7-luc gastruloid or tetO-Clock/Bmal1 pHes7-luc gastruloid without and with dox. Dox-containing medium was added at the indicated time points at the final dox concentration of 1000 ng/mL ( C, F, I, L ). Each kymograph is shown along the yellow lines in B, E, H, K . In situ hybridization of Uncx4 . 1 in the gastruloids after the live cell imaging ( D, G, J, M ). Scales = 250 µm. n = 2-4 biological replicates.

Article Snippet: The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) , anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody , or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C.

Techniques: In Vitro, Imaging, Concentration Assay, In Situ Hybridization, Live Cell Imaging

Journal: bioRxiv

Article Title: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids

doi: 10.1101/2020.10.30.362830

Figure Lengend Snippet: ( A ) Upregulated and downregulated DEGs in the indicated iPSM colonies treated with dox. ( B ) KEGG pathway analysis of the DEGs. Each pathway was indicated with each transformed p-value. The ranked pathways contained several common genes in the WNT, MAPK, and NOTCH signaling pathways. ( C ) UCSC genome browser views of RNA-seq data of the contiguous genes Vamp2, Per1, Hes7 , and Aloxe3 . The reads shown are normalized average reads per 10 million total reads in 10-bp bins. ( D ) mRNA expression of Vamp2, Per1, Hes7 , and Aloxe3 in the indicated iPSM colonies according to RNA-seq. ( E ) Validation of Vamp2 and Aloxe3 gene expression levels in the indicated iPSM colonies using qPCR. Colored boxes indicate 1000 ng/mL dox treatment for 2 hr (red) or no treatment (blue). Mean ± SD (n = 2 technical replicates). The averaged expression level of pHes7-luc iPSM colonies without dox was set to 1. Two-tailed t-test, *P < 0.05, **P < 0.01. ( F ) The premature expression of CLOCK/BMAL1 in the iPSM and gastruloids interfered with the segmentation clock oscillation and somitogenesis-like process.

Article Snippet: The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) , anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody , or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C.

Techniques: Transformation Assay, RNA Sequencing Assay, Expressing, Two Tailed Test

Journal: PLoS ONE

Article Title: Circadian Genes, xBmal1 and xNocturnin , Modulate the Timing and Differentiation of Somites in Xenopus laevis

doi: 10.1371/journal.pone.0108266

Figure Lengend Snippet: A compilation of results from microarray analyses of temporal expression of Notch, WNT, and circadian genes in the somites [34] and suprachiasmatic nucleus, liver, and heart [35] , [36] , [37] , [38] .

Article Snippet: Blots were incubated overnight at 4°C with a 1∶500 dilution of either xNocturnin antibody , Goat anti-Bmal1 (ARNTL) antibody (MyBioSource MBS422067), or anti-alpha A2 (DM1) tubulin (Neomarkers MS-581-P0).

Techniques: Microarray, Expressing

Journal: PLoS ONE

Article Title: Circadian Genes, xBmal1 and xNocturnin , Modulate the Timing and Differentiation of Somites in Xenopus laevis

doi: 10.1371/journal.pone.0108266

Figure Lengend Snippet: Both cells of a two celled embryos were injected with 1 ng Control Morpholino (Ctrl; 2 ng total), 1 ng or 500 pg of xBmal1 MO (Bmal1; 2 ng and 1 ng total) and 1 ng of xNocturnin MO (Noc; 2 ng total). Significant reduction of xBMAL1 protein (69Kd) was observed with injection of 2 ng or 1 ng xBmal1 MO compared to control MO injection (0.11 and 0.26 relative to control MO injected protein levels). An approximate 50% reduction of NOCTURNIN (43Kd, indicated) protein was observed when embryos were injected with a total of 1 ng xNocturnin MO (0.49 relative to control MO injected protein levels). The Nocturnin antibody also recognizes a larger (62Kd) band which likely represents a postranslationally modified form of xNOCTURNIN . Alpha tubulin (100Kd) was used as a loading control for each lane.

Article Snippet: Blots were incubated overnight at 4°C with a 1∶500 dilution of either xNocturnin antibody , Goat anti-Bmal1 (ARNTL) antibody (MyBioSource MBS422067), or anti-alpha A2 (DM1) tubulin (Neomarkers MS-581-P0).

Techniques: Injection, Control, Modification

Journal: PLoS ONE

Article Title: Circadian Genes, xBmal1 and xNocturnin , Modulate the Timing and Differentiation of Somites in Xenopus laevis

doi: 10.1371/journal.pone.0108266

Figure Lengend Snippet: The somite and circadian clocks both consist of negative feedback loops requiring the transcriptional activation (BMAL1, CLOCK) and repression (HES6/7, HER1; PERIOD1/2, CRY1/2) of clock components. The period of the somite clock is thought to be regulated by the balance of bHLH transcription factors present in the cell (“dimer cloud”) . Bmal1 may upset the balance by hetero-dimerizing with proteins in the dimer cloud (HES6/7, HER1) or by competing with HES6/7/HER1 for binding to the H-box (green arrow). NOCTURNIN may also impact the balance of proteins present in the dimer cloud by inhibiting translation of dimer cloud components (red line). BMAL1-CLOCK heterodimers are known to positively activate genes involved in myogenesis ( MyoD , MRF5 ). Perhaps, NOCTURNIN inhibits translation of repressors of myogenesis (blue arrow). We hypothesize that phosphorylation of CREB protein (CREB-P) may also act to coordinate components of the circadian and somite clock.

Article Snippet: Blots were incubated overnight at 4°C with a 1∶500 dilution of either xNocturnin antibody , Goat anti-Bmal1 (ARNTL) antibody (MyBioSource MBS422067), or anti-alpha A2 (DM1) tubulin (Neomarkers MS-581-P0).

Techniques: Activation Assay, Binding Assay, Phospho-proteomics