prostate epithelial cell growth kit  (ATCC)

Journal: bioRxiv

Article Title: Electrical Impedance Spectroscopy as a Tool to Detect the Epithelial to Mesenchymal Transition in Prostate Cancer Cells

doi: 10.1101/2024.09.29.615724

Figure Lengend Snippet: EIS experimental workflow for characterizing phenotype changes in PCCs. (1) Illustration of EMT in cancer cells. Initially, cells exhibit an epithelial phenotype characterized by the expression of E-cadherin and ZO-1. Then, the cells undergo downregulation of E-cadherin and ZO-1, transitioning to an intermediate phenotype. In this stage there is a shift in the protein expression profile with an upregulation of N-cadherin and vimentin leading to mesenchymal phenotype. (2) DU145, PC3, and LNCaP cells were obtained from cryogenic storage, thawed, and expanded in proliferation media. Cells were seeded with EMT-inducing media and allowed 5 days to incubate. (3) Cells were characterized using EIS and the 3DEP analyzer. A phenotype change was validated by a nuclei stain and immunofluorescence imaging. Figure created with Biorender.com .

Article Snippet: HPrECs were obtained from ATCC (PCS-440-010) and subcultured in prostate epithelial cell basal medium (ATCC, PCS-440-030) supplemented with prostate epithelial cell growth kit (ATCC, PCS-440-040) at 37°C in a humidified 5% CO 2 incubator.

Techniques: Expressing, Staining, Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: Electrical Impedance Spectroscopy as a Tool to Detect the Epithelial to Mesenchymal Transition in Prostate Cancer Cells

doi: 10.1101/2024.09.29.615724

Figure Lengend Snippet: Normalized EIS cell analysis of EMT treated PCCs. (A) Average spectra of PC3 EMT-cells and PC3 EMT+ cells. (B) Average spectra of DU145 EMT-cells and DU145 EMT+ cells. Average spectra of (C) PC3 EMT- and PC3 EMT+ cells and (D) DU145 EMT-and DU145 EMT+ cells compared to epithelial and mesenchymal controls. Average normalized impedance of (E) PC3 EMT-and PC3+ cells and (F) DU145 EMT-and DU145 EMT+ cells compared to epithelial and mesenchymal controls. Error bars represent standard error mean. n=3 for PC3, DU145, epithelial control, and mesenchymal control cells. Statistical analysis completed on pooled data sets; ** p < 0.05, *** p < 0.001, and **** p < 0.0001.

Article Snippet: HPrECs were obtained from ATCC (PCS-440-010) and subcultured in prostate epithelial cell basal medium (ATCC, PCS-440-030) supplemented with prostate epithelial cell growth kit (ATCC, PCS-440-040) at 37°C in a humidified 5% CO 2 incubator.

Techniques: Cell Analysis, Control

Journal: bioRxiv

Article Title: Electrical Impedance Spectroscopy as a Tool to Detect the Epithelial to Mesenchymal Transition in Prostate Cancer Cells

doi: 10.1101/2024.09.29.615724

Figure Lengend Snippet: Morphology assessment of EMT treated PCCs. Phase contrast images overlayed with Hoechst-stained nuclei of PC3, DU145, and LNCaP cells without (EMT-) and with EMT (EMT+) treatment. The white arrows indicate a representative cell exhibiting characteristic epithelial morphology under EMT-conditions and mesenchymal morphology under EMT+ conditions.

Article Snippet: HPrECs were obtained from ATCC (PCS-440-010) and subcultured in prostate epithelial cell basal medium (ATCC, PCS-440-030) supplemented with prostate epithelial cell growth kit (ATCC, PCS-440-040) at 37°C in a humidified 5% CO 2 incubator.

