Journal: Molecular Therapy Oncology

Article Title: HIF1A, BRG1, and p300 interaction confers paclitaxel-induced drug resistance by enabling the overexpression of ABCC genes

doi: 10.1016/j.omton.2025.201049

Figure Lengend Snippet: HIF1A-EP300-BRG1 functional crosstalk on the chromatin defines transcription of ABCC transporters in PTX-resistant breast and lung cancer cells (A–D) HIF1A is responsible for overexpression of ABCC3, ABCC5 and ABCC10 in PTX-resistant MDA-MB-231 and A549 cells. (A and B) mRNA level of ABCC3 , ABCC5 , and ABCC10 was compared in cells 72 h after their transfection with siCTRL or siHIF1A, siMAF, and siISL1 by real-time PCR in PTX-resistant MDA-MB-231 (A) and A549 (B) cells. Raw values were normalized first to housekeeping genes ( ACTB , GAPDH , and HPRT1 ), and then the ratio was assumed as 1 in control sample. The difference between two means was tested with Student’s t test, and statistically significant differences are marked as follows: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (C and D) Effect of HIF1A, MAF, and ISL1 transient silencing on ABCC3 and ABCC10 protein level was studied by western blot in PTX-resistant MDA-MB-231 (C) and A549 (D) cells. Histone H3 was used as a loading control. (E) The expression and intracellular localization of ABCC5 and ABCC10 in HIF1A proficient and deficient MDA-MB-231-PTX cells were analyzed by immunocytostaining followed by confocal microscopy. Green fluorescence corresponds to ABCC5 and ABCC10, DNA was stained with DAPI (blue), whereas lysosomes with LysoTracker (red). (F and G) The green fluorescence intensity, which corresponds to intracellular ABCC5 and ABCC10 protein abundance (F), and colocalization between ABCC5 or ABCC10 and lysosomes (G) were determined in arbitrary units (a.u.) with Leica Application Suite X. The difference between two means was tested with Student’s t test, and statistically significant differences are marked as follow: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (H) HIF1A causes lysosomal sequestration of PTX Oregon Green in lysosomes of PTX-resistant cancer cell lines. Colocalization of fluorescently labeled PTX and lysosomes was compared between HIF1A proficient and deficient cells. PTX is marked in green (Oregon Green 488), lysosomes in red (LysoTracker Deep Red), DNA in blue (DAPI). (I) The colocalization was determined in a.u. with Leica Application Suite X. The difference between two means was tested with Student’s t test, and statistically significant differences are marked as follows: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (J) The toxicity of anticancer therapeutics was compared in HIF1 proficient (siCTRL) and deficient (siHIF1A) cells. Cells were transfected 24 h before administration of drugs for the following 48 h. Cell viability was quantified with resazurin-based assay. Cell viability with drug alone/in control, drug untreated cells was assumed as 100%. (K) The impact of HIF1A silencing (siHIF1A), iABC (MK571, 25 μM) and their combination on PTX- or doxorubicin-induced apoptosis (0.25 μM for each drug) was compared by measuring caspase 3/7 activity with Caspase-Glo 3/7 Reagent. Caspase 3/7 activity was normalized to the relative fraction of living cells, which were quantified with resazurin assay.

Article Snippet: Non-small-cell lung cancer cell line A549 was purchased from ATCC.

Techniques: Functional Assay, Over Expression, Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot, Expressing, Confocal Microscopy, Fluorescence, Staining, Quantitative Proteomics, Labeling, Resazurin Assay, Activity Assay

