Journal: bioRxiv

Article Title: Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

doi: 10.64898/2025.12.24.696437

Figure Lengend Snippet: (A) Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) was performed using an anti-RNAPII (8WG16) antibody to evaluate recruitment of RNA polymerase II to the RRM2 promoter, with or without osimertinib (Osi) treatment. Relative enrichment was normalized to the ACTB promoter. (B) ChIP-qPCR using HA-tagged MYBL2-overexpressing cell lines was conducted to assess MYBL2 binding to the RRM2 promoter. Promoter occupancy is shown relative to input. Data are presented as mean ± s.e.m. (n = 3). ***p < 0.001. (C) RRM2 mRNA expression was quantified by RT-qPCR in PC-9 and HCC827 cells following EGFR knockdown using siRNA. Transcript levels were normalized to GAPDH. (D) The impact of EGFR knockdown on MYBL2 recruitment to the RRM2 promoter was evaluated by ChIP-qPCR in HA-MYBL2-expressing cells transfected with control or EGFR siRNA. Promoter enrichment was normalized to input. Data are shown as mean ± s.e.m. (n = 3). ***p < 0.001. All data are representative of at least three independent experiments.

Article Snippet: RRM1 (#10526-1-AP), RRM2 (11661-1-AP), RRM2B (#18005-1-AP), CHK1 (#25887-1-AP), CHK2 (#13954-1-AP), EGFR (#66455-1-Ig), beta Actin (#20536-1-AP), HA tag (#51064-2-AP), Histone H3 (#17168-1-AP), GAPDH (#60004-1-Ig), POLD1(#15646-1-AP), POLH (#28133-1-AP), MYBL2 (#18896-1-AP) and TNNT3 (#19729-1-AP) were purchased from Proteintech.

Techniques: Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Expressing, Quantitative RT-PCR, Knockdown, Transfection, Control

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet:

Article Snippet: Rabbit anti-NR1D1 (ChIP) , Proteintech , 14506-I-AP.

Techniques: Virus, Recombinant, Staining, Cytotoxicity Assay, cDNA Synthesis, Luciferase, Construct, Expressing, Plasmid Preparation, Software

Journal: Genes & Development

Article Title: Integrator orchestrates RAS/ERK1/2 signaling transcriptional programs

doi: 10.1101/gad.301697.117

Figure Lengend Snippet: Integrator is phosphorylated following MAPK induction, and phoshpho-ERK1/2 is recruited to EGF-responsive genes upon pathway activation. ( A , B ) Phospho-ERK1/2 ChIP-qPCR (ChIP combined with qPCR) with antibody-1 (Cell Signaling Technology, no. 4370) and antibody-2 (Invitrogen, no. 700012). The cells were collected before and after 20 min of EGF induction with or without the presence of MEK inhibitor (PD0325901). After ChIP, qPCR was performed with primers located in the TSS region of the NR4A1 , EGR1 , and FOSB genes. The primer pairs located in the TSS region of the EHD1 gene and ∼3 kb downstream from the transcription end site (TES) of the EGR1 gene were used as negative controls. The average from at least three independent experiments is shown. (*) P < 0.05; (**) P < 0.01, t -test. ( C , D ) Western blots of immunoprecipitation for exogenous expression of Flag-INTS11 in HeLa cells ( C ) and endogenous INTS11 in A375 (BRAF_V600E) cells ( D ). Phosphorylated INTS11 protein was detected by specific antibody against phosphothreonine (Cell Signaling, no. 9381).

Article Snippet: 4370 and 9101; and Invitrogen, catalog no. 700012), ERK1/2 (Cell Signaling Technology, no. 9102), MED1 (Bethyl Laboratories, A300-793A), MED12 (Bethyl Laboratories, A300-774A), phospho-p90RSK (Thr359) (Cell Signaling Technology, no. 8753), RSK1 (Cell Signaling Technology, no. 9333), phosphor-EGFR (pTyr1068) (Cell Signaling Technology, no. 3777), EGF receptor (Cell Signaling Technology, no. 4267), and phosphothreonine (Cell Signaling Technology, no. 9381).