Techniques: Staining

Journal: bioRxiv

Article Title: Electrical Impedance Spectroscopy as a Tool to Detect the Epithelial to Mesenchymal Transition in Prostate Cancer Cells

doi: 10.1101/2024.09.29.615724

Figure Lengend Snippet: Immunofluorescent staining of PCCs without and with EMT treatment (EMT- and EMT+, respectively). The staining highlights the expression of epithelial marker E-cadherin and mesenchymal marker vimentin both tagged with fluorescent labels. Quantification of fluorescent intensity for each marker is provided in the bar charts. Error bars represent standard error mean. n=3 for all conditions. Statistical analysis completed on pooled data sets; ** p < 0.05, and **** p < 0.0001.

Article Snippet: HPrECs were obtained from ATCC (PCS-440-010) and subcultured in prostate epithelial cell basal medium (ATCC, PCS-440-030) supplemented with prostate epithelial cell growth kit (ATCC, PCS-440-040) at 37°C in a humidified 5% CO 2 incubator.

Techniques: Staining, Expressing, Marker

Journal: Cancers

Article Title: GABA(A) Receptor Activation Drives GABARAP–Nix Mediated Autophagy to Radiation-Sensitize Primary and Brain-Metastatic Lung Adenocarcinoma Tumors

doi: 10.3390/cancers16183167

Figure Lengend Snippet: GABA(A) receptor activation potentiates radiation. ( A ) Illustration of a human-relevant ex vivo ‘chip’ employed to test AM-101 and docetaxel (DTX) efficacy. Lung adenocarcinoma cancer cells (H1792-GFP, green) can be co-cultured with primary human alveolar and pulmonary endothelial cells and exposed to air (air–liquid interface) on-chip. Cancer cells form clusters that grow and spread through the epithelial compartment of the chip over time . ( B ) Testing of AM-101 and DTX ex vivo or ‘on-chip’ reveals that AM-101 is as cytotoxic as DTX but at a significantly lower concentration. The chip is a 3-D ex vivo model, and, to achieve the cytotoxicity that AM-101 generates in 5 μM concentration, DTX is required in 10 mM concentrations. To determine p -values between two groups, one-way ANOVA with Tukey’s multiple comparisons test was performed. ** p < 0.001 and *** p < 0.0001. Images acquired from chips were subjected to background signal removal and analysis using Fiji (Image J). To generate bar graphs, acquired images were evaluated through background subtraction and signal thresholding. Subsequently, particle analysis was performed using a Fiji plugin to estimate the number of GFP+ cells per field of view under each testing condition. ( C ) A clonogenic assay was employed to examine the radio-sensitizing effect of AM-101 in H1792 cells. The survival curves showing surviving fraction of H1792 cells following radiation exposure at two separate doses with and without AM-101. Cell cultures were treated with either AM-101 (2.5 μM) combined with two separate doses of radiation (3 Gy and 6 Gy) versus DMSO (vehicle) and two separate doses of radiation (3 Gy and 6 Gy). H1792 cells in culture were treated with AM-101 (2.5 μM) or DMSO (vehicle) 1 h before radiation and maintained in the medium after irradiation. According to the experimental design the media containing AM-101 or DMSO in all groups was replaced with fresh media 72 h after treatment. Colony-forming efficiency was determined 14 days later, and survival curves were generated. The vehicle in this experiment is DMSO, since DMSO is used as the solvent to solubilize AM-101. ( D ) Schematic of the efficacy experiment in H1792 subcutaneous heterotopic bilateral xenograft tumors generated in NSG mice. Mice in vehicle or drug treatment groups received i.p., vehicle, AM-101 (2.5 mg/kg), or DTX (8 mg/kg), on day 36 post-implantation and then six injections once per day. Mice in radiation (RT) or combo groups received a single fraction of radiation (5 Gy) to left flank only at 2 h before vehicle or drug on the first day of treatment. ( E ) At experimental endpoint, tumors from left (L) and right (R) flanks of each mouse were resected. H1792 subcutaneous xenograft tumor growth in NSG mice from different treatment groups: vehicle, radiation (RT), AM-101 ± RT, DTX ± RT. Number of mice per treatment group: n = 6 for vehicle, n = 4 for RT; n = 7 for AM-101 and n = 7 for AM-101 + RT, n = 5 for DTX + RT and n = 6 for DTX. ( F ) Tumor volume of left and right flank tumors was measured over time using Vernier calipers. The tumor growth delay curves show the tumor volumes of mice treated with a vehicle, radiation (RT), AM-101, and AM-101 plus RT. Each point on the curve represents the mean tumor volume after treatment, with error bars indicating the standard error (SE). Statistical significance is indicated by p < 0.001.

