Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 1. Low levels of FOXA1 and high levels of O-GlcNAcylation in breast tumors are correlated with poor patient prognosis. (A) FOXA1 mRNA expression between pathological low-grade (grades 1 and 2) and high-grade (grade 3) breast tumor tissues from GEO datasets (n = 589, GSE25066\61304\425668) are shown. The box plots show the medians (black lines), 25th and 75th percentiles (boundaries), and minimum/maximum values (whiskers). The P value is indicated. (B) Kaplan-Meier curves of patients with breast cancer from GEO datasets (n = 589, GSE25066\61304\425668) grouped by FOXA1 mRNA expression in tumor tissues. Patients with FOXA1 expression above the median are indicated by the yellow line, and patients with gene expression below the median are indicated by the blue line. The 95% confidence interval is shown. OS, overall survival. (C) Representative IHC staining of FOXA1 and global O-GlcNAcylation in a tissue microarray containing 114 breast tumors and 10 adjacent samples. The patient no. (F10, G7) on the microarray is indicated. Histological scoring was calculated using Image Pro Plus. Scale bar, 250 μm. (D) Pearson’s correlation analysis of FOXA1 and global O-GlcNAcylation IHC scores among different progression stages of breast cancer tissues in the tissue microarray (n = 114). The violin plots show the 25th, 50th, and 75th percentiles. (E) Pearson’s correlation analysis of FOXA1 and O-GlcNAcylation IHC scores in 114 breast tumor tissues. (F) Kaplan-Meier curves of 114 patients with breast cancer grouped by FOXA1 protein expression in tumor tissues. Patients with FOXA1 and O-GlcNAcylation levels above the median are indi- cated by the yellow line, and patients with gene expression levels below the median are indicated by the blue line. The 95% confidence interval is shown. (G) Western blotting (WB) was performed in five breast cancer patient tumor samples. The relative intensity of OGT and OGA bands is shown. The pathological grade and the patient no. are indicated.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Expressing, Gene Expression, Immunohistochemistry, Microarray, Western Blot

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 2. FOXA1 is O-GlcNAcylated by OGT in breast cancer cells. (A) An opposite trend of FOXA1 expression and global O-GlcNAcylation levels was identified in breast cancer cells. Cells were incubated with/without 100 nM fulvestrant for 3 hours. Indicated proteins were detected by WB. (B) FOXA1 O-GlcNAcylation and FOXA1-OGT interactions were compared in breast cancer cells. After treatment of breast cancer cells with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours, FOXA1 co-IP was performed. (C) OGT gain and loss of function affects the O-GlcNAcylation level of FOXA1. MCF-7 and MDA-MB-231 cells were transfected with OGT heterogeneous expression or shRNA (shOGT) plasmids. After 48 hours, sWGA lectin pulldown was performed. CBB, Coomassie Brilliant Blue staining. (D) A Click iT O-GlcNAc enzymatic labeling system was used to confirm the O-GlcNAcylation of endogenous FOXA1 in breast cancer cells. (E) The CTD is crucial for FOXA1 O-GlcNAcylation. Left: Three truncated variants of FOXA1 (ΔFOXA1-A to ΔFOXA1-C) fused with an HA-tag were generated according to its key domains. DBD, DNA binding domain. Right: Three truncated FOXA1 expres- sion plasmids were transfected with the OGT expression plasmid in HEK-293 T cells. After 48 hours, HA-tag co-IP was performed. (F) The sites of FOXA1 O-GlcNAcylation were mapped using mass spectrometry. (G) FOXA1 protein sequences, including the O-GlcNAcylation sites T432, S441, and S443, were aligned across species using BLAST. Red letters indicate conserved serine/threonine residues. (H and I) FOXA1 is O-GlcNAcylated at T432, S441, and S443 in breast cancer cells. After treatment with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours, MCF-7 FOXA1 KO and MDA-MB-231 cells stably expressing HA-tagged FOXA1WT (WT) or HA-tagged FOXA13A

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Expressing, Incubation, Co-Immunoprecipitation Assay, Transfection, shRNA, Staining, Labeling, Generated, Binding Assay, Plasmid Preparation, Mass Spectrometry, Stable Transfection

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 3. O-GlcNAcylation regulates the stability and chromatin binding of FOXA1. (A) Site-specific O-GlcNAcylation expedites the degradation of FOXA1. Left: Cells were incubated with 50 μM cycloheximide (CHX) for up to 12 hours. The ubiquitination and HA-FOXA1 were detected. Right: After treatment with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours, cell lysates were immunoprecipitated with anti-HA-tag magnetic beads. (B) O-GlcNAcylation regulates the stability of FOXA1 through the ubiquitin- proteasome pathway. Cells were treated with 10 μM MG132 for 10 hours. HA-FOXA1 immunoprecipitation was performed. (C) Overall serine and threonine phosphor- ylation were analyzed by immunoprecipitation and WB. (D) O-GlcNAcylation enhances the chromatin binding of FOXA1. After treatment with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours, chromatin and cytoplasm proteins were detected for HA-FOXA1. (E) Fluorescent staining results showing the nuclear localization dependence of FOXA1 on O-GlcNAcylation. Breast cancer cells were treated with 50 μM OSMI-4 or TMG for 24 hours. The micrograph scale bar represents 25 μm. DAPI, 40,6-diamidino-2-phenyl- indole. (F) Molecular dynamic simulation of O-GlcNAcylated and nonglycosylated FOXA1 binding with DNA. Left: The overall model structure of FOXA1-DNA complex with the substrate DNA helix. Middle: The typical conformations of O-GlcNAcylated and nonglycosylated FOXA1 transcriptional activating domain (TAD)-DNA complex from MD simulation. Right: The interaction network around O-GlcNAc groups in the O-GlcNAcylated system and the nonglycosylated system. DT, DNA T base; DA, DNA A bases; DC, DNA C base; DG, DNA G base. The serial number of sense-strand starts with 1 as the 50-terminal, and the serial number of antisense strand starts from 29 as the 50-terminal. (G) The center of mass distance between the helix region (amino acids: 402 to 425) in FOXA1 CTD and the major groove (base pair: 6 to 14) of DNA as a function of time. Snapshots from the simulations of two systems are shown on the bottom.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Binding Assay, Incubation, Ubiquitin Proteomics, Immunoprecipitation, Magnetic Beads, Staining