Journal: Molecular Therapy Oncology

Article Title: HIF1A, BRG1, and p300 interaction confers paclitaxel-induced drug resistance by enabling the overexpression of ABCC genes

doi: 10.1016/j.omton.2025.201049

Figure Lengend Snippet: BRG1 and p300 associate with HIF1A and are enriched at HRE motifs in promoters of transcriptionally active ABCC genes (A) The heatmap distribution of H3K4me3, p300, and BRG1 in the window of 2.5 kbp around HIF1A binding motif (HRE) at the gene promoters in non-resistant and PTX-resistant MDA-MB-231. (B) The occurrence of H3K4me3, p300, and BRG1 at the promoters characterized by HIF1A bingeing motif with respect to PTX resistance. Histone modification and proteins were counted by computing overlapping intervals of their enriched regions in the genome and HRE-positive promoters. (C) The abundance of the mapped reads of the three considered chromatin features around HIF1A binding motif (±1 kbp) and at the MACS2-derived peaks of H3K4me3, p300, and BRG1 in non-resistant and PTX-resistant cells. (D) HIF1A physically interacts with EP300 and BRG1 in PTX-resistant A549 and MDA-MB-231 cells. HIF1A, EP300 and BRG1 were detected by western blot in HIF1A co-immunoprecipitates. IgG served as an isotypic control. (E) Diagrams presenting HIF1A binding motif (“KACGTGS”) at the promoters of ABCC10 , ABCC5 , and ABCC3 with respect to their transcription start site (TSS). (F) Column chart presents mapped reads of H3K4me3, p300, and BRG1 counted on HRE motif (±1 kbp) the promoters of ABCC10 , ABCC5 , and ABCC3 in resistant and non-resistant cells. (G) HIF1A is enriched at the promoters of ABCC3, ABCC5, and ABCC10 in PTX-resistant A549 and MDA-MB-231 phenotypes. The abundance of HIF1A at the gene promoters was compared and quantified by ChIP-qPCR in basal and PTX-resistant cell lines. Raw values were normalized to IgG. The difference between two means was tested with Student’s t test, and statistically significant differences are marked as follows: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Non-small-cell lung cancer cell line A549 was purchased from ATCC.

Techniques: Binding Assay, Modification, Derivative Assay, Western Blot, Control, ChIP-qPCR

Journal: Molecular Therapy Oncology

Article Title: HIF1A, BRG1, and p300 interaction confers paclitaxel-induced drug resistance by enabling the overexpression of ABCC genes

doi: 10.1016/j.omton.2025.201049

Figure Lengend Snippet: HIF1A-p300-BRG1 co-occur on the chromatin in PTX-resistant breast and lung cancers (A and D) BRG1-HIF1A and EP300-HIF1A colocalize on the chromatin in PTX-resistant A549 and MDA-MB-231 cell lines as evidenced by immunostaining and confocal microscopy. (A) Alexa Fluor 488-labeled HIF1A is marked in green, Alexa Fluor 594-labeled BRG1 is marked in red, whereas DAPI-stained DNA is marked in blue. (D) Alexa Fluor 488-labeled p300 is marked green, Alexa Fluor 594-labeled BRG1 is marked in red, whereas DAPI-stained DNA is marked in blue. (B, C, E, and F) The colocalization between HIF1A and BRG1 (B and C) as well as HIF1A and p300 (E and F) were determined in arbitrary units (a.u.) with Leica Application Suite X. The difference between two means was tested with Student’s t test, and statistically significant differences are marked as follows: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (G) HIF1A and BRG1 colocalization in tumor xenograft (MDA-MB-231-PTX) slides was evidenced by immunostaining and confocal microscopy. Alexa Fluor 488-labeled HIF1A is marked in green and Alexa Fluor 594-labeled BRG1 is marked in red. (H) HIF1A silencing uncouples chromatin co-distribution of p300 and BRG1, which was analyzed using immunostaining and confocal microscopy in 2D cell culture. Alexa Fluor 488-labeled p300 is marked in green, Alexa Fluor 594-labeled BRG1 is marked in red, whereas DAPI-stained DNA is marked in blue. (I–L) The colocalization (I and J) between BRG1 and p300 and p300 abundance (K and L) were determined in a.u. with Leica Application Suite X. The difference between two means was tested with Student’s t test, and statistically significant differences are marked as follows: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (M) HIF1A and BRG1 as well as HIF1A and p300 co-silencing do not act synergistically and phenocopy the effect of single HIF1A silencing on transcription of ABCC3 in PTX-resistant MDA-MB-231 and A549 cells. mRNA level was compared in cells 72 h after their transfection with siCTRL, siHIF1A alone, or in combination with siSMARACA4 and siEP300 by real-time PCR in PTX-resistant MDA-MB-231 and A549 cells. Raw values were normalized first to housekeeping genes (ACTB, GAPDH, and HPRT1), and then the ratio was assumed as 1 in control sample. The difference between means was tested using one-way ANOVA or Kruskal-Wallis test, and statistically significant differences are marked as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (N and O) HIF1A prevents PTX accumulation in deeper layers of PTX-resistant MDA-MB-231 spheroids. Spheroids (21 days old) were transfected with siHIF1A or siCTRL or a combination with siHIF1A and siBRG1, and 72 h later, treated with PTX Oregon Green for 48 h. Pictures were taken with confocal microscope Leica TCS SP8 (Leica Microsystems, Germany). Green fluorescence corresponds to PTX, blue to DNA stained with DAPI, whereas red to lysosomes stained with LysoTracker Deep Red. The fluorescence intensity plot at spheroid cross section was determined in a.u. with Leica Application Suite X.

