Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 is significantly expressed in PDAC patients and correlated with poor prognosis. ( A ) Analysis of NCBP2 transcript levels in PDAC and normal tissues based on the RNA-seq data from GEPIA 2.0 cohort. ( B ) Analysis of NCBP2 transcript levels in PDAC and normal tissues based on DNA microarray data from GEO cohorts (GSE15471, GSE28735, GSE16515). ( C , D ) Overall survival and disease-free survival curves for PDAC patients with high or low NCBP2 expression in GEPIA 2.0 cohort. ( E , F ) The RNA and protein expression level of NCBP2 in HPDE and five PDAC cell lines. ( G ) The expression level of NCBP2 in PDAC and para-carcinoma tissues from tissue microarray, * p < 0.05 and *** p < 0.001.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: RNA Sequencing, Microarray, Expressing

Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 promotes PDAC cell growth in vitro. ( A – C ). The cell counting, 3D sphere, and colony formation assay results in control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. ( D – F ). The cell counting, 3D sphere and colony formation assay results in control and NCBP2-overexpression AsPC-1 and BxPC-3 cells, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Cell Counting, Colony Assay, Control, Knockdown, Over Expression

Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 promotes PDAC cell growth in-vivo. ( A , B ) Tumor growth curves of xenograft models established from control or stable NCBP2-knockdown Panc 05.04 cells. ( C ) Assessment of tumor weight from control and NCBP2-knockdown groups. ( D , E ) The expression level of Ki-67 in tumor tissues from control and NCBP2-knockdown groups. ( F ) Body weight of nude mice in control and NCBP2-knockdown group, * p < 0.05, and ** p < 0.01.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vivo, Control, Knockdown, Expressing

Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 upregulates c-JUN to activate MEK/ERK signaling in a m 7 G-dependent manner. ( A ) KEGG analysis of RNA-seq data in PDAC patients with high or low NCBP2 expression from TCGA cohort. ( B ) The GSEA enrichment plot of “MAPK signaling pathway” in PDAC patients with high or low NCBP2 expression from TCGA cohort. ( C ) Immunoblotting for protein levels of total JNK/phosphorylated JNK (Thr183/Tyr185), total p38/phosphorylated p38 (Thr180/Tyr182), and total ERK/phosphorylated ERK (Thr202/Tyr204) in control and NCBP2-knockdown PDAC cells. Tubulin was used as the internal control. ( D ) Immunoblotting for protein levels of total/phosphorylated MEK and total/phosphorylated ERK (Thr202/Tyr204) in control and c-JUN-knockdown Panc 05.04 and PANC-1 cells. Tubulin was used as the internal control. ( E ) The mRNA expression levels of NCBP2 and c-JUN in control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. ( F ) Immunoblotting for protein levels of NCBP2 and c-JUN in control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. Tubulin was used as the internal control. ( G ) Polysome profiling results of the control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. ( H ) Gene-specific m 7 G qPCR results for the m 7 G methylation levels of c-JUN in PANC 05.04 and PANC-1 cells, *** p < 0.001.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Knockdown, Methylation

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Endogenous PAR1 expression in different human pancreatic ductal adenocarcinoma (PDAC) cells. Six human PDAC cell lines, namely MIA PaCa-2, HPAF-II, SU.8686, Capan-1, ASPC-1, and CFPAC-1, were cultured for PAR1 level screening. A RT-PCR analysis of endogenous PAR1 mRNA levels (*** p < 0.001). B Western blot analysis of PAR1 expression (~ 66 kDa) in whole-cell lysates among the six cell lines (*** p < 0.001). C The mean fluorescence intensity (MFI) of PAR1 surface expression by tumor cells was determined by a flow cytometric analysis using a phycoerythrin (PE)-anti-PAR1 antibody (Ab) versus an isotype control. Propidium iodide (PI) levels were used to examine apoptotic cells. D Immunocytofluorescence (IF) analysis of PAR1 expression patterns in PDAC cells using antihuman PAR1 with signal enhancement through m-IgGκ BP-FITC labeling (left panel). Quantified statistics of green fluorescent protein-positive (GFP + ) to DAPI. + cell ratio of IF results are also shown (right panel). Individual scale bars are shown. All data are presented as the mean ± SD. of three experiments. *** p < 0.001

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Fluorescence, Control, Labeling