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 4. O-GlcNAcylation regulates the interaction between FOXA1 and epigenetic regulation factors. (A) Silver staining of FOXA1WT and FOXA13A interactome. MCF- 7 FOXA1 KO cells stably expressing HA-FOXA1WT or HA-FOXA13A were immunoprecipitated with anti-HA-tag magnetic beads. The immunoprecipitated fractions were resolved using SDS-PAGE and silver stained. (B) Volcano plot of LFQ proteomics data of FOXA1WT and FOXA13A interactome in MCF-7 FOXA1 KO cells (n = 6 biologically independent experiments, two-sided unpaired Student’s t test). The FOXA1 partners mentioned in this study are labeled in bold. (C) STRING protein-protein interaction analysis and functional enrichment analysis of FOXA1 interactome in FOXA1WT or FOXA13A expressed MCF-7 FOXA1 KO cells. Protein LFQ intensity of FOXA1 partner is shown in heat maps (yellow, high; blue, low). (D) GO terms enriched for FOXA1 partners in FOXA1WT or FOXA13A expressed MCF-7 FOXA1 KO cells. (E) O-GlcNAcylation affects the interaction between FOXA1 and epigenetic regulation factors. After treatment with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours, MCF-7 FOXA1 KO cells stably resecue-expressing HA-FOXA1WT or HA-FOXA13A were immunoprecipitated with anti-HA-tag magnetic beads. The indicated protein levels were detected by WB. The relative intensity of indicated protein bands is shown. (F) MECP2 knockdown decreased the chromatin binding of FOXA1. MCF-7 FOXA1 KO cells were transfected with MECP2 shRNA (shMECP2). Scrambled shRNA (shScr) was used as a control. After treatment with TMG (50 μM) for 24 hours, chromatin proteins were extracted, and the levels of HA-FOXA1WT and HA-FOXA13A were detected by WB.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Silver Staining, Stable Transfection, Expressing, Immunoprecipitation, Magnetic Beads, SDS Page, Staining, Labeling, Functional Assay, Knockdown, Binding Assay, Transfection, shRNA, Control

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 5. FOXA1 O-GlcNAcylation governs metastasis-related genes expression through FOXA1 chromatin loci fluctuations. (A) The average enrichment profiles of FOXA1WT and FOXA13AChIP-seq signals at all gene regions in the reference genome hg19. The transcriptional start site (TSS) and termination site (TES) are indicated. n = 2 biologically independent ChIP-seq replicates. (B) Heat maps of the FOXA1WT and FOXA13AChIP-seq reads density at the peak center (± 2 kb). (C) Gene density map plot of the FOXA1WT and FOXA13A ChIP-seq peak locations over the whole human genome. (D) FOXA1WT and FOXA13A ChIP-seq peak annotation relative to known genomic elements. (E) ChIP-seq occupancy signal in FOXA1WT-biased, FOXA13A-biased and unbiased sites identified by MAnorm. Left: MA plots of all peaks from both FOXA1WT

