![Expression and knockdown of Girdin in PaCa cell line. (A) Expression of Girdin mRNA in normal pancreatic and PaCa cell lines. The reverse transcription-quantitative PCR experiments were calculated using a comparative quantitative method with calibration curves. The vertical axis represents the ratio of Girdin expression to that in the human pancreatic ductal epithelial cell lines. (B) Western blotting was performed using Girdin and GAPDH antibodies. The density of bands detected was measured, and the ratio of Girdin to GAPDH was plotted. (C) Transfection of PaCa cell lines with negative control siRNA or Girdin siRNA. Girdin was knocked down by transfection of Girdin siRNA for MIA PaCa-2, AsPC-1 and PANC-1. (D) The knockdown of Girdin in these cell lines was confirmed by western blotting. The data are presented as the mean ± SEM. All experiments were performed in triplicate and repeated three times. *P<0.01 [A: One-way ANOVA with Dunnett's multiple comparison test. B: Student's t-test (unpaired), two-tailed]. PaCa, pancreatic cancer; siRNA, small interfering RNA.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8027/pmc10398027/pmc10398027__or-50-03-08606-g01.jpg)
Journal: Oncology Reports
Article Title: Girdin regulates both migration and angiogenesis in pancreatic cancer cell lines
doi: 10.3892/or.2023.8606
Figure Lengend Snippet: Expression and knockdown of Girdin in PaCa cell line. (A) Expression of Girdin mRNA in normal pancreatic and PaCa cell lines. The reverse transcription-quantitative PCR experiments were calculated using a comparative quantitative method with calibration curves. The vertical axis represents the ratio of Girdin expression to that in the human pancreatic ductal epithelial cell lines. (B) Western blotting was performed using Girdin and GAPDH antibodies. The density of bands detected was measured, and the ratio of Girdin to GAPDH was plotted. (C) Transfection of PaCa cell lines with negative control siRNA or Girdin siRNA. Girdin was knocked down by transfection of Girdin siRNA for MIA PaCa-2, AsPC-1 and PANC-1. (D) The knockdown of Girdin in these cell lines was confirmed by western blotting. The data are presented as the mean ± SEM. All experiments were performed in triplicate and repeated three times. *P<0.01 [A: One-way ANOVA with Dunnett's multiple comparison test. B: Student's t-test (unpaired), two-tailed]. PaCa, pancreatic cancer; siRNA, small interfering RNA.
Article Snippet: Human PaCa cell lines [MIA PaCa-2 (cat. no. CRL-1420TM), SW 1990 (cat. no. CRL-2172TM), AsPC-1 (cat. no. CRL-1682TM), BxPC-3 (cat. no. CRL-1687TM), PANC-1 (cat. no. CRL-1469TM), Capan-2 (cat. no. HTB-80TM) ( )] and the human pancreatic duct epithelial cell line H6c7 (cat. no. PCS-600-010) were obtained from the American Type Culture Collection (ATCC).
Techniques: Expressing, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Negative Control, Comparison, Two Tailed Test, Small Interfering RNA
Journal: Annals of Medicine and Surgery
Article Title: Combination of cycling hyperthermia and echinacoside creates a synergistic curing effect on pancreatic cancer PANC-1 cells
doi: 10.1097/MS9.0000000000003545
Figure Lengend Snippet: Effect of Ech alone or in combination with TC-HT on the cell viability. (A) MTT viability assay of PANC-1 cells treated with different concentrations of Ech or in combination with TC-HT treatment. (B) MTT viability assay of H6c7 normal human pancreatic cells treated with 20 μM Ech or in combination with the same TC-HT treatment. Data represent the mean ± standard deviation ( n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (*** P < 0.001).
Article Snippet: H6c7 human pancreatic duct epithelial cell line was obtained from Kerafast, Inc. (Absolute Biotech, Boston, MA, USA) and maintained in keratinocyte serum-free medium (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) supplemented with human recombinant epidermal growth factor, bovine pituitary extract (Invitrogen; Thermo Fisher Scientific, Inc.), and 1% (v/v) penicillin and streptomycin.