Article Snippet: BEAS-2B cells were grown in airway epithelial cell basal medium (ATCC, Manassas, FL, USA) supplemented with bronchial epithelial cell growth kit (ATCC).

Techniques: Activation Assay, Ex Vivo, Cell Culture, Concentration Assay, Particle Size Analysis, Clonogenic Assay, Irradiation, Generated, Solvent

Journal: bioRxiv

Article Title: PROS1 released by human lung basal cells upon SARS-CoV-2 infection facilitates epithelial cell repair and limits inflammation

doi: 10.1101/2024.09.11.612489

Figure Lengend Snippet: A. Formation of pseudostratified epithelium with tight junctions (yellow) and cilia (magenta). B. H&E staining of pseudostratified epithelial transverse section with cilia (white arrows); 20X zoomed in. C. PAS staining of the pseudostratified epithelium showing the mucin in red-purple (yellow arrows); 20X zoomed in. D. Lateral view of the 3D section of epithelium from confocal microscopy Z-stacks, showing TJP1 layer (yellow) and the cilia (magenta). E. Infection of the epithelial ALI cultures with different strains of SARS-CoV-2. The 0h represent the virus in the inoculum, the 2h represents the virus in the first wash of epithelium after incubation with the inoculum, and timepoints 24-96h measure the virus that was produced and released from the cells over time. F. UMAP visualization of 36,640 cultured epithelial cells. Each cell is represented by an individual point and is coloured by cluster identity. G. Split UMAP visualization to depict the differences in cluster distribution and TPPP3, TP63 and MUC5AC gene expression between in control vs SARS-Cov-2 infected epithelia. The clusters that were most affected by the SARS-CoV-2 were basal and secretory cells, as shown by the expression of TP63 (basal cell marker) and MUC5AC (secretory cell marker). H. Split UMAP visualization of ALI cultured epithelia (n= 36,640 cells) data integrated with published airway epithelia dataset (n = 63,319 cells) with samples from nasal, tracheal and bronchial epithelium of healthy donors and patients with mild to severe SARS-CoV-2 infection (Yoshida et al 2022).

Article Snippet: Human primary bronchial/tracheal epithelial cells (ATCC, PCS-300-010; Donor: Hispanic/latino man, 14 years old), passage 2, were expanded in T25 flasks in Airway Epithelial Cell Basal Medium (ATCC, PCS-300-030), supplemented with Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) and 10 U/mL Penicillin and 10 μg/mL streptomycin (Gibco, 15140-122).

Techniques: Staining, Confocal Microscopy, Infection, Virus, Incubation, Produced, Cell Culture, Expressing, Control, Marker

Journal: bioRxiv

Article Title: PROS1 released by human lung basal cells upon SARS-CoV-2 infection facilitates epithelial cell repair and limits inflammation