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Expressing, ChIP-sequencing

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 6. The chromatin-binding switch of O-GlcNAcylated FOXA1 is stimulated by MECP2. (A and B) O-GlcNAcylated FOXA1 chromatin loci shows transcriptional suppression characteristics. Average enrichment profiles of published MCF-7 cells (A) ATAC-seq (GSE179666), RNA-Pol II ChIP-seq (GSE54693), (B) H3K27me3 (GSE96363), H3K4me1 (GSE86714), H3K4me3 (GSE97481), and H3K27ac (GSE97481) ChIP-seq reads at differential quantitative FOXA1WT and FOXA13A ChIP-seq peaks are shown. (C) Heat maps of the FOXA1WT and FOXA13A ChIP-seq signal density at hg19 genome-wide CpG islands. (D) Average published MCF-7 cells genome-wide DNA methylation (GSE54693) profiles at CpG islands (left) and typical/super enhancer elements (right) of differential quantitative FOXA1WT and FOXA13A ChIP-seq peaks. (E) Heat map representation of endogenic MECP2 ChIP-seq (in FOXA1WT and FOXA13A expressed MCF-7 FOXA1 KO cells, respectively) signal enrichment at differential quantitative FOXA1WT and FOXA13A sites. (F) Cells were transfected with MECP2 shRNA (shMECP2). Average enrichment profiles of FOXA1 ChIP-seq reads at FOXA1WT and FOXA13A- biased sites are shown. (G) The box plots showing the mRNA expression changes of FOXA1-targeting genes associated with FOXA1WT and FOXA13A-biased sites. n = 2 biologically independent RNA-seq replicates. (H) Predicted regulatory network between FOXA1 and the corresponding downstream genes in different O-GlcNAcylation state. Functional enrichment is indicated. (I) Integrative Genomics Viewer tracks showing FOXA1, MECP2, RNA-Pol II ChIP-seq and DNA methylation, ATAC-seq signal at the promoter regions of the representative genes EPB41L3 and COL9A2. (J) Certain FOXA1 targeting genes were validated by qPCR and ChIP-qPCR. (K) Methylation status of EPB41L3 and COL9A2 gene promoter regions in the indicated cells were measured by methylation-specific qPCR. (L) Cells were transfected with MECP2 shRNA (shMECP2). Certain FOXA1 targeting genes were validated by qPCR and ChIP-qPCR. For (J) to (L), cells were pretreated with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours. n = 4 biologically independent experiments.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Binding Assay, ChIP-sequencing, Genome Wide, DNA Methylation Assay, Transfection, shRNA, Expressing, RNA Sequencing, Functional Assay, ChIP-qPCR, Methylation

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 7. FOXA1 O-GlcNAcylation elevates the metastatic potential of breast cancer cells. (A to C) The motility (A), migration and invasion ability (B), and adhesion ability (C) of breast cancer cells were analyzed. Cells were treated with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours. Mitomycin C was used to inhibit the impact of cell proliferation. Representative and quantified results of the wound-healing (A; scale bar, 200 μm), Transwell (B, scale bar, 100 μm) and adhesion (C; scale bar, 100 μm) assays in FOXA1WT- or FOXA13A-expressing breast cancer cells are shown. (A and B) n = 3 biologically independent experiments. (C) n = 6 biologically independent experiments. (D and E) The migration and invasion abilities were analyzed and quantified by Transwell assays in FOXA1WT- or FOXA13A-expressing breast cancer cells (MCF-7 FOXA1 KO and MDA-MB-231; scale bar, 100 μm). Cells were transfected with MECP2 (D) or EPB41L3 (E) shRNA (shMECP2 and shEPB41L3) or scrambled shRNA (control) for 48 hours before the assays. Mitomycin C was used to inhibit the impact of cell proliferation. n = 3 biologically independent experiments. (F) FOXA1 immunoprecipitation was performed in five breast cancer patient tumor samples, and immunoprecipitated fractions were analyzed by WB. The pathological grade and the patient no. are indicated.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Migration, Expressing, Transfection, shRNA, Control, Immunoprecipitation

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 8. FOXA1 O-GlcNAcylation promotes oncogenesis and metastasis of breast cancer in vivo. (A and B) Elimination of FOXA1 O-GlcNAcylation diminished xe- nograft tumor formation in vivo. WT MCF-7, MCF-7 FOXA1 KO, FOXA1WT, or FOXA13A MCF-7 FOXA1 KO cells were injected subcutaneously into the axillae of nude mice (n = 5 for each group). Mice were euthanized after 24 days, and their tumor masses were excised (A), measured and weighed (B). (C) HA-FOXA1 immunoprecipitation was performed with anti-HA-tag magnetic beads using xenograft tumor samples in each group, and the immunoprecipitated fractions were analyzed by WB. (D) Xenograft tumor samples were subjected to H&E, EPB41L3, and Ki67 staining. One representative experiment of n = 3 independent experiments is shown. Scale bar, 100 μm. (E) Schematics for tail vein injection of breast cancer cells in nude mice to generate experimental pulmonary metastasis. (F) MCF-7, MCF-7 FOXA1 KO, FOXA1WT, or FOXA13A

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: In Vivo, Injection, Immunoprecipitation, Magnetic Beads, Staining

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 9. Schematic working model of FOXA1 O-GlcNAcylation–mediated breast cancer metastasis. O-GlcNAcylation at T432/S441/S443 shapes the FOXA1 interac- tome, especially triggers the recruitment of transcriptional repressor MECP2, consequently stimulates FOXA1 chromatin-binding sites switch to chromatin loci of adhe- sion-related genes, including EPB41L3 and COL9A2, and thus promotes breast cancer proliferation and metastasis both in vitro and in vivo.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Binding Assay, In Vitro, In Vivo

Journal: OncoTargets and therapy

Article Title: Forkhead box protein A1 is a prognostic predictor and promotes tumor growth of gastric cancer

doi: 10.2147/OTT.S91035

Figure Lengend Snippet: FOXA1 knockdown inhibits gastric cancer cell proliferation and induces apoptosis in SGC-7901 cells. Notes: ( A ) and ( B ) FOXA1 shRNA significantly inhibited the levels of FOXA1 mRNA and protein in SGC-7901 cells; n = three independent experiments. ( C ) SGC-7901 cell proliferation measured by BrdU incorporation was inhibited by FOXA1 knockdown; n = three independent experiments; n = three independent repeats with similar results. ( D ) Apoptosis assays demonstrated that FOXA1 knockdown increased the percentage of apoptotic SGC-7901 cells; n = three independent repeats with similar results. ( E ) Western blot analyses indicated that FOXA1 knockdown increased the expression of cleaved Caspase-3 protein in SGC-7901 cells; n = three independent experiments. * P <0.05 by t -test. Abbreviations: FOXA1, forkhead box protein A1; NT, non-targeting; shRNA, short hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FITC, fluorescein isothiocyanate.

