Journal: bioRxiv

Article Title: NFYA regulates two sequential genome-wide transcriptional activation events during oocyte to embryo transition

doi: 10.64898/2026.03.30.715371

Figure Lengend Snippet: (A) Left upper panel: schematic diagram of the experimental design. Left lower panel: the embryo development rate upon DMSO or G&V treatment (Ganetespib 5nM and VER155008 5μM). The data are presented as mean ± SEM. [DMSO, n = 67; G&V (1C-B), n = 64; G&V (4C-B), n = 47. N represents the total number of embryos from two independent experiments]. Right panel: representative images of blastocyst stage embryos after chaperones inhibition (G&V). Scale bar,100 μm. Red arrows indicate representative arrested embryos in G&V (1C-B) group. (B) Scatter plots comparing gene expression profiles of late-2cell embryos treated with G&V or DMSO. The x and y axis of the dot plots are Log 2 CPM from RNA-seq. Fold change > 2, FDR < 0.05. ZGA genes, n=2,773. (C) Venn diagram showing the overlapped downregulated ZGA genes between NFYA depleted (NFYA-dTAG) and G&V treated L2C embryos. P values (Fisher’s exact test, two-sided) for overlapped genes are also shown. (D) Upper panel: schematic diagram of the experimental design. Lower left panel: ovarian morphology of 6 days in vitro cultured P4 ovaries with DMSO and G&V treatment. DDX4 stained oocytes. The white arrowhead indicates representative growing oocytes. Scale bar, 50 μm. Lower right panel: numbers of growing oocytes (DDX4 positive oocytes bigger than 25 μm in diameter that were surrounded by cuboidal granulosa cells) in each ovary after 6 days culture with DMSO and G&V treatment. Quantitative data are shown as mean ± SEM; ∗∗p < 0.01, Student’s t test. (E) Scatter plots comparing the gene expression profiles of growing oocytes (GOs) from 6 days in vitro cultured P4 ovaries with G&V or DMSO. The x and y axis of the dot plots are Log 2 CPM from RNA-seq. Fold change > 2, FDR < 0.05. PFA genes, n=2,463. (F) Venn diagram showing the overlapped downregulated PFA genes between Nfy a-cKO and G&V treated GOs. P values (Fisher’s exact test, two-sided) for overlapped genes are also shown. (G) Model illustrating the role of NFYA in PFA and ZGA. Before PFA (primordial), the PFA genes are silenced. Subsequently (primary and secondary), NFYA binding promotes open chromatin and activates PFA genes through promoter and distal binding. Loss of NFYA leads to reduced chromatin accessibility, defective PFA, and early follicular degeneration. Before ZGA (1C), the ZGA genes are silenced. Subsequently (L2C), NFYA activates ZGA genes through promoter and enhancer binding. Loss of NFYA leads to defective ZGA and predominantly embryo arrest at 2-cell stage. (H) NFYA pre-occupies and regulates a set of genes, including chaperones and histone genes, common in both PFA and ZGA through conserved promoter binding.

Article Snippet: Ganetespib (MCE, HY-15205) and VER155008 (Selleckchem, S7751) were reconstituted in DMSO to a 100 mM or 150 mM stocks, respectively.

Techniques: Inhibition, Gene Expression, RNA Sequencing, In Vitro, Cell Culture, Staining, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70s Regulate Circadian Rhythm under various conditions. A Immunoprecipitation assays were performed to compare the interaction strength between wild-type PER and Hsp70Ba under various temperature conditions. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and rabbit anti-Actin (ABclonal, #AC026). B Statistics of ( A ). N = 3. Statistical differences were assessed using one-way ANOVAs and post-hoc Tukey tests (P < 0.05). C Representative double plot actograms showing average locomotor activity for the respective genotypes under heat shock conditions. White background indicates light, and black indicates darkness. D Analysis of circadian rhythm persistence in Hsp70 mutants and w 1118 controls following heat shock. P-values from two-sided Fisher’s exact test are indicated (***P < 0.001)

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Immunoprecipitation, Western Blot, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70 Protein Interacts with PER, Affecting PER Phosphorylation and Cellular Localization. A Relative per expression at different time points in Hsp70 mutants and the control group. B Antibody specificity evaluation in western blot analysis for PER protein. C PER protein detection by western blots at ZT3, ZT9, ZT15, and ZT21. B - C Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and mouse anti-β-Tubulin (ABclonal, Cat#AC021). D Quantification of results in ( C ). N = 5. Statistical differences were measured using an unpaired Student’s t-test. **P < 0.01, ***P < 0.001. E Statistics of the cellular localization of PER in s-LNvs and l-LNvs clusters of clock neurons in indicated genotypes. C represents cytoplasm, and N represents nucleus. F Immunoprecipitation experiments to detect the interaction between PER, TIM, and Hsp70s. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and mouse anti-β-Tubulin (ABclonal, Cat#AC021). Samples were prepared in S2 cells, with β-tubulin used as an internal control. G Calculation of average TIM binding by PER molecule. N = 3. Significance of differences was determined using one-way ANOVAs and post-hoc Tukey tests. H Immunoprecipitation experiments to detect the interaction between PER and Hsp70s in tim 01 and w 1118 controls. Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and anti-β-Tubulin (ABclonal, Cat#AC021). β-tubulin was used as an internal control. ( I ) Calculation of average Hsp70 binding by PER molecule. N = 5. The t-test was used to calculate the significance of differences. **P < 0.01

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Immunoprecipitation, Binding Assay