Techniques: Activation Assay, ChIP-qPCR, Western Blot, Immunoprecipitation, Expressing

Journal: bioRxiv

Article Title: FOXA1 is required for ErbB2 expression and luminal differentiation in HER2-positive breast cancer

doi: 10.1101/2024.04.16.589460

Figure Lengend Snippet: Co-expression of FOXA1 and ErbB2 in HER2 positive breast cancer cells. A. Uniform Manifold Approximation and Projection (UMAP) of human breast tumor cells. B . UMAP (upper panel) and split violin plots for ESR1 , ErbB2 and FOXA1 . C. Pearson correlation coefficient between ESR1 and ErbB2 among ER-positive or HER2-positive epithelial cells. P- value was calculated from a down-sampled dataset with 1 to 10 % to avoid zero inflation of scRNA-seq data. D. UMAP plots and co-expression pattern of ESR1 , ErbB2 , and FOXA1 in epithelial cells. E. Bar plot showed co-expression of two features simultaneously (Criteria that expression level more than 0.5 is used to assign positive cells). F. H&E staining FFPE tissue conducted pre-CytAssist is shown. G. Cell type specific marker genes are expressed as log 2 (normalized UMI counts). H. Spatial distribution of cells idented by unsupervised clustering based on differential gene expression analysis. I. Violin plot illustrating the selected marker genes expression of each cell type. The box centerlines depict the medians, and the edges depict the first/third quartiles. J. Pearson correlation coefficient between FOXA1 and ERBB2 of each cell type.

Article Snippet: Quantitative RT-PCR was performed with the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) using a Step One Plus Real- Time PCR System (Applied Biosystems) and the following TaqMan primer sets: FOXA1 , Hs04187555_m1; ErbB2 , Hs01001580_m1; GATA3 , Hs00231122_m1.

Techniques: Expressing, Staining, Marker, Gene Expression

Journal: bioRxiv

Article Title: FOXA1 is required for ErbB2 expression and luminal differentiation in HER2-positive breast cancer

doi: 10.1101/2024.04.16.589460

Figure Lengend Snippet: FOXA1 regulates ErbB2 expression. A. Histogram showing the relative expression levels of FOXA1 from qRT-PCR in control and FOXA1KD-SKBR3 cells. B. Immunofluorescence images of FOXA1 (green) in control and FOXA1KD-SKBR3 cells. Scale bars is 10μm. C. Histogram showing the relative expression levels of ErbB2 from qRT-PCR in control and FOXA1KD-SKBR3 cells. D. Western blots showing the indicated protein levels in control and FOXA1KD-SKBR3 cells. E. Immunofluorescence images of HER2 (green) and pHER2 (red) in control and FOXA1KD- SKBR3 cells. DAPI was used to stain the nucleus. Scale bars is 10μm. F. Immunofluorescence images of FOXA1 (green) in MCF7 and SKBR3 cells. Scale bars is 10μm. G. Histogram showing the relative expression levels of FOXA1 from qRT-PCR among MCF10A, MCF7, and SKBR3 cells. H. Donut charts showing the distribution of FOXA1 ChIP-seq peaks in functional genomic regions according to Homer gene annotation. I. Venn diagram showing the overlapped functional genomic regions between FOXA1 binding regions from SKBR3 and MCF7 breast cancer cells. J-L. Genome tracks of ErbB2 and ESR1 gene showing the identified peaks over pre-defined promoter region (H3K4Me3) and regulatory region (H3K27Ac). FOXA1 binding motifs matched with ChIP-seq peak regions were marked with a red triangle. The screenshot was generated using the UCSC Genome Browser or IGV. M. Dot plot of the Oncogenic signature gene sets significantly (adjusted p-value < 0.05, Benjamini-Hochberg correction) activated (left) or suppressed (right) in the FOXA1KD-SKBR3 cells compared to the control. N. Volcano plot showing the log2FC and adjusted P-values of all genes (light grey) from the differential test between control and FOXA1KD-SKBR3 cells. The red dots represent ERBB2_UP.V1_UP leading-edge subset genes and the blue dots represent ERBB2_UP.V1_DN leading-edge subset genes. Dots that have adjusted P-value 0 were marked with jittered value ± 5 at y = 300 for visualization. Blue dotted lines indicate |log2FC| = 2, adjusted P-value = 0.05. O. Heatmap showing the relative expression of ERBB2_UP.V1_DN leading-edge subset genes and ERBB2_UP.V1_UP leading-edge subset genes, respectively. Bar graphs represent the mean±SEM. *** denotes p<0.0005, **** denotes p<0.00005.

