Journal: Molecular Cancer

Article Title: Regulation of DNA methyltransferase 1 transcription in BRCA1-mutated breast cancer: a novel crosstalk between E2F1 motif hypermethylation and loss of histone H3 lysine 9 acetylation

doi: 10.1186/1476-4598-13-26

Figure Lengend Snippet: H3K9ac and E2F1-mediated transcriptional regulation of DNMT1. Ai , RT-PCR showing GCN5, PCAF and E2F1 levels before and after knockdown by shRNAs, and normalized to β-actin expression. Aii , the results from three independent experiments are represented as mean ± SD. Bi , EdU labeling showing proliferation of GCN5, PCAF and E2F1-silenced and control cells. Blue, Hoechst 33342 labeling of cell nuclei; Red, EdU labeling of nuclei of proliferative cells. Bii , the EdU incorporation rate was expressed as the ratio of EdU positive cells to total Hoechst33342 positive cells. Ci and Cii , analysis of histone modification H3K9ac and transcription factor E2F1 enrichment around the E2F1 motif within the CpG islands after the deletion of GCN5, PCAF or E2F1. D , The interaction of E2F1 and GCN5 or PCAF were examined by the immunoprecipitation of cell extracts with an antibody to E2F1, and co-immunoprecipitation of E2F1, GCN5 and PCAF by western blot analysis. Results of Figure A-D were obtained in BRCA1-mutated breast cancer cells, and the same results were also obtained in 293 T cells and non-BRCA1 mutated breast cancer cells. Ei-Ev , the DNMT1 expression levels after deletion of H3K9ac and E2F1 around the E2F1 motif in 293 T cells, and 15 primary non-mutated and BRCA1-mutated breast cancer and their normal breast cells. Each experiment was repeated four times for 293 T cells and primary breast cells of each patient. Bar graphs show mean ± SD. * P < 0.05 vs. Control.

Article Snippet: The shRNA lentiviral particles of GCN5 (sc-37946-V), PCAF (sc-36198-V) and E2F1 (sc-29297-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Knockdown, Expressing, Labeling, Control, Modification, Immunoprecipitation, Western Blot

Journal: Scientific reports

Article Title: Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia.

doi: 10.1038/s41598-025-86292-5

Figure Lengend Snippet: Fig. 1. The expression of Kat2b located in the cytosol, centrosome, and basal body increased during ciliogenesis, and depletion of Kat2b causes delays in ciliogenesis. (a) qRT-PCR data showing a gradual increase in the level of Kat2b transcript during primary cilia formation. (b) Western blot data showing an increase in Kat2b protein expression during ciliogenesis. (c) Representative figures of Kat2b localization in NIH/3T3 cells by serum starvation time (0 h, 6 h, 12 h, and 24 h). In serum starvation 12 h and 24 h, two panels were displayed depending on presence of primary cilia NIH/3T3 cells were transfected with expressing GFP-tagged full-length-Kat2b. After 24 h, cells were incubated under serum starvation conditions, followed by staining with endogenous acetylated α-tubulin for primary cilia (red) and pericentrin (PCNT) for centrosome and basal body (yellow). Nuclei were stained with DAPI (blue). (d) Quantification of the subcellular localization of Kat2b. Counted cell numbers were as follows: serum starvation for 6 h, n = 133; 12 h, n = 129; and 24 h, n = 124. Data were collected from three independent experiments. (e) Validation of transcriptional Kat2b level reduction in stable Kat2b knockdown NIH/3T3 cell line using qRT-PCR. (f) Validation of translational Kat2b level reduction in stable Kat2b KO-NIH3T3 cells or control KO-NIH3T3 using western blot analysis. (g) Representative figures of ciliated NIH/3T3 cells transfected in stable Kat2b KO and control cells. After serum- free treatment, cells were fixed and stained using ICC in a time-course manner. Acetylated α-tubulin was stained for primary cilia (green). Nuclei were stained with DAPI (blue). Primary cilia are indicated by white arrows. Scale bar: 10 μm. (h) Quantification of the percentage of ciliated cells in Kat2b-depleted cells compared to control shRNA cells. Counted cell numbers were as follows: controls with serum starvation for 6 h, n = 685; 12 h, n = 665; 24 h, n = 589. Kat2b-depleted cells with serum starvation for 6 h, n = 789; 12 h, n = 579; 24 h, n = 649. Data were collected from three independent experiments.

