Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of differentiation heterogeneity in the OCI-AML8227 cell line model. This cell line produces leukemic blasts expressing immature myeloid markers (CD34) as well as differentiated monocytic markers (CD64 and CD14). CD38 is a transient myeloid differentiation marker, which is initially expressed in early myeloid progenitor cells. b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 96 hours along an 8-point dose curve with venetoclax. Cell viability was assessed by CellTiter Aqueous colorimetric assay. c. AUC values from the dose-response curves for each immunophenotypic population. Significance was evaluated using a two-tailed t-test. d–i. Cell surface expression of CD34, CD38, CD64, and CD14 in OCI-AML8227 cells cultured in triplicate for 72 hours with 1 μM venetoclax or an equivalent volume of DMSO. Cells were analyzed either as immunomagnetically fractionated populations (CD34-enriched and CD34-depleted) or as unfractionated cells. Significance was evaluated using ordinary one-way ANOVA followed by Holm-Šidák post-test correction. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Marker, Cell Culture, Colorimetric Assay, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a, b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 30 minutes or 6 hours following treatment with 1 μM venetoclax or an equivalent volume of DMSO. Proteins were extracted from cell pellets and either separated by liquid chromatography and analyzed by tandem mass spectrometry for protein identification and quantification or enriched for phosphorylated species prior to LC–MS/MS analysis to profile phosphorylation-dependent signaling. Relative (a) global protein and (b) phosphoprotein abundance at 6 hours following venetoclax treatment in CD34-enriched and CD34-depleted populations were analyzed using proteomic network analysis.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Cell Culture, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Predicted transcription factor activity was evaluated in single-cell RNA-seq data presented in . Significantly enriched transcription factors were identified and denoted as enriched in progenitor (blue), monocytic (gold), or both (green) populations. Gray dots represent transcription factors that did not reach statistical significance (FDR < 0.05). b. Predicted activity of PU.1 visualized by each time point and drug condition. c. Pseudotime values generated in were used to determine each cell’s relative position along the myeloid differentiation trajectory. Predicted activity of PU.1 is visualized along its pseudotime trajectory in each drug condition. d. OCI-AML8227 cells were cultured with DMSO or 1 μM venetoclax and processed at either 0 or 72 hours for single-cell ATAC-seq. Differential peaks were identified from open chromatin regions in each condition. Significant peaks were compared between monocytic and progenitor clusters. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). e, f. Transcription factor motif analysis was performed on (e) upregulated and (f) downregulated regions in monocytic cells relative to progenitor cells as described in panel d. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). g. Venn diagram displaying the overlap of significantly dysregulated transcription factors identified from single-cell RNA-seq analysis in panel a compared to enriched motifs identified in panels e and f.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Activity Assay, RNA Sequencing, Generated, Cell Culture