Article Snippet: The non-targeting vector (TR30012, OriGene, Beijing, People’s Republic of China) or the FOXA1-specific short hairpin RNA (shRNA) (TR312942, OriGene) was transfected into gastric cancer cells using Lipofectamine 2000 following the manufacturer’s instructions (catalog number: 11668-027, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Knockdown, shRNA, BrdU Incorporation Assay, Western Blot, Expressing

Journal: OncoTargets and therapy

Article Title: Forkhead box protein A1 is a prognostic predictor and promotes tumor growth of gastric cancer

doi: 10.2147/OTT.S91035

Figure Lengend Snippet: FOXA1 knockdown inhibits cell proliferation and induces apoptosis in MGC-803 cells. Notes: ( A ) and ( B ) FOXA1 shRNA significantly inhibited the level FOXA1 mRNA and protein in MGC-803 cells; n = three independent experiments. ( C ) MGC-803 cell proliferation measured by BrdU incorporation was inhibited by FOXA1 knockdown; n = three independent experiments. ( D ) Apoptosis assays demonstrated that FOXA1 knockdown increased the percentage of apoptotic MGC-803 cells; n = three independent repeats with similar results. ( E ) Western blot analyses indicated that FOXA1 knockdown increased the expression of cleaved Caspase-3 protein in MGC-803 cells; n = three independent experiments. * P <0.05 by t -test. Abbreviations: FOXA1, forkhead box protein A1; NT, non-targeting; shRNA, short hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FITC, fluorescein isothiocyanate.

Article Snippet: The non-targeting vector (TR30012, OriGene, Beijing, People’s Republic of China) or the FOXA1-specific short hairpin RNA (shRNA) (TR312942, OriGene) was transfected into gastric cancer cells using Lipofectamine 2000 following the manufacturer’s instructions (catalog number: 11668-027, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Knockdown, shRNA, BrdU Incorporation Assay, Western Blot, Expressing

Journal: OncoTargets and therapy

Article Title: Forkhead box protein A1 is a prognostic predictor and promotes tumor growth of gastric cancer

doi: 10.2147/OTT.S91035

Figure Lengend Snippet: FOXA1 knockdown suppresses tumor growth in nude mice. Notes: ( A ) SGC-7901 cells that were transfected with FOXA1 shRNA or NT shRNA were injected subcutaneously into nude mice. Tumor growth curves showed that SGC-7901 cells with FOXA1 knockdown (n=6) exhibited a significant slower growth as compared with control cells (n=6). * P <0.05 by ANOVA. Scale bar: 1 cm. ( B ) Representative immunostaining of Ki-67 and quantitative analysis of Ki-67 positive cells showed that FOXA1 knockdown significantly reduced cell proliferation; n=6, * P <0.05 by t -test. Scale bar: 20 μm. ( C ) Representative staining of TUNEL and quantitative analysis of TUNEL positive cells revealed that FOXA1 knockdown significantly increased the number of apoptotic cells; n=6, * P <0.05 by t -test. Scale bar: 20 μm. Abbreviations: FOXA1, forkhead box protein A1; NT, non-targeting; shRNA, short hairpin RNA; ANOVA, analysis of variance; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The non-targeting vector (TR30012, OriGene, Beijing, People’s Republic of China) or the FOXA1-specific short hairpin RNA (shRNA) (TR312942, OriGene) was transfected into gastric cancer cells using Lipofectamine 2000 following the manufacturer’s instructions (catalog number: 11668-027, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Knockdown, Transfection, shRNA, Injection, Control, Immunostaining, Staining, TUNEL Assay

Journal: OncoTargets and therapy

Article Title: Forkhead box protein A1 is a prognostic predictor and promotes tumor growth of gastric cancer

doi: 10.2147/OTT.S91035

Figure Lengend Snippet: Downregulation of FOXA1 reduces the expression of YAP in gastric cancer cells. Notes: SGC-7901 and MGC-803 cells that were transfected with FOXA1 shRNA or NT shRNA were subjected to qRT-PCR and Western blot for YAP expression. ( A ) and ( B ) FOXA1 knockdown significantly reduced the level of YAP mRNA in both SGC-7901 and MGC-803 cells. ( C ) and ( D ) Inhibition of FOXA1 clearly decreased the expression of YAP protein in both SGC-7901 and MGC-803 cells; n = three independent repeats with similar results. * P <0.05 by t -test. Abbreviations: FOXA1, forkhead box protein A1; YAP, Yes-associated protein; qRT-PCR, quantitative reverse transcription polymerase chain reaction; NT, Non-targeting; shRNA, short hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The non-targeting vector (TR30012, OriGene, Beijing, People’s Republic of China) or the FOXA1-specific short hairpin RNA (shRNA) (TR312942, OriGene) was transfected into gastric cancer cells using Lipofectamine 2000 following the manufacturer’s instructions (catalog number: 11668-027, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Transfection, shRNA, Quantitative RT-PCR, Western Blot, Knockdown, Inhibition, Reverse Transcription, Polymerase Chain Reaction