Techniques: MTT Viability Assay, Standard Deviation
Figure S2 ). Representative images of nine FOVs showing response to CBZ in parental AsPC-1 OLs. Scale bar: 328 μm. Data in the bar chart show organoid MIP size as the mean ± SEM of measurements in triplicate from 96-well plates. ∗ p < 0.02 ; n.s., not significant by ordinary one-way ANOVA with Tukey’s multiple comparisons test. Total N OL = 446. (C) Row z-scored changes in average MIP area with CBZ or KET normalized to DMSO treatment from primary screening show PDACO-specific responses to the selective inhibition of CYP3A5 or pan-inhibition of CYP3A. MIP size was determined by batch image analysis. N ORG·DMSO = 1359; N ORG·CBZ = 1774; N ORG·KET = 2323. (D) Immunoblot showing the basal levels of CYP3A5 in PDACOs and OLs. Immunoblots indicating CYP3A5 induction after exposure to CBZ are included in
Figure S3 . (E) Quantitative characterization of the CBZ response in PDACOs and OLs across independent experiments. The measurements were extracted from an independent validation experiment (with quadruplicate wells) and pooled with organoid data from the primary screening obtained by supervised segmentation. N ORG = 10743. The histograms report the average fold change in organoid MIP area (mean ± SEM) after treatment with 30 μM CBZ. DMSO control bars for each line are shown in gray to illustrate the organoid shrinkage that occurred in lines treated with CBZ (bars in color). Quantitative classification of the organoid shrinkage response by tiers of residual MIP defines sensitivity as major (≤25% residual MIP), minor (up to 50%), or mild (≤70%), as compared with resistant or non-responder organoids (≥70% residual MIP). ∗∗∗∗ p < 0.0001 , ∗∗∗ p < 0.001 ∗∗ p < 0.01 , ∗ p < 0.05 ; n.s ., not significant (by Mann–Whitney test). (F) Scatterplot of z-scores for organoid responses to CBZ of PDACO and OLs compared to normalized levels of CYP3A5 protein at the basal level as quantified by immunoblot analysis. Protein levels were normalized to PDM30 to enable comparison across the set. R-squared of regression analysis (R) = 0.1 ( p < 0.28 ). The inset shows the same analysis for the PDM24/38/30 subset and OL controls. Spearman correlation (R sp ) = −1 ( p < 0.02 ) and R-squared of regression analysis (R) = 0.97 ( p < 0.002 ). Resistance scores (R, in red) and sensitivity scores (S, in blue) are indicated. See
Figures S4 A and S4B for comparison. (G) AUC scores extracted from datasets pooled together (
Figure 2 E), depict the response to CBZ for collectives of organoids. All PDACO lines responded. The effect on the PDM24/38/30 subset shows proportionality with the levels of CYP3A5 protein, with PDM24 displaying 16% modification relative to the original population structure, followed by 49% in PDM38 and 67% in PDM30. (H) Representative confocal images of nine FOVs illustrating the pharmaco-typing of CBZ in PDM24 (a hyposensitive PDACO with low levels of CYP3A5), as compared with PDM30 (a hypersensitive PDACO with the highest levels of CYP3A5) or PDM38 which also displayed sensitivity to CBZ in a dose-response manner. Insets show individual segmented organoids in pseudo-color. Scale bar: 328 μm. (I) Demography profiles tracking the major responsiveness of PDM30 to CBZ across increasing dosages of this compound. PDM30 demography profile shows statistical significance ( p < 0.0001 by Chi-square test of equal frequences). (J) Dose–response (DR) analysis for CBZ and full PDACO cohort (
Figure S4 A), using FC values on organoid MIPs plotted as the mean ± SEM from three technical replicates in 96-well plates. N ORG = 11524. Fitting curves and CBZ IC 50 were calculated with MIPs normalized to DMSO and across the CBZ DR range. The rightmost panel displays a segment of the fitting curves comprised between 15 μM and 30 μM CBZ dosages for the stratification of the PDACO with respect to their response to CBZ. " width="100%" height="100%">
Journal: iScience
Article Title: Pancreatic cancer organoid-screening captures personalized sensitivity and chemoresistance suppression upon cytochrome P450 3A5-targeted inhibition
doi: 10.1016/j.isci.2024.110289