Article Snippet: Non-small-cell lung cancer cell line A549 was purchased from ATCC.

Techniques: Immunostaining, Confocal Microscopy, Labeling, Staining, Cell Culture, Transfection, Real-time Polymerase Chain Reaction, Control, Microscopy, Fluorescence

Journal: Biochemistry and Biophysics Reports

Article Title: Investigating the ABTS-based antioxidant potential and antiproliferative activity of pulp, skin and seeds extracts from Nephelium lappaceum , Manilkara zapota , and Meliccocus oliviformis on human prostate and colon cancer cells

doi: 10.1016/j.bbrep.2025.102257

Figure Lengend Snippet: Effects of the different fruit extracts on the proliferation of the colon cancer cell line HCT116 and the prostate cancer cell line DU145 . DU145 (A) or HCT116 (B) were seeded in a 12-well plate and allowed to grow for 12 h. Then, 500 μg/mLof PBS-resuspended extract derived from the pulp, seeds, and skin of Meliccocus oliviformis , Nephelium lappaceum , and Manilkara zapota were added and cell proliferation was monitored by analyzing the surface area occupied by the cells in each well (% confluence) from photos taken at regular intervals (every 3 h) by the Incucyte live cell imaging system over a period of three days. Data shown are the average ± SD of three independent experiments.

Article Snippet: The HCT116 (colic), and DU145 (prostatic) human cancer cell lines were purchased from ATCC (American Type Culture Collection).

Techniques: Derivative Assay, Live Cell Imaging

Journal: Biochemistry and Biophysics Reports

Article Title: Investigating the ABTS-based antioxidant potential and antiproliferative activity of pulp, skin and seeds extracts from Nephelium lappaceum , Manilkara zapota , and Meliccocus oliviformis on human prostate and colon cancer cells

doi: 10.1016/j.bbrep.2025.102257

Figure Lengend Snippet: Effects of the different fruit extracts on the viability of the colon cancer cell line HCT116 and the prostate cancer cell line DU145 . DU145 (A) or HCT116 (B) were seeded in a 12-well plate and allowed to grow for 12 h. Then, 500 μg/mL of PBS-resuspended extract derived from the pulp, seeds, and skin of Meliccocus oliviformis , Nephelium lappaceum , and Manilkara zapota were added. 24h later, cell viability was assessed using the MTT assay. Data shown are the average ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: The HCT116 (colic), and DU145 (prostatic) human cancer cell lines were purchased from ATCC (American Type Culture Collection).