Article Snippet: To perform establishing stable Kat2b knock-out NIH3T3, we used PCAF shRNA Plasmid (Santa cruz, sc-36199-SH) and Control shRNA Plasmid-A (Santa cruz,, sc-108060) as control cell line.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Incubation, Staining, Biomarker Discovery, Knockdown, Control, shRNA

Journal: Scientific reports

Article Title: Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia.

doi: 10.1038/s41598-025-86292-5

Figure Lengend Snippet: Fig. 3. The depletion of Kat2b impairs the recruitment of IFT and Hh components to primary cilia. (a, b) IFT components 6 h after serum starvation (early ciliogenesis). (a) Western blot data showed that protein levels of IFT components decreased in Kat2b silenced NIH/3T3 cells. (b) Fluorescence staining data revealed that the intensity of IFT components located in primary cilia was decreased in the stable Kat2b deleted cell. Scale bar: 2 μm (c-d) IFT components 24 h after serum starvation (maturated stage of ciliogenesis). (c) Western blot data show that protein levels of IFT components decreased in Kat2b silenced NIH/3T3 cells. (d) Immunocytochemistry data revealed that fluorescence intensity of IFT components located in primary cilia. Scale bar: 2 μm (e) Graphs showed fluorescence intensity of IFT25 and IFT52 under serum starvation 6 h, 24 h in stable Kat2b KO NIH3Te and control NIH3T3. (f) Immunocytochemistry data display that the fluorescence intensity of IFT25 and IFT52 (green) at primary cilia (red) in Kat2b KO MEF cells at 6 h and 24 h after serum starvation, respectively. Scale bar: 2 μm. (g) Graphs showed fluorescence intensity of IFT25 and IFT52 under serum starvation 6 h, 24 h in MEF cells. (h) Representative images displayed accumulation of IFT52 within cilia when treated garcinol and serum withdrawal 6 h, and below graph showed fluorescence intensity at cilia.

Article Snippet: To perform establishing stable Kat2b knock-out NIH3T3, we used PCAF shRNA Plasmid (Santa cruz, sc-36199-SH) and Control shRNA Plasmid-A (Santa cruz,, sc-108060) as control cell line.

Techniques: Western Blot, Fluorescence, Staining, Immunocytochemistry, Control

Journal: Scientific reports

Article Title: Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia.

doi: 10.1038/s41598-025-86292-5

Figure Lengend Snippet: Fig. 4. Kat2b knockdown impairs the recruitment of Hh components to primary cilia. (a) Immunocytochemistry data confirmed that the fluorescence intensity of Smo (green) was decreased in Kat2b silenced cell. Acetylated α-tubulin and γ-tubulin were stained for primary cilia and basal body (red). Nuclei were stained with DAPI (blue). Scale bar: 2 μm Right graph showed fluorescence intensity of Smo within primary cilia. (b, c) qRT-PCR and western data showed that mRNA and protein levels of Gli1 expression were decreased when Kat2b was depleted by siRNA transfection. Gli1 was used as an activation marker of the Hh signaling pathway. NIH/3T3 cells were treated with serum starvation for 24 h and 500 nM of SAG for activation of Hh signaling. (d) Immunocytochemistry data of Smo protein (green) at primary cilia (red) in Kat2b KO MEF cells with serum starvation for 24 h and 400 nM of SAG. Scale bar: 2 μm. Right graph showed fluorescence intensity of Smo within primary cilia. (e) qRT-PCR data showing that transcriptional Gli1 expression was decreased in Kat2b KO MEF cells. (f) Representative images displayed accumulation of Smo within primary cilia when treated garcinol 6 h before SAG treatment and serum withdrawal 24 h, and right graph showed mean of fluorescence intensity within cilia.

Article Snippet: To perform establishing stable Kat2b knock-out NIH3T3, we used PCAF shRNA Plasmid (Santa cruz, sc-36199-SH) and Control shRNA Plasmid-A (Santa cruz,, sc-108060) as control cell line.