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of the targeted CRISPR screen. A stable Cas9-expressing OCI-AML8227 cell line was generated by electroporation and sorted for GFP expression. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression. Image was created with BioRender. b. Live cell counts determined by forward/side scatter gating, excluding DAPI-stained cells. Counts were normalized to the average of their respective DMSO-treated controls. Blue lines and asterisks indicate comparisons with the safe-harbor locus AAVS1 (control) whereas black lines and asterisks indicate intra-sample comparisons. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. c–h. Live cell counts for each immunophenotypic population, normalized as described in panel b. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. i–j. Representative flow cytometry plots showing CD64-FITC and CD38-APC-Fire surface expression in OCI-AML8227 cells transduced with guide RNAs targeting AAVS1 or SPI1 following treatment with DMSO or 1 μM venetoclax. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: CRISPR, Expressing, Generated, Electroporation, Transduction, Cell Culture, Flow Cytometry, Staining, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression as described in . Quantification of total cell counts in additional knockout models of OCI-AML8227 cells. Total cell counts were normalized to the average of their respective DMSO-treated controls. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Sensitivity to venetoclax was unchanged by the experimental knockouts compared to control AAVS1 . b–g. Live cell counts for each immunophenotypic population were calculated by multiplying their proportions of live single cells from flow cytometric analysis by their normalized values in panel a. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Few changes were observed in knockouts relative to control. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Transduction, Electroporation, Cell Culture, Flow Cytometry, Expressing, Knock-Out, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. OCI-AML8227 cells were cultured in triplicate for 72 hours along an 8-point dose curve of DB2313. Cell viability was assessed by CellTiter Aqueous colorimetric assay. b. OCI-AML8227 cells were treated in triplicate with an 8×8 dose matrix of DB2313 and venetoclax for 72 hours prior to viability assessment by CellTiter Aqueous colorimetric assay. Zero interaction potency (ZIP) synergy scores were calculated on the average values for each drug dose. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. c–e. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 0.1 μM venetoclax, 0.5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Live cell counts were determined by forward/side scatter gating and exclusion of DAPI-stained cells, then normalized to DMSO-treated controls. Cells were analyzed by flow cytometry for CD34, CD38, CD64, and CD14 surface expression. Quantification of live cell counts for the remaining cell surface markers is shown in . Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. f–h. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 1 μM venetoclax, 5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Analysis was performed as described in panels c–e. i. Transcriptional signatures of nine primary AML samples selected for drug sensitivity evaluation are shown. One sample was excluded from downstream analyses due to widespread cell death (18-00105). j. Primary AML blasts from eight patients with stem-monocytic AML were cultured in triplicate for 72 hours along a 7-point dose curve with venetoclax, DB2313, or equimolar amounts of the drug combination. Viability was assessed using the Guava/EMD Millipore platform after a short incubation with Guava Nexin Reagent (Annexin V–PE + 7-AAD). ZIP synergy scores were calculated from averaged viability data across replicates for each drug dose in primary AML blasts shown in panel i. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. k. Venetoclax dose-response curves for each patient at a fixed dose of 1.25 μM DB2313, which corresponds to maximal synergy in panel j. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Cell Culture, Colorimetric Assay, Staining, Flow Cytometry, Expressing, Incubation

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: p38 MAPK inhibition decreases cell proliferation and survival in prostate cancer cells. ( a ) Western blots from DU145, PC3, VCaP, LNCaP, V16D and MR49F cells showing AR expression. ( b ) Indicated cell lines were treated with 10 µM SB203580 (SB) under normoxia (21% O 2 ) (left) or hypoxia (0.2% O 2 ) (right) for 150 h. Confluency was measured with the IncuCyte Live Cell Imaging system after confirming proportionality to cell numbers. Each data point represents an independent experiment, bars represent the mean value ± S.E.M. ( c ) V16D cells were transfected with siRNA targeting MAPK11, MAPK14, or non-targeting negative control (NTC). Expression of target genes was assessed by qPCR (left) or western blotting (right). ( d ) Cells as in (c) were placed in normoxia (21% O 2 ) or hypoxia (0.2% O 2 ) for 72 h and confluency was measured with the IncuCyte Live Cell Imaging system. ( e ) V16D cells were treated with various doses of SB203580 (5, 10, 20 µM) for 72 h under normoxia (21% O 2 ) or hypoxia (0.2% O 2 ). Single cells were seeded for clonogenic survival in triplicate and surviving fraction calculated from colony formation 14 days later. Data points represent independent experiments and bars represent the mean value ± S.E.M. (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Inhibition, Western Blot, Expressing, Live Cell Imaging, Transfection, Negative Control

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: p38 MAPK inhibition decreases Hsp27 phosphorylation, AR activity and expression of AR target genes under normoxia and hypoxia. ( a ) Prostate cancer cell lines (DU145, PC3, VCaP, LNCaP, V16D, MR49F) were treated with 10 µM SB203580 for one hour and subjected to western blotting (left). V16D cells were treated with 25 µM of anisomycin for one hour as a positive control for p38 MAPK activation. Densitometry analysis was performed to calculate fold change of p-Hsp27 (Ser82) relative to Hsp27 for each cell line. ( b ) V16D cells were transfected with a plasmid encoding luciferase driven by the AR promoter. 24 h later, cells were treated with 1 nM DHT, 10 µM SB203580, or 10 µM enzalutamide (Enza) in 10% charcoal treated (CT)-FBS media (androgen depleted) and incubated under normoxia (21% O 2 ) or hypoxia (0.2% O 2 ) for 48 h. Relative light units (RLU) from luciferase was measured after addition of luciferin. ( c–e ) V16D cells were treated with 10 µM SB203580, or 10 µM enzalutamide for 48 h and relative mRNA expression of KLK3 ( c ), NKx3.1 ( d ), and FKBP5 ( e ) was measured by RT-qPCR normalized to the average of HPRT1 and GUSB gene expression. Fold change was calculated relative to the 10% CT-FBS negative normoxia control. Data points represent an independent experiment and bars represent the mean value ± S.E.M. (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Inhibition, Phospho-proteomics, Activity Assay, Expressing, Western Blot, Positive Control, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Incubation, Quantitative RT-PCR, Gene Expression, Control

