Fig 2: Silencing of NF-YC affects localization of NF-Y. (A) Western blot analysis using anti-NF-YA and anti-NF-YC antibodies in both trophozoites (T) and 48 h cysts (C) in control and silenced NF-YC cells. Silencing of NF-YC did not alter protein levels of NF-YA. Anti-actin antibody was used as a control. (B) Immunostaining with anti-NF-YA antibody in silenced NF-YC trophozoites, and 48 h cyst are shown (green). In NF-YC silenced parasites, localization of NF-YA is altered in trophzoites and cysts. DNA was stained with DAPI (blue). Scale bar for trophozoites and cysts are 25 µm. (C) Results of immunostaining with anti-NF-YC antibody in silenced NF-YC cysts (48 h) are presented (green) and show very low signal levels. DNA was stained with DAPI (blue). Bars, 25 µm.

Fig 3: Loss of NF-YA delays regenerative myogenesis in vivo.a Representative immunostaining for eMyHC (red) and laminin (green) on TA muscle sections of NF-YAfl/fl and NF-YAcKO mice 5, 15 and 25 days after CTX injury. Nuclei were identified by DAPI staining. Scale bar: 100 µm. b Relative mRNA levels of myogenic markers Pax7, Myf5, Myogenin, eMyHC in whole 5-days injured TA muscles of NF-YAfl/fl and NF-YAcKO mice measured by RT-qPCR. Transcript levels of NF-YAfl/fl and NF-YAcKO uninjured mice have been arbitrarily set at 1. Data represent mean ± s.d. (one-Way Anova: Pax7 F(3,8)=9.66, p = 0.0049; Myf5 F(3,8)=44.63, p < 0.0001; MyoG F(3,8)=9,99, p = 0.0044; eMyHC F(3,8)=170.8, p < 0.0001; n = 3 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control; ##p < 0.01, ####p < 0.0001 vs NF-YAfl/fl). c Western blot analysis performed on whole protein extracts of uninjured (control) and 5-days injured TA muscles of NF-YAfl/fl and NF-YAcKO mice. Immunoblots represent protein levels of Pax7, eMyHC, Myogenin and Tubulin. n = 3 experiments. d Western blot analysis of eMyHC on whole protein extracts of 15-days injured TA muscles of NF-YAfl/fl and NF-YAcKO. Tubulin was used as loading control. n = 3 experiments.

Fig 4: NF-YA specifically bound to CCAAT/ATTGG box upstream of the SOX2 promoter in cervical cancer cells. (A) The diagram of SOX2 promoter upstream with the binding sites of NF-YA factor. (B) The qChIP assay is shown in NF-YA-overexpressing SiHa and C33A cells. The cell lysate was immunoprecipitated by NF-YA or SOX2 antibodies and IgG antibody (as the negative control). (C) EMSA was performed by incubating nuclear lysate from NF-YA-overexpressing SiHa and C33A cells with biotin-labelled DNA fragment containing an NF-YA binding sequence and the EBNA extract served as the positive control. (D) SOX2 was silenced by specific shRNA targeting the SOX2 CDS region in NF-YA-overexpressing SiHa cells, and the tumour growth curve was shown. (E) The expression of NF-YA, SOX2 and Ki-67 was detected by IHC in 10 SCC samples, and the relationship was determined using the Pearson correlation. (F) Proposed model of the NF-YA-mediated up-regulation of SOX2 in cervical progression. Data are presented as the mean ± SD of experiments in triplicate. The symbols represent the following: *, P < 0.05; **, P < 0.01

Fig 5: NFYAv2 enhances gluconeogenesis by transcriptional activation of PCK1. (A,B) qRT-PCR (A) and western blot analysis (B) of the expression levels of PCK1 in wild-type, NFYAv2OE, and NFYA-deficient SK-Hep1 cells under normal or glucose deprivation conditions after 10 h of culture. (C) Western blot analysis of the expression levels of PCK1, GLUD1, GOT2, and NFYA in wild-type and NFYAv2OE SK-Hep1 cells under normal or glucose deprivation conditions with treatment with NAC (100 µM) or dimethyl a-KG (5 mM) after 10 h of culture. (D) The location of NFYA binding sites on the PCK1 promoter region (upper panel). CUT&RUN assay to evaluate the association of NFYA with the promoter of PCK1 in wild-type, NFYAv2OE, and NFYA-deficient SK-Hep1 cells under glucose deprivation conditions after 5 h of culture. Input and eluted chromatins were subjected to qRT-PCR analysis using promoter-specific primer. Data represents the % input of the immunoprecipitated chromatin for each gene (lower panel). (E) Representative fluorescence images of living cells (green) detected with Calcein-AM and dead cells (red) detected with Propidium iodide (PI) in NFYAv2OE SK-Hep1 cells under normal or glucose deprivation conditions with treatment with 3-MPA (5 mM) after 10 h of culture. (F) The bar graph shows the percentage of dead cells among the more than 600 cells counted in each condition. (G) Intracellular ATP level in wild-type, NFYAv2OE, and NFYA-deficient SK-Hep1 cells under glucose deprivation conditions after 5 h of culture. All data are shown as mean and SEM. Unpaired two-tailed t-test was performed. (n.s.) not significant; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.