Techniques: Knockdown, Immunocytochemistry, Fluorescence, Staining, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Activation Assay, Marker

Journal: Scientific reports

Article Title: Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia.

doi: 10.1038/s41598-025-86292-5

Figure Lengend Snippet: Fig. 5. The acetyltransferase domain of Kat2b in the cytosol is significant for localization of IFT components at primary cilia. (a) Graphical view of domains of Kat2b and designed mutant Kat2b. (b) Immunocytochemistry images confirmed the localization of the designed mutant Kat2b construct. DsRed tagged Kat2b constructs were investigated vector transfected cell and nuclear localization signal domain deleted Kat2b positioned only cytosol. (c) Immunoblotting analysis verified cytosolic and nucleic distribution under transfection with Kat2b constructs in NIH/3T3 cell. α-tubulin represented as cytosol marker and histone H3 as nuclear marker. (d) Immunofluorescence images showed that IFT 52 localization depending on Kat2b existence in the nucleus and function of acetylation. shControl NIH/3T3 cells transfected by empty vector, and stable Kat2b depleted NIH/3T3 cells, as shKat2b, transfected by empty vector and mutant Kat2b constructs. Nuclei were stained with DAPI (blue), transfected vector constructs were EGFP (green), acetylated α-tubulin and γ-tubulin were stained for primary cilia (red), and IFT52 were indicated yellow. White arrows indicate IFT components located in the cilia axoneme. The proportion of IFT52 distribution by categories are displayed below the graphs; ciliary axoneme, basal body, and not on the cilia. The graph on the bottom right indicated fluorescence intensity of ciliary IFT52. Scale bar: 2 μm.

Article Snippet: To perform establishing stable Kat2b knock-out NIH3T3, we used PCAF shRNA Plasmid (Santa cruz, sc-36199-SH) and Control shRNA Plasmid-A (Santa cruz,, sc-108060) as control cell line.

Techniques: Mutagenesis, Immunocytochemistry, Construct, Plasmid Preparation, Transfection, Western Blot, Marker, Immunofluorescence, Staining, Fluorescence

Journal: Scientific reports

Article Title: Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia.

doi: 10.1038/s41598-025-86292-5

Figure Lengend Snippet: Fig. 6. Kat2b knockout mice show decreased IFT25 in primary cilia and renal abnormalities. (a) Kat2b mRNA expression data using qRT-PCR validated that the level of Kat2b was decreased in the kidney of Kat2b KO mice. (b) Western blots also showed decreased Kat2b expression in KO mice. (c) Two kidney weights per total body weight demonstrate that there were no significant differences between Kat2b KO mice and wild-type mice. (d) Western blot data showed that acetylated α-tubulin level decreased in the renal tissue of Kat2b KO mice. (e) H&E staining of kidney paraffin sections from 16 weeks Kat2b knockout mice showed glomerular cyst, loosen glomeruli, and dilated tubules. Black arrowheads indicate glomeruli. The black dotted line defines the area of the dilated tubule. Scale bar: 100 μm. (f) Confocal microscopy analysis revealed that the fluorescence intensity of IFT25 (red) at primary cilia decreased in the renal tubule from 16 weeks Kat2b knockout mice compared to the wild-type. Acetylated α-tubulin and γ-tubulin were stained for primary cilia (green). Nuclei were stained with DAPI (blue). The White dotted line defines the area of the renal tubule. Right graph showed relative mean of IFT25 fluorescence intensity within primary cilia. Scale bar: 20 μm.

Article Snippet: To perform establishing stable Kat2b knock-out NIH3T3, we used PCAF shRNA Plasmid (Santa cruz, sc-36199-SH) and Control shRNA Plasmid-A (Santa cruz,, sc-108060) as control cell line.

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Western Blot, Staining, Confocal Microscopy, Fluorescence

Journal: Scientific reports

Article Title: Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia.

doi: 10.1038/s41598-025-86292-5

Figure Lengend Snippet: Fig. 7. Summary illustration of this study. Red ovals indicate KAT2B; green circles indicate IFT-B particles; blue circles indicate SMO; light grey circles indicate α-tubulin. The color intensity indicates the abundance. (a, b) During ciliogenesis, the basal expression level and subcellular localization of Kat2b in the cytosol, centrosome, and basal body gradually increased. Kat2b interacts with α-tubulin and regulates the acetylation level of α-tubulin via its catalytic activity. When Kat2b was depleted, the rate of cilium assembly was delayed, causing less acetylation level of α-tubulin, and the recruitment of IFT to be impaired. (c) When the Hh signaling pathway was activated using SAG treatment, the recruitment of SMO along the cilium is less under the absence of Kat2b.

Article Snippet: To perform establishing stable Kat2b knock-out NIH3T3, we used PCAF shRNA Plasmid (Santa cruz, sc-36199-SH) and Control shRNA Plasmid-A (Santa cruz,, sc-108060) as control cell line.

Techniques: Expressing, Activity Assay