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: Androgen and hypoxia activate p38 MAPK and Hsp27. ( a ) V16D cells were exposed to 0 nM, 1 nM, or 10 nM DHT in charcoal-stripped media for 6 h in normoxia (21% O 2 ) or hypoxia (0.2% O 2 ). 25 µM of anisomycin for one hour was used as a positive control for p38 MAPK activation. Total cell lysates were subjected to western blotting. Densitometry analysis of protein bands was performed to calculate fold change of p-p38 (Tyr182) ( b ) and p-Hsp27 (Ser82) ( c ) relative to normoxia control with 0 nM DHT and normalized to eIF4E. Data points represent independent experiments and bars represent the mean value ± S.E.M. (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Positive Control, Activation Assay, Western Blot, Control

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: p38 MAPK inhibition prolongs survival of mice bearing CRPC xenografts. Subcutaneous V16D xenografts were established and treatment with vehicle or SB203580 (10 mg/kg) started when tumors reached 200 mm 3 , in a 5 days on and 2 days off schedule until endpoint. Kaplan-Meier survival curves are shown for the time to reach tumor volume endpoint (1000 mm 3 ) from treatment start. Tumor growth curves from individual mice are shown in .

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Inhibition

Journal: Cancer immunology research

Article Title: Soluble CD80 protein delays tumor growth and promotes tumor infiltrating lymphocytes

doi: 10.1158/2326-6066.CIR-17-0026

Figure Lengend Snippet: A, CD80-Fc induces transcription of EGR1/2/3/4. CD3+ cells were isolated by negative selection from the blood of healthy human donors, activated with PHA for 72 hours, and then either untreated or incubated for two hours with isotype control mAb, agonist CD28 mAb, TROY-Fc, or CD80-Fc. RNA was then isolated, converted into cDNA, and the DNA analyzed by qRT-PCR for EGR1/2/3/4. Values were normalized to β-actin expression. Results are shown as fold change in expression level compared to untreated T cells. Statistical analysis was performed using Student’s t test. Data are from one of three independent experiments. B, CD3+CD28+ Jurkat cells were treated for 30 min at 37OC with isotype mAb, agonist CD28 mAb, TROY-Fc, or CD80-Fc. Cells were then fixed and permeabilized, and stained with mAbs to pMAPK or pNF-κB followed by anti-rabbit IgG-FITC. C, Jurkat cells were untreated or treated for 30 min with agonist anti-CD28 mAb and then lysed and analyzed by western blot for phosphorylated MAPK and NFκB. Data for B and C are representative of one of two and one of three independent experiments, respectively.

Article Snippet: Membranes were blocked with 5% milk in TBST. pNF-κB and pMAPK were detected with anti-pNF-KB antibody (clone 93H1; 1:1000 in 10ml of 2.5% milk/TBST) or anti-pMAPK antibody (clone D13.14.4E; 1:2000 in 10ml of 2.5% milk/TBST) (both from Cell Signaling) followed by goat-anti-rabbit-HRP (Biolegend; 1:10000 in 10ml of 2.5% milk/TBST).

Techniques: Isolation, Selection, Incubation, Control, Quantitative RT-PCR, Expressing, Staining, Western Blot

Journal: Oncology Reports

Article Title: Hypoxia induces radioresistance, epithelial-mesenchymal transition, cancer stem cell-like phenotype and changes in genes possessing multiple biological functions in head and neck squamous cell carcinoma

doi: 10.3892/or.2022.8269

Figure Lengend Snippet: Top significantly dysregulated associated genes a .