Journal: Molecular cell

Article Title: Redirecting the pioneering function of FOXA1 with covalent small molecules.

doi: 10.1016/j.molcel.2024.09.024

Figure Lengend Snippet: Figure 1. Tryptoline acrylamide WX-02-23 covalently engages cysteine-258 (C258) in FOXA1 in human prostate cancer cells (A) Chemical structures of tryptoline acrylamide stereoprobes. (B) Cysteine-directed ABPP data for FOXA1 cysteines from 22Rv1 cells treated with stereoprobes (20 mM, 3 h). n = 4. Mean ± SEM. (C) Volcano plot of IA-DTB blockade by WX-02-23 versus WX-02-43 (same ABPP experiments shown in B). Blue circles indicate >66.7% engagement by WX-02- 23. Average values from 2 independent experiments. p values: two-tailed Student’s t test. (D) Chemical structures of alkyne stereoprobes. (E) Protein-directed ABPP data from 22Rv1 cells showing proteins stereoselectively enriched by CJR-6A versus CJR-6B (x axis) and proteins for which CJR-6A enrichment is competed by WX-02-23 versus WX-02-43 (y axis). Stereoselectively enriched proteins that displayed >66.7% blockade of enrichment by WX-02-23 are in blue. n = 4 biological replicates. Data are averaged. (F) Protein-directed ABPP data showing concentration-dependent blockade of CJR-6A reactivity with proteins substantially and stereoselectively engaged by WX-02-23 in cysteine- (Figure 1C) and protein-directed (Figure 1E) ABPP experiments. Data are normalized to DMSO. n = 2–3 per group. Mean ± SEM. (G) Gel-ABPP data showing stereoselective reactivity of FOXA1_C258 with alkyne stereoprobes CJR-6A (6A) versus CJR-6B (6B) and WX-01-10 (10) versus WX- 01-12 (12; see Figure S1E) in HEK293T cells recombinantly expressing 2xStrep-FLAG epitope-tagged WT-FOXA1 or C258A-FOXA1 (or mock cells). Data are representative of two independent experiments.

Article Snippet: Sheard chromatin was diluted to achieve 0.1 % SDS and incubated with FOXA1 antibody (Novus Biologicals, NBP2-45354) pre-conjugated on Dynabeads (Thermo, Protein G, 10004D) overnight at 4 C. Beads were washed with sonication buffer (2x, without SDS), the sonication buffer with higher salt (1x, 500 mM NaCl, without SDS), LiCl wash buffer (1x, 20 mM Tris pH7.5, 1 mM EDTA, 250 mM LiCl, 0.5 % NP-40, 0.5 % Nadeoxycholate), and TE supplemented with 50 mM NaCl.

Techniques: Two Tailed Test, Concentration Assay, Expressing, FLAG-tag

Journal: Molecular cell

Article Title: Redirecting the pioneering function of FOXA1 with covalent small molecules.

doi: 10.1016/j.molcel.2024.09.024

Figure Lengend Snippet: Figure 2. Stereoprobes engage FOXA1_C258 in a DNA-dependent manner (A) Homology model of a FOXA1 FKHD-DNA complex bound to a canonical DNA-binding motif. Representative cancer-related mutations are shown in green (missense) or gray (in-frame deletion). (B) Lollipop plot of FOXA1 mutations from compiled data of breast and prostate cancer studies (http://www.cbioportal.org/). (C) Sequence alignment of human FOXA1, FOXA2, and FOXA3 around the Wing2 region.

Article Snippet: Sheard chromatin was diluted to achieve 0.1 % SDS and incubated with FOXA1 antibody (Novus Biologicals, NBP2-45354) pre-conjugated on Dynabeads (Thermo, Protein G, 10004D) overnight at 4 C. Beads were washed with sonication buffer (2x, without SDS), the sonication buffer with higher salt (1x, 500 mM NaCl, without SDS), LiCl wash buffer (1x, 20 mM Tris pH7.5, 1 mM EDTA, 250 mM LiCl, 0.5 % NP-40, 0.5 % Nadeoxycholate), and TE supplemented with 50 mM NaCl.

Techniques: Binding Assay, Sequencing

Journal: Molecular cell

Article Title: Redirecting the pioneering function of FOXA1 with covalent small molecules.

doi: 10.1016/j.molcel.2024.09.024

Figure Lengend Snippet: Figure 3. Stereoprobes enhance FOXA1-DNA interactions in vitro (A) Schematic for NanoBRET-based oligonucleotide-binding assay. (B) Sequences of TAMRA-conjugated DNA oligonucleotides (FOXA1-binding motif in red) for NanoBRET. (C) NanoBRET binding curves. Average ± SEM from 2 independent experiments. (D) Stereoprobe effects on NLuc-FOXA1 interactions with oligo-1 measured by NanoBRET (normalized to DMSO). Average ± SEM; n = 6. Two-tailed t test ****p <0.0001 compared with DMSO control. (E) Gel-ABPP data confirming stereoselective and site-specific engagement of NLuc-WT-FOXA1 by stereoprobes. HEK293T cells expressing NLuc-WT- or NLuc-C258A-FOXA1 (or NLuc control) proteins were treated with WX-02-23 (23) or WX-02-43 (43) (20 mM, 2 h), followed by CJR-6A (5 mM, 1 h). C, DMSO control. Data are representative of two independent experiments. (F) Concentration-dependent effects of WX-02-23 on NanoBRET signal for NLuc-WT-FOXA1 with oligo-1 (average ± SEM, n = 4). Data presented as the relative maximum percentage increase of NanoBRET signal in WX-02-23-treated samples over DMSO-treated control samples (where DMSO-treated controls are set to 100%).