doi: 10.1101/2024.09.11.612489

Figure Lengend Snippet: A. Left: Expression of MERTK (yellow) in basal cells of healthy pseudostratified epithelium. 20X magnification, scale = 100μm. Middle, Z stack image projection, 63X magnification, scale = 50μm. Right, split channels for Acetylated tubulin (cilia), DAPI (nuclei) and MERTK. Arrows indicate basal cells expressing MERTK. B. Left: Expression of TYRO1 (yellow) in basal cells of healthy pseudostratified epithelium 20X magnification, scale = 100μm. Middle, Z stack image projection, 63X magnification, scale = 50μm. Right, split channels for Acetylated tubulin (cilia), DAPI (nuclei) and MERTK. C. Proportions of cell clusters in control ALI cultures at 72 hours, cultures stimulated with PROS1, cultures infected with SARS-CoV-2, and cultures infected with SARS-CoV2 and treated with PROS1 dissected by scRNAseq, that showed differences between treatments. The proportion of cell clusters per each conditions were obtained by generating a data frame (as.data.frame), using the epithelial object as for idents, and splitting by the SampleID metadata column that represented each condition (Supplemental Table 5). The data were visualised using the png and ggplot functions. The genes listed per each cluster represent top 10 DE gene markers, with a minimum log fold threshold of 0.25. D. Single-cell trajectory of cultured epithelial cells with RNA Velocity analysis visualized on UMAP embeddings. The direction of arrows infers the path of cell trajectory based on spliced versus unspliced RNA counts and suggests a differentiation path from CXCL10/11 high basal cells, that were increased during infection, to S100A2 pos KRT high basal cells, characterising infected cultures treated with PROS1. E. Integration of ALI scRNAseq dataset with published dataset of COVID19 (Yoshida et al., 2021) showed that KRT14 and KRT6A (markers of PROS1 stimulated epithelium) were increased in basal cells clusters (red circles) in patients with mild COVID-19.

Article Snippet: Human primary bronchial/tracheal epithelial cells (ATCC, PCS-300-010; Donor: Hispanic/latino man, 14 years old), passage 2, were expanded in T25 flasks in Airway Epithelial Cell Basal Medium (ATCC, PCS-300-030), supplemented with Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) and 10 U/mL Penicillin and 10 μg/mL streptomycin (Gibco, 15140-122).

Techniques: Expressing, Control, Infection, Cell Culture

Journal: bioRxiv

Article Title: PROS1 released by human lung basal cells upon SARS-CoV-2 infection facilitates epithelial cell repair and limits inflammation

doi: 10.1101/2024.09.11.612489

Figure Lengend Snippet: A. UCSC Genome Browser visualization of PROS1 gene, the H3K27Ac regions are indicated as active regulatory elements. Multiple transcription factors are reported to bind to PROS1 promoter (ORegAnno annotation), including the STAT1 (indicated) which is involved in interferon signalling and viral responses. Other transcription factors identified included : GATA2, TFAP2C, SMARCA4, CEBPA, TRIM28,CTCF, E2F4, ETS1, FOXA1, FOS, GATA3, HNF4A, TRIM28, MITF, RBL2, SPI1. B. Multiple proteins are predicted to regulate the PROS1 production in epithelial cells. This was done using the ligand-receptor expression analysis (NicheNet), on an object containing only the epithelial cells from the sequenced BALF dataset (Liao et al., 2020). Amongst multiple proteins that could potentially regulate PROS1 expression, either the top regulators (56 ligands with score >0.001) or type I and type III interferons are shown. C. Visualisation of top-10 reactome pathways, ordered by p-value ranking, of epithelial cells infected with virus. Reactome pathway activity inferred by evaluation of differentially expressed genes upregulated in epithelial cells infected with virus vs all other conditions with a minimum log.fc of 0.25 and a p-value < 0.05 based on Wilcoxon Rank Sum Test D. Average-expression heatmap visualising scaled expression of selected DE IFN-inducible genes, alongside putative cluster marker genes, and PROS1 in ALI cultured epithelial cell clusters. DE marker genes for each cluster with a minimum log.fc of 0.25 were identified based on Wilcoxon Rank Sum Test x test with a p-value < 0.05 number (Supp Tab 2). IFN-inducible genes were selected from this list (Supp Tab 2). E. Violin plots illustrating alra-imputed expression values of PROS1 in ALI cultured epithelial cell clusters. Black dot represents median expression value. F. Infection of epithelium with SARS-CoV-2 resulted in Interferon lambda 1 secretion 72 hours after infection. Each group is represented by multiple transwell systems that were used as control or infected with SARS-CoV-2, Mock N= 6, SARS-CoV-2 infected epithelia N= 9. Statistical comparison was performed using unpaired T test between different conditions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 . G. Stimulation of ALI epithelial cultures with 10 ng/mL IFN-β overnight upregulated PROS1 release. Each group is represented by multiple transwell systems that were used as control or stimulated with IFN-β, N= 4. Statistical comparison was performed using unpaired T test between different conditions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 . H. Epithelial ALI cultures stimulated with IFN-β for 24 hours, and then cultured without IFN-β for another 72 hours. The PROS1 (magenta) in IFN-β pre-stimulated cells at 72 hours was present in ciliated cell (Middle Z axis), as compared to the control cultures where it was limited to the basal cells on the surface of the transwell (Left Z axis). PROS1 (magenta), Acetylated tubulin (yellow), DAPI (blue).