Article Snippet: The expression of hypoxia-responsive genes was analyzed with a panel of TaqMan ® Gene Expression assays [cadherin 1 (CDH1; Hs01023895_m1); cadherin 2 ( CDH2 ; Hs00983056_m1); vimentin (VIM; Hs00958111_m1); fibronectin 1 (FN1; Hs01549976_m1); FOXC2 (Hs00270951_s1); TWIST1 (Hs04989912_s1); CD44 (Hs01075864_m1); SOX2 (Hs04234836_s1); NANOG (Hs02387400_g1); GLUT3 (Hs00359840_m1); CA9 (Hs00154208_m1); caspase 14 ( CASP14 ; Hs00201637_m1); serpin family E 1 ( SERPINE1 ; Hs00167155_m1); lysyl oxidase ( LOX ; Hs00942480_m1); amphiregulin ( AREG ; Hs00950669_m1); epiregulin ( EREG ; Hs00914313_m1); all labelled with FAM and purchased from Thermo Fisher Scientific, Inc.] and amplified using a TaqMan real-time PCR protocol according to manufacturer's instructions (Thermo Fisher Scientific, Inc.).

Techniques: Sequencing, Binding Assay

Journal: Oncology Reports

Article Title: Hypoxia induces radioresistance, epithelial-mesenchymal transition, cancer stem cell-like phenotype and changes in genes possessing multiple biological functions in head and neck squamous cell carcinoma

doi: 10.3892/or.2022.8269

Figure Lengend Snippet: Validation of microarray analysis. Reverse transcription-quantitative PCR analysis was used to analyze the mRNA expression levels of hypoxia-responsive genes in the LK0858, LK0863 and UT-SCC-14 cells using microarray. The relative amounts of SERPINE1, CA9, CASP14, LOX, GLUT3, AREG and EREG mRNA was calculated using the 2 −ΔΔCq method and amplification of both GAPDH and β-actin were used as an internal standard. The cumulative, relative mRNA levels from the LK0858, LK0863 and UT-SCC-14 cells are presented as the mean ± SD relative to cells cultured in normoxia. *P<0.05. The data was analyzed using an unpaired Student's t-test. SERPINE1, serpin family E 1; CA9, carbonic anhydrase 9; CASP14, caspase 14; LOX, lysyl oxidase; GLUT3, glucose transporter 3; AREG, amphiregulin; EREG, epiregulin.

Article Snippet: The expression of hypoxia-responsive genes was analyzed with a panel of TaqMan ® Gene Expression assays [cadherin 1 (CDH1; Hs01023895_m1); cadherin 2 ( CDH2 ; Hs00983056_m1); vimentin (VIM; Hs00958111_m1); fibronectin 1 (FN1; Hs01549976_m1); FOXC2 (Hs00270951_s1); TWIST1 (Hs04989912_s1); CD44 (Hs01075864_m1); SOX2 (Hs04234836_s1); NANOG (Hs02387400_g1); GLUT3 (Hs00359840_m1); CA9 (Hs00154208_m1); caspase 14 ( CASP14 ; Hs00201637_m1); serpin family E 1 ( SERPINE1 ; Hs00167155_m1); lysyl oxidase ( LOX ; Hs00942480_m1); amphiregulin ( AREG ; Hs00950669_m1); epiregulin ( EREG ; Hs00914313_m1); all labelled with FAM and purchased from Thermo Fisher Scientific, Inc.] and amplified using a TaqMan real-time PCR protocol according to manufacturer's instructions (Thermo Fisher Scientific, Inc.).

Techniques: Biomarker Discovery, Microarray, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Amplification, Cell Culture

Journal: Oncology Reports

Article Title: Hypoxia induces radioresistance, epithelial-mesenchymal transition, cancer stem cell-like phenotype and changes in genes possessing multiple biological functions in head and neck squamous cell carcinoma