Article Snippet: Sheard chromatin was diluted to achieve 0.1 % SDS and incubated with FOXA1 antibody (Novus Biologicals, NBP2-45354) pre-conjugated on Dynabeads (Thermo, Protein G, 10004D) overnight at 4 C. Beads were washed with sonication buffer (2x, without SDS), the sonication buffer with higher salt (1x, 500 mM NaCl, without SDS), LiCl wash buffer (1x, 20 mM Tris pH7.5, 1 mM EDTA, 250 mM LiCl, 0.5 % NP-40, 0.5 % Nadeoxycholate), and TE supplemented with 50 mM NaCl.

Techniques: In Vitro, Binding Assay, Two Tailed Test, Control, Expressing, Concentration Assay

Journal: Molecular cell

Article Title: Redirecting the pioneering function of FOXA1 with covalent small molecules.

doi: 10.1016/j.molcel.2024.09.024

Figure Lengend Snippet: Figure 4. WX-02-23 redirects the pioneering activity of FOXA1 in prostate cancer cells (A) Density plots showing correlations of FOXA1 ChIP-seq for compound treatments (20 mM for each stereoprobe; 10 nM for PladB, 3 h) versus DMSO control in 22Rv1 cells. PCC, Pearson’s correlation coefficient. (B) Number of FOXA1 binding sites with substantial changes induced by compound treatment relative to DMSO. (C) ATAC-seq changes induced by compound treatments (3 h, 20 mM for each stereoprobe) in 22Rv1 cells showing substantial (|log2 fold change [FC]| > 1) and significant (p < 0.05) changes. p values were determined by two-tailed Welch’s t test (n = 3). (D) The number of ATAC sites substantially and significantly changed for each compound treatment group. (E) De novo motif discovery analysis on ATAC-seq sites grouped by WX-02-23-induced changes (see Figure 4C). Top enriched sequence is shown (see Fig- ure S4E for others). (F) Proportion of ATAC-seq sites occupied by FOXA1 ChIP-seq peaks, grouped by substantial and significant WX-02-23-induced ATAC-seq changes (un- changed, |log2FC| < 1 and p > 0.05). (G) FOXA1 ChIP-seq signal at ATAC-seq sites grouped by WX-02-23-induced ATAC-seq changes. p values: two-tailed Welch’s t test between DMSO and WX- 02-23.

Article Snippet: Sheard chromatin was diluted to achieve 0.1 % SDS and incubated with FOXA1 antibody (Novus Biologicals, NBP2-45354) pre-conjugated on Dynabeads (Thermo, Protein G, 10004D) overnight at 4 C. Beads were washed with sonication buffer (2x, without SDS), the sonication buffer with higher salt (1x, 500 mM NaCl, without SDS), LiCl wash buffer (1x, 20 mM Tris pH7.5, 1 mM EDTA, 250 mM LiCl, 0.5 % NP-40, 0.5 % Nadeoxycholate), and TE supplemented with 50 mM NaCl.

Techniques: Activity Assay, ChIP-sequencing, Control, Binding Assay, Two Tailed Test, Sequencing

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 3. siRNA-mediated knockdown of Foxa1 inhibits FOXA1-mediated activation of the Odf2 promoter. Overexpression of FOXA1 (+ FOXA1) induced transcriptional upregulation of the firefly luciferase reporter controlled by the Odf2 promoter (2.2-pGL3) compared to the control (2.2-pGL3 + phRL) in both cycling cells and serum-starved cells. Co-transfection of the scrambled non-target siRNA (+ FOXA1 + control siRNA) did not significantly change the activity of the firefly luciferase. Co-transfection of either one of the three Foxa1 siRNA duplexes (+ FOXA1 + Foxa1 siRNA A, B, or C, respectively) repressed activation of the reporter vector in both cycling cells and serum-starved cells. Significant transcriptional repression by Foxa1 siRNA C (in cycling cells: p = 0.039532*, in serum-starved cells: p = 0.014283*, Student’s T-test, two-tailed, homoscedastic). Three biological replicates each.

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Knockdown, Activation Assay, Over Expression, Luciferase, Control, Cotransfection, Activity Assay, Plasmid Preparation, Two Tailed Test