Techniques: Derivative Assay, MTT Assay, Comparison

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine and thimerosal inhibit the formation of endogenous TopBP1 condensates. ( A ) Representative immunofluorescence images of TopBP1 foci in HCT116 cells incubated with quinacrine (40 µM) and/or SN-38 (300 nM) for 2 h, and ( B ) the corresponding quantification. ( C ) Representative immunofluorescence images of TopBP1 foci in HCT116 cells incubated with thimerosal (20 µM) and/or SN-38 (300 nM) for 2 h, and ( D ) the corresponding quantification. The experiment was repeated three times with similar results. Data from the three independent replicates were pooled and displayed as superplots in panels (B) and (D), error bars represent the standard error of the mean. ( E ) Representative immunofluorescence images of PML nuclear bodies in HCT116 cells incubated with quinacrine (40 µM) and/or SN-38 (300 nM) for 2 h and ( F ) the corresponding quantification. “−” denotes the absence of the indicated compound (control condition). CellProfiler 4.2.8 was used to quantify TopBP1 and PML nuclear foci (>300 cells analyzed per condition). Statistical significance was assessed using one-way ANOVA followed by Šídák’s post hoc test. ns, non-significant; * P -value <.05; **P-value <.01; and ****P-value <.0001. Scale bars, 10 µm for TopBP1 images and 20 µm for PML images.

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Immunofluorescence, Incubation, Control

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine and thimerosal inhibit ATR/Chk1 signaling activation in cell-free extracts and in living cells. ( A ) Immunoblot showing phosphorylated Chk1 (pChk1) levels after incubation of nuclear extracts from optoTopBP1-HEK293 cells with increasing concentrations of quinacrine (QC) or ( B ) thimerosal (TH) for 30 min before induction of TopBP1 condensation at 37°C. The experiments were reproduced three times for QC in panel (A) and once for TH in panel (B). ( C ) Fractionation experiment to monitor TopBP1 association with chromatin in HCT116 cells incubated with quinacrine (40 µM) and/or SN-38 (300 nM) for 2 h. The experiments were reproduced three times. ( D ) Immunoblot showing the effect of SN-38 and/or quinacrine on histone marks in HCT116 cells. H3K4me3, H3K9Ac, and H3K9me3 levels were assessed to evaluate changes in chromatin state. Representative immunofluorescence images of pChk1 signals obtained with a Celigo Imaging Cytometer in HCT116 cells incubated with SN-38 (300 nM) and increasing concentrations of quinacrine (4.4, 13.3, and 40 µM) ( E ) and the corresponding quantification ( F ) or increasing concentrations of thimerosal (2.2, 6.7, and 20 µM) for 2 h ( G ) and the corresponding quantification ( H ). Error bars represent the standard deviation from three independent experiments; ns, non-significant; * P -value <.05; ** P- value <.01; and **** P -value <.0001 (one-way ANOVA; SN-38 alone versus SN-38 with increasing doses of quinacrine or thimerosal).

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Activation Assay, Western Blot, Incubation, Fractionation, Immunofluorescence, Imaging, Cytometry, Standard Deviation

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine and thimerosal effects on RPA and H2AX phosphorylation. Immunoblots to assess the phosphorylation (p) level of the indicated ATR substrates in HCT116 cells incubated with SN-38 (300 nM) and/or quinacrine (4.4, 13.3, and 40 µM) ( A ) or thimerosal (2.2, 6.7, and 20 µM) ( B ) for 2 h and the corresponding quantifications ( C, D ) displayed as fold change over vinculin, normalized to non-treated cells. Bars represent mean ± standard error of the mean from two biological replicates. Two-dimensional flow cytometry analysis of pRPA32 (Ser33)/DAPI ( E ) and yH2AX (Ser139)/DAPI ( F ) fluorescence signals by flow cytometry after the extraction of soluble proteins from HCT116 cells, incubated with SN-38 (300 nM) and/or quinacrine (40 µM) or thimerosal (20 µM), as indicated.

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Phospho-proteomics, Western Blot, Incubation, Flow Cytometry, Fluorescence, Extraction