doi: 10.3892/or.2022.8269

Figure Lengend Snippet: Effect of CA9, SERPINE1, AREG and EREG downregulation on the radiation response in head and neck squamous cell carcinoma cells. UT-SCC-14 cells were transiently transfected with either non-targeting siRNA or siRNA interfering with expression of (A) CA9, (B) SERPINE1, (C) AREG and (D) EREG. Cells transfected with either non-targeting siRNA or siRNA targeting CA9 or SERPNE1 were exposed to hypoxia for 24 h prior transfection and placed back under hypoxic (1% O 2 ) conditions after transfection. The protein expression was evaluated 48 h post-transfection using western blotting. β-actin was used as the loading control. In parallel experimental settings, cells transfected with CA9 or SERPINE1 siRNA were irradiated at 2, 4 or 6 Gy 24 h post-transfection, followed by re-exposure to hypoxic conditions, whereas cells transfected with AREG or EREG siRNA were exposed to normoxic conditions (21% O 2 ) after irradiation. The efficiency of AREG and EREG downregulation with specific siRNA was assessed via reverse transcription-quantitative PCR. After 9 days, the cytotoxic/cytostatic effect on cell proliferation was determined by a crystal violet assay. Cell proliferation is presented as the percentage of the untreated controls, and the data are presented as the mean ± SD from three experiments performed in triplicate. All results are shown as the mean ± SD. *P<0.05 vs. non-targeting siRNA. The data was analyzed either using one-way ANOVA followed by Bonferroni's multiple comparison test or an unpaired Student's t-test. SERPINE1, serpin family E 1; CA9, carbonic anhydrase 9; AREG, amphiregulin; EREG, epiregulin; siRNA, small interfering RNA; ns, not significant.

Article Snippet: The expression of hypoxia-responsive genes was analyzed with a panel of TaqMan ® Gene Expression assays [cadherin 1 (CDH1; Hs01023895_m1); cadherin 2 ( CDH2 ; Hs00983056_m1); vimentin (VIM; Hs00958111_m1); fibronectin 1 (FN1; Hs01549976_m1); FOXC2 (Hs00270951_s1); TWIST1 (Hs04989912_s1); CD44 (Hs01075864_m1); SOX2 (Hs04234836_s1); NANOG (Hs02387400_g1); GLUT3 (Hs00359840_m1); CA9 (Hs00154208_m1); caspase 14 ( CASP14 ; Hs00201637_m1); serpin family E 1 ( SERPINE1 ; Hs00167155_m1); lysyl oxidase ( LOX ; Hs00942480_m1); amphiregulin ( AREG ; Hs00950669_m1); epiregulin ( EREG ; Hs00914313_m1); all labelled with FAM and purchased from Thermo Fisher Scientific, Inc.] and amplified using a TaqMan real-time PCR protocol according to manufacturer's instructions (Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Expressing, Western Blot, Control, Irradiation, Reverse Transcription, Real-time Polymerase Chain Reaction, Crystal Violet Assay, Comparison, Small Interfering RNA

Journal: Cells

Article Title: Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder ( Paralichthys olivaceus ): Isolation, Culture Conditions, and Myogenic Protein Expression.

doi: 10.3390/cells12182325

Figure Lengend Snippet: Figure 6. Differentiation of OFMCs into myotubes. (a) Representative images of undifferentiated (myoblasts) and differentiated cells (myotubes) incubated with 2% horse serum. Magnification, 10×; Scale bar—100 µm. (b) MyoD, myogenin, and desmin protein expression levels in undifferentiated and differentiated cells. Results are means ± standard deviation of three independent experiments. ** p < 0.05 vs. corresponding control group (D-0). CON, control; D, day; UD, undifferentiation, D-0. (c) FITC-phalloidin and DAPI were used to visualize actin and nucleus staining, and the colocalization of phalloidin and DAPI staining is indicated in the merged images. Magnification 10×; scale bar—100 µm. green- cytoskeleton staining, blue-nucleus staining.

Article Snippet: The primary antibodies were MyoD (E-1) (Santa cruz cat number: sc-377186; mouse), myogenin (Santa cruz cat number: sc-12732; mouse), and desmin (Santa cruz cat number: sc-23879; mouse); anti-β-actin (Santa cruz cat number: sc-47778; mouse) antibody was used as the control.