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Suppression of PAR1-expressing MIA PaCa-2 and CFPAC-1 cells by PAR1CAR-T cells in vitro. A A standard 24-h MTT cytotoxicity assay using three replicates ( n > 3) with increasing effector/tumor (E/T; effector: PAR1CAR-T cells) ratios of 0, 0.1, 1, 5, 10, and 20 against pancreatic ductal adenocarcinoma (PDAC) cell lines of MIA PaCa-2, CFPAC-1, and HPAF-II. Cytotoxic activities were compared to those of non-transduced CD3 + T-cell-treated cells, and mock-transduced T-cell-treated cells served as the control PAR1CAR-T cells ( n > 3; * p < 0.05 and *** p < 0.001). B Real-time monitoring of cytotoxic activities used for comparison between non-transduced CD3 + T cell-treated and mock-transduced T cell-treated cells, and 1% Triton-X-100-treated cells served as a positive control. Real-time monitoring of PAR1CAR-T-cell-treated cells revealed specific growth inhibition of PAR1-expressing CFPAC-1 (low levels; n > 3; * p < 0.05 and ** p < 0.01) and MIA PaCa-2 cells (high levels; n > 3; * p < 0.05, ** p < 0.01, and *** p < 0.001) compared to PAR1 non-expressing HPAF-II cells, as observed using the x-CELLigence System. Data are presented as the mean ± SD of three independent experiments

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, In Vitro, Cytotoxicity Assay, Control, Comparison, Positive Control, Inhibition

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Transforming growth factor (TGF)-β-mediated PAR1 upregulation enhances pancreatic ductal adenocarcinoma (PDAC) cell responsiveness to PAR1CAR-T-cell-specific suppression. A Human PDAC cell lines (HPAF-II, CFPAC-1, and MIA PaCa-2) were exposed to TGF-β (18 ng/mL), and cells were collected at indicated times over 48 h. PAR1 expression was measured by flow cytometry. Results revealed original and enhanced levels of PAR1 by quantifying the mean fluorescent intensity (MFI) (left panel), expression fold-changes (right panel), and cell fold-changes (middle panel) over incubation times. B Standard 24-h cytotoxic activities of PAR1CAR-T cells toward tumor cells were measured using MTT assays with increasing effector/tumor (E/T) ratios of 0, 0.1, 1, 5, 10, and 20 against HPAF-II, CFPAC-1, and MIA PaCa-2 cells following 18 ng/mL TGF-β stimulation (18 ng/mL) for 48 h. Cytotoxic activities were compared to those of non-transduced CD3 + T-cell-treated cells, and mock-transduced T-cell-treated cells served as control PAR1CAR-T cells ( n > 3; * p < 0.05 and *** p < 0.001, respectively). Results are the mean ± SD of three independent experiments

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, Flow Cytometry, Incubation, Control

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Relationship between transforming growth factor (TGF)-β-modulated PAR1 and regulatory T cell (Treg) function and pancreatic ductal adenocarcinoma (PDAC) cell response to PAR1CAR-T cell targeting. A Western blotting results of 24-h stimulation with TGF-β on the MIAPaCa-2 and HPAF-II PDAC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. B Analysis of tissue factor (TF) and thrombin expressions in individual cell lines treated for 24 h with the TGF-β growth factor. C Effect of adding cancer-associated fibroblasts (CAFs) and different phenotypes of cytokine-independent T cells on cell viability according to different treatments. Different co-culture combinations also resulted in various tumor-derived TGF-β levels. D A schematic diagram shows the role of immuno-mediated TGF-β affecting Treg function and transformation in PDAC treated with PAR1CART cells

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Western Blot, Control, Co-Culture Assay, Derivative Assay, Transformation Assay

Journal: Journal of medicinal chemistry

Article Title: Clobetasol propionate is a heme-mediated selective inhibitor of human cytochrome P450 3A5

doi: 10.1021/acs.jmedchem.9b02067

Figure Lengend Snippet: (a) Expression of CYP3A5 across all cancers within the PanCancer dataset from The Cancer Genome Atlas (TCGA). Expression is derived from 10,534 tumor samples across 33 cancers and is ranked by median values. The pancreatic adenocarcinoma cohort (PAAD) is highlighted in red. (Sample IDs, the expansion of the cohort abbreviations, and the full CYP3A5 expression data are located in Supplementary Data 2; the stepwise normalization protocol is available in Protocol Exchange44). (b) CYP3A5 expression in pancreatic adenocarcinoma cell lines compared to that in the noncancerous pancreatic cell line HPNE. *** P ≤ 0.0001, ns = not significant (P ≥ 0.05); t statistic–derived P values adjusted by the Benjamini-Hochberg false discovery rate. (c) Expression of the xenobiotic-metabolizing CYPs from all pancreatic adenocarcinoma and control cell lines. Units of expression for all panels are log2 (normalized CPM+1).