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine in combination with FOLFIRI displays additive and synergistic effects in 2D and 3D spheroid cell culture models (next page). Viability matrix (blue) and synergy matrix (red) of human HCT116 ( A ) and murine CT26 cells ( B ) incubated with increasing concentrations of quinacrine and FOLFIRI. FOLFIRI concentration for HCT116 cells: 5FU (from 0.009 to 0.148 µM), SN-38 (from 0.077 to 1.235 nM); and for CT26 cells: 5FU (0.083 to 1.333 µM), SN-38 (from 0.694 to 11.11 nM). Quinacrine concentration from 0.188 to 3 µM. Cell viability was assessed with the SRB assay (2D). Viability matrix (green) and cytotoxicity matrix (orange) of HCT116 ( C ) and CT26 ( D ) cells incubated with the same quinacrine and FOLIFRI concentrations; values were obtained with the “Dead + Total” cell viability application of the Celigo Imaging Cytometer (Nexcelom) using Hoechst/propidium iodide staining. Viability and synergy matrices of 3D spheroid models of HCT116 ( E ) and CT26 ( F ) cells (same quinacrine and FOLIFRI concentrations as before); viability was measured with the 3D-CellTiterGlo assay. Viability and synergy matrices of the SN-38-resistant HCT116-SN6 (SN6) ( G ) and HCT116-SN50 (SN50) ( H ) cell lines in 2D cultures; FOLFIRI: 5FU (from 0.083 to 1.133 µM and from 0.75 to 12 µM), SN-38 (from 0.694 to 11.11 nM and from 6.25 to 100 nM); quinacrine: from 0.188 to 3 µM (for both cell lines). Viability and synergy matrices of the SN-38-resistant HCT116-SN6 (SN6) ( I ) and HCT116-SN50 (SN50) ( J ) cell lines in 3D spheroid cultures; FOLFIRI: 5FU (from 0.0625 to 0.08 µM and from 0.25 to 4 µM), SN-38 (from 0.52 to 8.33 nM and from 2.08 to 33.33 nM); quinacrine: from 0.188 to 3 µM (for both cell lines). Cell viability was quantified with the 3D-CellTiterGlo assay. Representative brightfield images of spheroids were obtained with a Celigo imaging cytometer (Nexcelom). The synergy matrix was calculated as described in the “Materials and methods” section. Cells were incubated for 96 h (2D culture) and for 7 days (3D cultures).

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Cell Culture, Incubation, Concentration Assay, Sulforhodamine B Assay, Imaging, Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: Long non-coding RNA enhances SARS-CoV-2-mediated apoptosis through epigenetic repression of angiotensin-converting enzyme 2

doi: 10.1016/j.jbc.2025.110812

Figure Lengend Snippet: SARS-CoV-2 and other coronaviruses consistently upregulate EAS1. A , RNA-seq analysis of EAS1 expression in Caco-2 cells infected with SARS-CoV-2 and Calu-3 cells infected with SARS-CoV at the indicated time points (n = 2 biologically independent samples). B , EAS1 expression in Calu-3, A549, and ACE2-overexpressing A549 (ACE2-A549) cells 24 h post-infection (hpi) with SARS-CoV-2 (n = 3). C , EAS1 levels in ACE2-A549 cells infected with SARS-CoV-2 (MOI = 0.1) for 24 h (n = 3). D , EAS1 expression in ACE2-A549 cells 24 hpi with SARS-CoV-2 (USA-WA1/2020) (n = 3). E , EAS1 levels in a blood vessel-liver organoid co-culture model 4 days post SARS-CoV-2 infection (n = 3). F , Time course of EAS1 expression in Calu-3 cells infected with SARS-CoV (Delta ORF6-mutant, MOI = 5) (n = 3). G , EAS1 expression in Calu-3 2B4 cells infected with MERS-CoV (HCoV-EMC) (n = 3). H , EAS1 levels in MRC5 cells infected with HCoV-229E (n = 3). I , EAS1 expression in A549 cells under hypoxia (1%O 2 ) for 42 h (n = 3). J , qPCR analysis of EAS1 in HepG2, A549, and immortalized HUVEC cells under hypoxia (1% O 2 ) for 24 and 48 h (n = 4 biologically independent experiments, each performed in triplicate). K , qPCR analysis of EAS1 in primary human hepatocytes, AT II cells, and HUVECs from four distinct donors under hypoxia (1% O 2 ) for 24 h (n = 3 independent experiments, each in triplicate). Data are mean ± SD. p values were determined by a two-tailed paired Student's t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Human Hepatoma cell line HepG2 (ATCC HB-8065) and human lung cancer cell line A549 (ATCC CCL-185) were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11995065) containing 10% fetal bovine serum (FBS, Biowest, S1810).