Article Snippet: Human primary bronchial/tracheal epithelial cells (ATCC, PCS-300-010; Donor: Hispanic/latino man, 14 years old), passage 2, were expanded in T25 flasks in Airway Epithelial Cell Basal Medium (ATCC, PCS-300-030), supplemented with Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) and 10 U/mL Penicillin and 10 μg/mL streptomycin (Gibco, 15140-122).

Techniques: Expressing, Infection, Virus, Activity Assay, Marker, Cell Culture, Control, Comparison

Journal: bioRxiv

Article Title: PROS1 released by human lung basal cells upon SARS-CoV-2 infection facilitates epithelial cell repair and limits inflammation

doi: 10.1101/2024.09.11.612489

Figure Lengend Snippet: A. Confocal 3D scans showing the monocytes on top of the epithelial barrier, at 72 hours cocultures of ALI epithelia with monocytes. B. Lateral view of the 3D scan showing the monocytes on top of epithelial cells, not passing through the tight junctions. C. Dot-plot illustrating that monocytes can be isolated from cocultures with ALI epithelium by expression of CD45 and CD14 D-E. Upon contact with epithelium blood monocytes increased MERTK expression as illustrated MFI of MERTK (D) and by % of MERTK positive cells (E). Each dot represents monocytes from 3 different patients on 3 different ALI epithelial systems (N=3). Statistical analysis was performed using One-Way Anova with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data is presented as bar plot +/- SD of mean. F. SARS-Cov-2 Infected bronchial cells produced MCSF and CCL2, but no GM- CSF, at 72 hours. Each group is represented by multiple transwell systems that were used as control or infected with SARS-CoV-2, Mock N= 6, SARS-CoV-2 infected epithelia N= 9. Data is presented as bar plot with +/- SD of mean. Statistical comparison was performed using unpaired T test between different conditions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 G. The IFN-β stimulated epithelium did not affect GM-CSF production. IFN-β pre-treated cocultures had lower levels of GM-CSF compared to controls at 72 hours (left graph). IFN-β stimulation resulted in higher M-CSF production from the epithelium at 24 hours. At 72 hour, the cocultures pre-treated with IFN-β also had higher M-CSF levels than the controls (right). Each group is represented by multiple transwell systems that were used as control or stimulated with 10 ng/mL IFN-β (24 hours stimulated cultures N=4, 72 hours cocultures N= 3). Data is presented as bar plot with mean +/- SD. Statistical comparison was performed using unpaired T test between different conditions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Human primary bronchial/tracheal epithelial cells (ATCC, PCS-300-010; Donor: Hispanic/latino man, 14 years old), passage 2, were expanded in T25 flasks in Airway Epithelial Cell Basal Medium (ATCC, PCS-300-030), supplemented with Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040) and 10 U/mL Penicillin and 10 μg/mL streptomycin (Gibco, 15140-122).

Techniques: Isolation, Expressing, Infection, Produced, Control, Comparison