Figure Lengend Snippet: Suppression of 3D growth via CBZ responds to CYP3A5 expression in a PDACO-dependent and dose-dependent manner (A and B) Visualization and quantitative characterization of the response to 30 μM CBZ in AsPC-1, 3A5 −/− , and H6c7 OLs (see Figure S2 ). Representative images of nine FOVs showing response to CBZ in parental AsPC-1 OLs. Scale bar: 328 μm. Data in the bar chart show organoid MIP size as the mean ± SEM of measurements in triplicate from 96-well plates. ∗ p < 0.02 ; n.s., not significant by ordinary one-way ANOVA with Tukey’s multiple comparisons test. Total N OL = 446. (C) Row z-scored changes in average MIP area with CBZ or KET normalized to DMSO treatment from primary screening show PDACO-specific responses to the selective inhibition of CYP3A5 or pan-inhibition of CYP3A. MIP size was determined by batch image analysis. N ORG·DMSO = 1359; N ORG·CBZ = 1774; N ORG·KET = 2323. (D) Immunoblot showing the basal levels of CYP3A5 in PDACOs and OLs. Immunoblots indicating CYP3A5 induction after exposure to CBZ are included in Figure S3 . (E) Quantitative characterization of the CBZ response in PDACOs and OLs across independent experiments. The measurements were extracted from an independent validation experiment (with quadruplicate wells) and pooled with organoid data from the primary screening obtained by supervised segmentation. N ORG = 10743. The histograms report the average fold change in organoid MIP area (mean ± SEM) after treatment with 30 μM CBZ. DMSO control bars for each line are shown in gray to illustrate the organoid shrinkage that occurred in lines treated with CBZ (bars in color). Quantitative classification of the organoid shrinkage response by tiers of residual MIP defines sensitivity as major (≤25% residual MIP), minor (up to 50%), or mild (≤70%), as compared with resistant or non-responder organoids (≥70% residual MIP). ∗∗∗∗ p < 0.0001 , ∗∗∗ p < 0.001 ∗∗ p < 0.01 , ∗ p < 0.05 ; n.s ., not significant (by Mann–Whitney test). (F) Scatterplot of z-scores for organoid responses to CBZ of PDACO and OLs compared to normalized levels of CYP3A5 protein at the basal level as quantified by immunoblot analysis. Protein levels were normalized to PDM30 to enable comparison across the set. R-squared of regression analysis (R) = 0.1 ( p < 0.28 ). The inset shows the same analysis for the PDM24/38/30 subset and OL controls. Spearman correlation (R sp ) = −1 ( p < 0.02 ) and R-squared of regression analysis (R) = 0.97 ( p < 0.002 ). Resistance scores (R, in red) and sensitivity scores (S, in blue) are indicated. See Figures S4 A and S4B for comparison. (G) AUC scores extracted from datasets pooled together ( Figure 2 E), depict the response to CBZ for collectives of organoids. All PDACO lines responded. The effect on the PDM24/38/30 subset shows proportionality with the levels of CYP3A5 protein, with PDM24 displaying 16% modification relative to the original population structure, followed by 49% in PDM38 and 67% in PDM30. (H) Representative confocal images of nine FOVs illustrating the pharmaco-typing of CBZ in PDM24 (a hyposensitive PDACO with low levels of CYP3A5), as compared with PDM30 (a hypersensitive PDACO with the highest levels of CYP3A5) or PDM38 which also displayed sensitivity to CBZ in a dose-response manner. Insets show individual segmented organoids in pseudo-color. Scale bar: 328 μm. (I) Demography profiles tracking the major responsiveness of PDM30 to CBZ across increasing dosages of this compound. PDM30 demography profile shows statistical significance ( p < 0.0001 by Chi-square test of equal frequences). (J) Dose–response (DR) analysis for CBZ and full PDACO cohort ( Figure S4 A), using FC values on organoid MIPs plotted as the mean ± SEM from three technical replicates in 96-well plates. N ORG = 11524. Fitting curves and CBZ IC 50 were calculated with MIPs normalized to DMSO and across the CBZ DR range. The rightmost panel displays a segment of the fitting curves comprised between 15 μM and 30 μM CBZ dosages for the stratification of the PDACO with respect to their response to CBZ.
Article Snippet: H6c7, human pancreatic duct epithelial cell line , Kerafast , Cat# ECA001-FP.
Techniques: Expressing, Inhibition, Western Blot, Control, MANN-WHITNEY, Comparison, Modification
Journal: iScience
Article Title: Pancreatic cancer organoid-screening captures personalized sensitivity and chemoresistance suppression upon cytochrome P450 3A5-targeted inhibition
doi: 10.1016/j.isci.2024.110289
Figure Lengend Snippet:
Article Snippet: H6c7, human pancreatic duct epithelial cell line , Kerafast , Cat# ECA001-FP.
Techniques: Recombinant, Membrane, Saline, Electron Microscopy, Staining, Lysis, Extraction, Protease Inhibitor, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Software, High Content Screening