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 4. FOXA1 binds to the sequence TGTTTAC situated at positions -1768 to -1775 of the Odf2 promoter. (A) Reporter gene assay to narrow down the FOXA1-binding site. Reporter vectors comprising specific parts of the Odf2 promoter were investigated for their activation by FOXA1. FOXA1-mediated transcriptional activation was always related to that of the respective vector, without FOXA1 co-expression. The strongest activation was observed for the reporter vectors 7.6 (~ 70x), 22.1 (~ 16x), and 7.1 (~ 4x) indicating that the FOXA1-binding site is positioned in the region − 1282 to − 1805. Three biological replicates. (B) Scheme of the Odf2 promoter fragments cloned upstream of the firefly reporter gene (e1 = exon1, i1 = intron1). The consensus binding site (cbs) for FOX TFs is located at position − 1768 to − 1775 (*). (C) Chromatin immune-precipitation by FOXA1 and qPCR of the binding-site sequence revealed significant enrichment. NIH3T3 cells were transfected with the Odf2 promoter reporter vector 2.2-pGL3 as bait. Co-transfection of the Foxa1 expression plasmid (Foxa1 + 2.2- pGL3/anti-FOXA1) resulted in an ~ 26 × enrichment of the FOXA1-binding site compared to the control IgGs (Foxa1 + 2.2-pGL3/control IgG) (p**, p = 0.007838). Precipitation of the endogenous FOXA1 by anti-FOXA1 antibodies (2.2-pGL3/anti-FOXA1) caused an ~ 4 × enrichment of the binding sequence compared to the control (2.2-pGL3/control IgG) that is, however, not significant (p = 0.137344).

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Sequencing, Reporter Gene Assay, Binding Assay, Activation Assay, Plasmid Preparation, Expressing, Clone Assay, Transfection, Cotransfection, Control

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 5. Expression of Fox TFs in NIH3T3 cells. (A) Amplification of Fox transcripts by first (a) and secondary/nested RT-PCR (b). Whereas in the first RT-PCR reaction no amplification products could be identified, the nested PCR performed on the first RT-PCR products as templates revealed products of the expected length of 449 bp (Foxo3a), 631 bp (Foxo1), 525 bp (Foxa1), and 674 bp (Foxj1). Correct amplification products were verified by sequencing. The original gel is presented in Supplementary Figure S3. (B) Immunological detection of endogenous FOXA1 in NIH3T3 nuclei (a, red), nuclear stain with DAPI (b, blue), and merge image (c). Bars are of 5 µm.

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Nested PCR, Sequencing, Staining

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 6. Knockdown of FOXA1::GFP by Foxa1 siRNA duplexes. (A) Western blots demonstrating the reduction in FOXA1::GFP by Foxa1 siRNA. Foxa1::gfp was co-transfected with either control siRNA (control) or one of the Foxa1 siRNA duplexes A, B, or C (Foxa1 siA, B, or C). 48 h post-transfection cells were harvested, and the cell lysates were analysed by Western blotting. Detection of FOXA1::GFP (green, ~ 77 kDa, asterisk) and α-Tubulin (red) on the same blot. The original blots are presented in Supplementary Figure S2. (B) Efficient reduction of FOXA1::GFP by Foxa1 siRNA-mediated knockdown. The quantity of FOXA1::GFP was related to the quantity of α-Tubulin in the same lane and the fold changes in the relative quantities calculated to the average of the relative quantity in control siRNA transfected cells. Three biological replicates for each RNA duplex. Student’s T-test two-tailed, homoscedastic: siRNA A p = 0.412464, siRNA B p = 0.004772**, siRNA C p = 0.002733**.

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Knockdown, Western Blot, Transfection, Control, Two Tailed Test

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 7. Decreased expression of Foxa1, Odf2/ODF2, and CP110 by siRNA-mediated Foxa1 knockdown. (A) Knockdown of Foxa1 and Odf2 transcripts by Foxa1 siRNA. NIH3T3 cells were transfected with either the scrambled control siRNA (control siRNA) or the Foxa1 siRNA (Foxa1 siRNA) and transcription of Foxa1 and Odf2 quantified by RT-PCR. The relative expression of Foxa1 or Odf2 was calculated to both housekeeping genes, Gapdh and Hprt, by ΔCt. Their relative expression following siRNA-mediated depletion of Foxa1 was compared to the control siRNA using the ΔΔCt-method and calculated as 2-ΔΔCt. Significant reduced expression of Foxa1 (p = 0.02727533*) and Odf2 (p = 0.00062325+++) related to the control siRNA. qRT-PCR was performed with at least three biological replicates, each measured in triplicate. Student’s T-test two-tailed, homoscedastic. (B) Western blot showing expression of ODF2 in NIH3T3 cells transfected with either the scrambled control siRNA (control siRNA, biological replicates #1 to #3) or the Foxa1 siRNA C (biological replicates #1 to #6). Cells were harvested 48 h post-transfection and cultivation in serum-deprived medium. Detection of ODF2 (~ 100 kDa) and ß-Actin (~ 42 kDa) simultaneously on the same blot using the fluorescent-labeled secondary antibody anti-rabbit IgGCW800. An unspecific band was observed > 100 kDa. (C) Foxa1 siRNA caused a reduction in ODF2 protein. The relative quantity of ODF2 was obtained by calculating the ratio between the quantity of ODF2 and ß-Actin in each lane, and relating the relative quantities to the average of the relative quantity obtained in control siRNA-transfected cells. Significant reduction of ODF2 expression by Foxa1 siRNA C (p = 0.000631***). Six biological replicates each and a total of n loadings: control n = 7, siRNA B n = 7, siRNA C n = 9. (D) Western blot showing expression of CP110 in control siRNA, and Foxa1 siRNA C transfected cells. (control siRNA: 4 biological replicates, Foxa1 siRNA: 5 biological replicates). CP110 (< 130 kDa) and ß-Actin (~ 40 kDa) were both detected simultaneously on the same blot (in green). (E) FOXA1 knockdown caused a decreased quantity of CP110 (p = 0.002232**). The relative quantity of CP110 was calculated as described for ODF2 quantification using the quantity of ß-Actin as internal standard. For quantification six biological replicates for both, the control and siRNA were used and a total of n loadings were quantified: control n = 7, siRNA n = 8. Always Student’s T-test two-tailed, homoscedastic. The original blots are presented in Supplementary Figure S2.