Article Snippet: Quantitative RT-PCR was performed with the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) using a Step One Plus Real- Time PCR System (Applied Biosystems) and the following TaqMan primer sets: FOXA1 , Hs04187555_m1; ErbB2 , Hs01001580_m1; GATA3 , Hs00231122_m1.

Techniques: Expressing, Quantitative RT-PCR, Control, Immunofluorescence, Western Blot, Staining, ChIP-sequencing, Functional Assay, Binding Assay, Generated

Journal: bioRxiv

Article Title: FOXA1 is required for ErbB2 expression and luminal differentiation in HER2-positive breast cancer

doi: 10.1101/2024.04.16.589460

Figure Lengend Snippet: Heatmap representing the signal enrichment of control and FOXA1KD-SKBR3 cells. ATAC- seq reads in 2-kb regions centered on the TSS sites of annotated promoter-TSS sites of protein- coding genes. Down-regulated (upper panel) and up-regulated (lower panel) in FOXA1KD- SKBR3 cells than control respectively. B. Boxplot showing the average scores per region from ATAC-seq for control and FOXA1KD-SKBR3 cells. Wilcoxon rank sum test was performed, and the P-value was labeled at the plot. C-D. Dot plot showing the significantly (adjusted p- value < 0.05, Benjamini-Hochberg correction) enriched Hallmark gene sets by GSEA (C) and ORA (D) in the FOXA1KD-SKBR3 cells compared to control. Genes corresponded with DARs annotated only as Promoter-TSS were used for analysis. E. Volcano plot showing the log2FC and adjusted P-values of all peaks which genes annotated as promoter-TSS from the differential test between control and FOXA1KD-SKBR3 cells. The red dots represent HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION leading-edge subset genes. Blue dotted lines indicate |log2FC| = 0.5, adjusted P-value = 0.05. F. Scatter plot showing the correlation of the log2FC values between RNA-seq and ATAC-seq. Displayed R value determined by Pearson correlation. G. Scatter plot showing the correlation of the log2FC values of HALLMARK_EMT_genes between RNA-seq and ATAC-seq. Displayed R value determined by Pearson correlation. H. Dot plot showing the significantly enriched Hallmark gene sets by ORA performed for DEGs (|FC| > 3) in the FOXA1KD-SKBR3 cells compared to the control. Heatmap showing the expression of 41 enriched genes in Hallmark_EMT gene set. I. UMAP dimensional reduction of all epithelial cells. ErbB2 / FOXA1 -high and -low groups were divided according to the corresponding gene expression level. J. Feature plot of cells that each high or low group for ErbB2 and FOXA1 . K. Dot plot showing the significantly enriched Hallmark gene sets by ORA performed for DEGs (|FC| > 0.25 utilized MAST) in the ErbB2 / FOXA1 -high compared to the -low group. Heatmap showing the expression of 20 enriched genes in Hallmark EMT gene set. L. Violin plot illustrating the EMT module score of each cell type. The box centerlines depict the medians, and the edges depict the first/third quartiles.