Article Snippet: To set about assessing CYP3A5 expression in pancreatic cancer models, we obtained eight commercially available pancreatic ductal adenocarcinoma (PDAC) cell lines (AsPC-1, HPAF-II, PANC-1, MIA PaCa-2, SU.86.86, Capan-2, CFPAC-1, and Panc 02.13) and one noncancerous immortalized pancreatic cell line (hTERT-HPNE) from the American Type Culture Collection (ATCC).

Techniques: Expressing, Derivative Assay, Control

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: A) Quantitative real-time PCR analysis of SLC7A11 and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Western Blot, Control, Marker, Staining, Immunohistochemistry, Biomarker Discovery

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: A-B) Schematic diagram of 3D co-culture spheroid outgrowth assay and quantification of 3D co-culture spheroid outgrowth post-transfection with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool. Labels: PDAC ns CAF ns = non-silencing controls; PDAC ns CAF slc = SLC7A11 knockdown in CAFs only; PDAC slc CAF ns = SLC7A11 knockdown in PDAC cells only; PDAC slc CAF slc = SLC7A11 knockdown in both PDAC cells and CAFs. Representative photos are shown above each bar with the core circled in white dashed lines and outgrowth in red dashed lines (bars in photos = 300μm). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Schematic diagram of 3D co-culture growth assay using stable shRNA cell lines and D) representative photos (bars in photos = 200μm) and quantification of 3D co-culture spheroid growth. Labels: PDAC scr CAF scr = scramble-shRNA controls; PDAC scr CAF slc = SLC7A11-shRNA seq 1 in CAFs only; PDAC slc CAF scr = SLC7A11-shRNA seq 1 in PDAC cells only; PDAC slc CAF slc = SLC7A11-shRNA seq 1 in both PDAC cells and CAFs. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ****p≤0.0001, n=4-6; One-way ANOVA). Replicate numbers in panel B refer to independent experiments performed using MiaPaCa-2 combined with CAF cells isolated from different PDAC patients. Replicate numbers in panels D refer to replicate spheroids performed using MiaPaCa-2 PDAC cells combined with an immortalised CAF line.

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Co-Culture Assay, Transfection, Control, Knockdown, Growth Assay, shRNA, Isolation

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: A) Quantification of Pancreatic Intraepithelial Neoplasia (PanINs) 1A-3 from KC mice (n=7) and KC mice with SLC7A11 conditional KO under Pdx1 -promoter (KC Slc7a11 fl/fl ; n=7) at 70 days of age (mean±s.e.m.). B) Kaplan-Meier analysis showing survival percentage of KPC (n=26) and KPC Slc7a11 fl/fl mice (n=24) mice. C) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section (n=5 mice per group). Scale bars = 400μm. D) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Scale bars = 400μm. Asterisks indicate significance (*p≤0.05; n=5 mice per group; student t-test). E-F) Live cell counts (mean±s.e.m.) of (E) KPC PDAC cells and (F) KPC CAFs 72h post-transfection with control-siRNA (ns-siRNA) or mouse SLC7A11-siRNA pool (SLC7A11 pool). Asterisks indicate significance (*p≤0.05, **p≤0.01; n=3; one-way ANOVA).

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Staining, Transfection, Control

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: All orthotopic tumours were co-injections of PDAC cells and CAFs. A) Orthotopic pancreatic tumours were treated with STAR nanoparticles + control-siRNA or SLC7A11 siRNA single sequence (SLC7A11 single seq) in the regimen shown. Representative photos of immunohistochemistry for SLC7A11 in tumour tissue at the model endpoint are shown. Graph shows optical density (staining intensity) calculated from average pixel intensity measurements from 3 representative images per tumour, using ImageJ. Circles indicate individual mice, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05; One-way ANOVA). B) Treatment regimen for therapeutic model analysed in panels (C-D). Circles and triangles in all dot plots in panels C-D represent individual mice. C) Tumour volume at therapeutic model endpoint, as assessed by calliper measurement ex vivo (mean±s.e.m.). Asterisks indicate significance (*p≤0.05; One-way ANOVA). D) Representative photos of metastases confirmed by H&E staining following detection at model endpoint by ex vivo luminescence imaging of organs. Graph shows metastatic sites per mouse (mean±s.e.m.) for each treatment group. Scale bars in all figures = 200μm.

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Control, Sequencing, Immunohistochemistry, Staining, Ex Vivo, Imaging