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Expressing, Knockdown, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Western Blot, Labeling

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 8. FOXA1 is mandatory for primary cilia formation. (A) Detection of primary cilia by immunodecoration of ARL13B (in red, arrows point to primary cilia). Merged images including nuclear counterstain with DAPI (in blue). Bars are of 10 µm. (B) siRNA-mediated depletion of Foxa1 led to a decrease in ARL13B-positive primary cilia. NIH3T3 cells were transfected either with the scrambled negative control siRNA, or one of either Foxa1 siRNA duplexes A, B, or C. 24 h post-transfection the medium was exchanged for serum starvation medium for cilia induction. Cells were fixed either after 24 h or 48 h in serum starvation medium and ARL13B decorated immunologically. ARL13B-positive primary cilia were manually counted. All experiments were performed in triplicates and n cells were counted: Foxa1 siRNA A (24 h) n = 1,599, FoxA1 siRNA B (24 h) n = 1,544, FoxA1 siRNA C (24 h) n = 1,564, control (24 h) n = 1,593, FoxA1 siRNA A (48 h) n = 1,593, FoxA1 siRNA B (48 h) n = 1,540, FoxA1 siRNA C (48 h) n = 1,590, control (48 h) n = 1,583. Student’s T-test two-tailed, homoscedastic, to control 24 h (+, siRNA A p = 0.004569, siRNA B p = 0.011402, siRNA C p = 0.010086), or to control 48 h (*, siRNA A p = 0.031788, siRNA B p = 0.008451, siRNA C p = 0.004429). p < 0.05 *, p < 0.01 **.

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Transfection, Negative Control, Control, Two Tailed Test

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 9. Interaction between FOXA1 and cJUN. (A, B) Co-immune precipitation of FOXA::GFP did not reveal direct interaction with cJUN. Cells were transfected with expression plasmids encoding Foxa1::gfp, and Mekk1 (A) or Foxa1::gfp solely (B), and FOXA1::GFP captured by immobilized anti-GFP antibodies. Bead-bound proteins were eluted by boiling in SDS-sample buffer and the eluate, and aliquots of the input, the insoluble pellet, and the wash solutions were separated on denaturing SDS-gels. FOXA1::GFP was detected with anti-GFP antibodies (in red) and cJUN with anti-cJUN antibodies (in green). The unphosphorylated cJUN of ~ 39 kDa is present in A and B, whereas the phosphorylated cJUN of ~ 43 kDa was detected only when Mekk1 was co-transfected (in A). Although the endogenous cJUN was detectable in the input fractions it did not co-precipitate with FOXA1::GFP (eluate). The original blots are presented in Supplementary Figure S2. (C) Co-localization of FOXA1::GFP and cJUN in transfected NIH3T3 cells. Antibody specificity of the anti- FOXA1 antibody was proven by expression of FOXA1::GFP (a, in green) and anti-FOXA1 antibody decoration (b, in red). Co-transfection of cJun with Foxa1::gfp followed by anti-cJUN antibody decoration (f, in red) demonstrated overlapping FOXA1::GFP fluorescence (e, in green) with anti-cJUN decoration (h, merged image). Nuclear staining (c, g, in blue) and merged images (d, h). Scales bares of 5 µm.

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Transfection, Expressing, Cotransfection, Fluorescence, Staining

Journal: Scientific reports

Article Title: FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

doi: 10.1038/s41598-022-25966-w

Figure Lengend Snippet: Figure 10. FOXA1 is a transcriptional activator of Odf2, which ultimately promotes cilia formation. FOXA1 binds to the sequence TGTTTAC at positions − 1768 to − 1775 of the Odf2 promoter and promotes transcription of Odf2. The JNK-pathway via cJUN, and C/EBPα are also involved in the transcriptional activation of Odf2, although their binding sites have not been determined. Positive feedback was found between cJUN and FOXA1 in the transcriptional activation of the Odf2 promoter, although a direct interaction could not be detected. According to the ChIP-seq dataset from the ENCODE Transcription Factor Targets Datasets, FOXA1 targets many more essential ciliary genes, including CP110, which we have shown is indeed regulated by FOXA1. The finally generated ODF2 protein, which is mandatory for the generation of primary cilia, eventually promotes cilia formation together with other ciliary proteins. The structure of the ODF2 protein (Mus musculus) was predicted by AlphaFold (AlphaFold DB version 2022-11-01, created with the AlphaFold Monomer v2.0 pipeline, licence CC-BY-4.0), and made available by EMBL-EBI (Wellcome Genome Campus, Hinxton, UK). The primary cilium was detected by immunological decoration of both, acetylated α-Tubulin (green) and ARL13B (red), shown here as merged image (yellow), and the nucleus stained with DAPI (blue) (inset).

Article Snippet: Foxa1 knockdown was achieved using three unique 27mer siRNA duplexes (Origene, SR415184A, SR415184B, SR415184C, all used at a concentration of 10 nM), and a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM).

Techniques: Sequencing, Activation Assay, Binding Assay, ChIP-sequencing, Generated, Staining