Article Snippet: Quantitative RT-PCR was performed with the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) using a Step One Plus Real- Time PCR System (Applied Biosystems) and the following TaqMan primer sets: FOXA1 , Hs04187555_m1; ErbB2 , Hs01001580_m1; GATA3 , Hs00231122_m1.

Techniques: Control, Labeling, RNA Sequencing, Expressing, Gene Expression

Journal: bioRxiv

Article Title: FOXA1 is required for ErbB2 expression and luminal differentiation in HER2-positive breast cancer

doi: 10.1101/2024.04.16.589460

Figure Lengend Snippet: Top enriched TF binding motifs identified by HOMER known motif analysis associated with down-regulated (A) or up-regulated (B) peak regions from ATAC-seq. C. Volcano plot showing the log2FC and adjusted P-values of all expressed genes (light grey) from the differential test between control and FOXA1KD-SKBR3 cells. The red dots represent TEAD family genes. Dots which have adjusted P-value 0 were marked with jittered value ± 5 at y = 300 for visualization. Blue dotted lines indicate |log2FC| = 2, adjusted P-value = 0.05. D. Western blots showing the indicated protein levels in control and FOXA1KD-SKBR3 cells. E. Donut charts showing the TEAD-binding sites mapping to functional genomic regions according to Homer gene annotation. F. Heatmap representing the increased enriched motif signal in control and FOXA1KD-SKBR3 cells. ATAC-seq reads against TEAD family binding sites. G. Dot plot showing the Hallmark ORA performed for 245 genes that enriched in TEAD family binding motif in FOXA1KD-SKBR3 cells compared to control. H. Volcano plot showing the log2FC and adjusted P-values of all genes (light grey) from the differential test between control and FOXA1KD-SKBR3 cells. The red dots represent enriched genes of Hallmark_EMT ORA. Blue dotted lines indicate |log2FC| = 2, adjusted P-value = 0.05. I. Immunofluorescence images of pan-TEAD or YAP in breast cancer cell lines. Scale bars is 10μm. J. Proximity Ligation Assay (PLA) assay of TEAD/YAP in MCF10A, SKBR3, and FOXA1KD-SKBR3 cells. Nuclei stained with DAPI (blue). Scale bars is 10μm. K. Genome tracks of TRPS1 gene showing the identified peaks over pre-defined promoter region (H3K4Me3) and enhancer (H3K27Ac). The screenshot was generated using the UCSC Genome Browser or IGV. L. Volcano plot showing the log2FC and adjusted P-values of all genes (light grey) from the differential test between control and FOXA1KDSKBR3 cells. The blue dots represent TRPS1 gene. Blue dotted lines indicate |log2FC| = 2, adjusted P-value = 0.05. M. Western blots showing the TRPS1 protein levels in control and FOXA1KD-SKBR3 cells. N. Immunofluorescence images of TRPS1in MCF10A, SKBR3, and FOXA1KD-SKBR3 cells. Nuclei stained with DAPI (blue). Scale bars is 10μm. O. Proximity Ligation Assay (PLA) assay of TRPS1/YAP in MCF10A, SKBR3, and FOXA1KD-SKBR3 cells. Nuclei stained with DAPI (blue). Scale bars is 10μm. P. UMAP of basal and luminal subtypes breast tumor cells were represented (upper panel) and for TRPS1 (middle panel). Boxplot showing the expression level of TRPS1 among breast cancer subtype in scRNA-seq data (bottom panel). Q. Working Model explaining the role of FOXA1 in ErbB2 expression and in luminal characteristics in HER2-positive breast cancer.

Article Snippet: Quantitative RT-PCR was performed with the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) using a Step One Plus Real- Time PCR System (Applied Biosystems) and the following TaqMan primer sets: FOXA1 , Hs04187555_m1; ErbB2 , Hs01001580_m1; GATA3 , Hs00231122_m1.

Techniques: Binding Assay, Control, Western Blot, Functional Assay, Immunofluorescence, Proximity Ligation Assay, Staining, Generated, Expressing