Techniques: Incubation, Expressing, Standard Deviation, Control, Staining

Journal: Journal of Virology

Article Title: MERS-CoV and SARS-CoV-2 infection in diverse human lung organoid-derived cultures

doi: 10.1128/jvi.01098-25

Figure Lengend Snippet: Generation of donor-derived lung organoids (hLO), organoid-derived monolayers (hLOm), and ALI (hLO ALI) cultures. ( A ) Schematic of hLO generation and derivation of hLOm and hLO-ALI cultures, followed by infection with SARS-CoV-2 or MERS-CoV. Schematic created using BioRender. ( B ) Identification of lung cell markers. hLOm, hLO, and hLO ALI cells were fixed in 4% paraformaldehyde, permeabilized and stained with antibodies against acetylated tubulin (cilia), KRT5 (Basal Stem Cells, white arrows), Muc5AC (mucus and mucus producing cells, Goblet Cells), CC10 (Club Cells), ZO-1 (zona occludens-1, tight junctions), PDPN1 (podoplanin-1, AT-1 cells, white arrows), SFTPC (surfactant protein C, AT-2 cells), and DAPI (nuclei, blue). Cells were imaged on a confocal microscope using a 63× objective. Scale bars correspond to 50 µm. ( C ) Gene expression of lung markers FoxJ1 , TUBB4B , TJP1 , TP63 , KRT5 , NGRF , Muc5AC , SCGB1A1 , PDPN1 , AQP5 (aquaporin 5, AT-1 cells), SFTPC and SLC34A2 (AT-2 cells) in hLO, hLOm, and hLO ALI was determined by RT-qPCR assays. Samples were assayed in technical duplicates, dCt was normalized to GAPDH, and expression was calculated by 2 −dCt . The mean of three independent samples is presented, and error bars are standard error of the mean (SEM).

Article Snippet: Podoplanin PDPN1 (E-1) AF647 , Santa Cruz Biotechnology , SC-376695.

Techniques: Derivative Assay, Infection, Staining, Microscopy, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Cell death & disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis.

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: Fig. 6 An inverse correlation between ETV7 and TNFR1 in BC patients. Three samples from BC patients with invasive ductal carcinoma were analyzed by IHC. Tissues were stained with antibodies against ETV7 and TNFR1 proteins and studied at ×10 magnification. A ×20 magnification insert is shown for each image. Top panels: tissues from a HER2-positive BC; middle panel: tissues from a triple-negative BC; bottom panels: tissues from a luminal A BC.

Article Snippet: Then, the samples were diluted and incubated with 2 μg the appropriate antibody targeting ETV7 (Santa Cruz, TEL2, E-1), pSTAT3 (Cell Signaling Technologies, 124H6), H3K9me3 (Cell Signaling Technologies, 13969P), H3K4me3 (Abcam, ab8580), H3ac (Abcam, ab47919) or IgG (Santa Cruz Biotechnologies, mouse or rabbit according to the antibody used) and Dynabeads with protein G or A (Life Technologies) overnight at 4 °C in a rotator.

Techniques: Staining

Journal: Cell death & disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis.

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: Fig. 7 ETV7 can compete with STAT3 in the regulation of the TNFRSF1A gene influencing the NF-κB regulatory pathway. A The canonical STAT3/TNF-α/NF-κB regulatory pathway. STAT3 binds to its regulatory element in the first intron of the TNFRSF1A gene and induces its expression by recruiting chromatin remodelers that result in an “active” state. Consequently, the TNF-α receptor 1 is produced. TNF-α molecules bind the TNFR1 receptor and activate the NF-κB signaling pathway. B In the context where ETV7 expression is increased, ETV7 can displace STAT3 from its binding sites on the Intron 1 of TNFRSF1A and directly represses its expression by altering the deposition of histone marks. This ETV7-mediated repression leads to the reduced activation of NF-κB signaling and, hence, reduces the expression of pro- inflammatory genes.

Article Snippet: Then, the samples were diluted and incubated with 2 μg the appropriate antibody targeting ETV7 (Santa Cruz, TEL2, E-1), pSTAT3 (Cell Signaling Technologies, 124H6), H3K9me3 (Cell Signaling Technologies, 13969P), H3K4me3 (Abcam, ab8580), H3ac (Abcam, ab47919) or IgG (Santa Cruz Biotechnologies, mouse or rabbit according to the antibody used) and Dynabeads with protein G or A (Life Technologies) overnight at 4 °C in a rotator.

Techniques: Expressing, Produced, Binding Assay, Activation Assay