Techniques: RNA Sequencing, Expressing, Infection, Co-Culture Assay, Mutagenesis, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Long non-coding RNA enhances SARS-CoV-2-mediated apoptosis through epigenetic repression of angiotensin-converting enzyme 2

doi: 10.1016/j.jbc.2025.110812

Figure Lengend Snippet: HIF-1α mediates the induction of EAS1 by SARS-CoV-2. ( A ) HIF1A mRNA levels in A549 and ACE2-A549 cells 24 hpi with SARS-CoV-2 (n = 3); ( B ) HIF1A expression in ACE2-A549 cells infected with SARS-CoV-2 (USA-WA1/2020) for 24 h (n = 3); ( C ) HIF1A levels in Calu-3 cells infected with SARS-CoV-2 or SARS-CoV (n = 2); ( D ) HIF1A expression in ACE2-A549 cells infected with SARS-CoV-2 (MOI = 0.1) for 24 h (n = 3); ( E ) Pearson correlation analysis of HIF1A and EAS1 expression across human tissues from the GTEx database, results are shown as (Pearson Correlation Coefficient, p -value); ( F ) Western blot analysis of HIF-1α protein levels after knockdown (KD) or overexpression (OE) in HepG2, A549, and immortalized HUVEC cells (n = 3 independent experiments, each in twice); ( G ) qPCR analysis of EAS1 expression (n = 3, each in triplicate); ( H ) Enrichment levels of H3K27ac and H3K4me1 at EAS1 genomic loci and transcriptional activity analysis, data were extracted from the UCSC Genome Browser (GRCh38/hg38 assembly); ( I ) DNA-binding motif of HIF1A from the JASPAR database; ( J ) Schematic of the HIF1α-binding site within the first intron of EAS1, indicating locations for ChIP-qPCR primers and CRISPRi and CRISPRa sgRNA; ( K ) ChIP-qPCR analysis of HIF1α binding at the EAS1 enhancer following HIF1A KD or OE (n = 3, each in triplicate); ( L ) qPCR analysis of EAS1 expression after targeted epigenetic repression or activation (dCas9-VP64 or dCas9-KRAB) of the enhancer region (n = 3, each in triplicate). Data are mean ± SD. p values were determined by a two-tailed paired Student's t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Human Hepatoma cell line HepG2 (ATCC HB-8065) and human lung cancer cell line A549 (ATCC CCL-185) were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11995065) containing 10% fetal bovine serum (FBS, Biowest, S1810).

Techniques: Expressing, Infection, Western Blot, Knockdown, Over Expression, Activity Assay, Binding Assay, ChIP-qPCR, Activation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Long non-coding RNA enhances SARS-CoV-2-mediated apoptosis through epigenetic repression of angiotensin-converting enzyme 2

doi: 10.1016/j.jbc.2025.110812

Figure Lengend Snippet: Inhibition of EAS1 attenuates SARS-CoV-2 infection-related apoptosis. A-B , flow cytometry analysis of apoptosis (Annexin V/PI staining) in HepG2, A549, and immortalized HUVEC cells treated with TNFα (HepG2-20ng, A549/immortalized HUVEC-10ng) for 24 h after EAS1 KD (n = 3). C-F , Western blot analysis of cleaved PARP1 and cleaved caspase 3 in EAS1 KD cells treated with TNFα (HepG2-20ng, A549/immortalized HUVEC-10ng), LPS (20 μg), or hypoxia (1%O 2 ) for 24 h (n = 3). G-K , Western blot analysis of apoptosis markers in primary human hepatocytes, AT II cells, and primary HUVECs with EAS1 KD under TNFα treatment for 24 h (representative of n = 1 experiment). L-O , Western blot analysis of apoptosis markers in EAS1 KD cells upon concomitant ACE2 KD and TNFα treatment (n = 3). Data are mean ± SD. Statistical significance was determined by a two-tailed paired Student's t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Human Hepatoma cell line HepG2 (ATCC HB-8065) and human lung cancer cell line A549 (ATCC CCL-185) were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11995065) containing 10% fetal bovine serum (FBS, Biowest, S1810).

Techniques: Inhibition, Infection, Flow Cytometry, Staining, Western Blot, Two Tailed Test