Review

human pdac cell lines bxpc 3 (ATCC)

ATCC is a verified supplier

ATCC manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

ATCC human pdac cell lines bxpc 3
Human Pdac Cell Lines Bxpc 3, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/human pdac cell lines bxpc 3/product/ATCC
Average 99 stars, based on 5080 article reviews

human pdac cell lines bxpc 3 - by Bioz Stars, 2026-03

99/100 stars

Images

Image Search Results

FAK is associated with the immunosuppressive microenvironment in pancreatic cancer. (A) The cell type annotation of 33,794 cells using t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation, categorizing them into a total of 8 types, including epithelial, myeloid, T cells, macrophage, fibroblast, B cell and mast cells, and plasma. (B) T-SNE plots showing average expression of gene markers for all cell clusters. (C) T-SNE plots showing the cell distribution originated from adjacent noncancerous pancreatic tissue (ANPT) and pancreatic ductal adenocarcinoma (PDAC). (D) Bar plot showing the overall cell composition of normal and tumor samples, colored by cell types. (E) The expression of FAK in various cells. (F) T-SNE plots showing the various subpopulations of epithelial cells. (G) The expression levels of FAK various subpopulations of epithelial cells. (H) Cell-cell communication from FAK high cells and FAK low cells to T cells. (I) Representative images and quantitation of the expression of FAK and CD8 in PDAC. Scale bars, 50 µm.

Journal: Oncoimmunology

Article Title: Inhibition of FAK promotes pancreatic cancer immunotherapy by mediating CXCL10 secretion to enhance CD8 + T cell infiltration

doi: 10.1080/2162402X.2025.2539442

Figure Lengend Snippet: FAK is associated with the immunosuppressive microenvironment in pancreatic cancer. (A) The cell type annotation of 33,794 cells using t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation, categorizing them into a total of 8 types, including epithelial, myeloid, T cells, macrophage, fibroblast, B cell and mast cells, and plasma. (B) T-SNE plots showing average expression of gene markers for all cell clusters. (C) T-SNE plots showing the cell distribution originated from adjacent noncancerous pancreatic tissue (ANPT) and pancreatic ductal adenocarcinoma (PDAC). (D) Bar plot showing the overall cell composition of normal and tumor samples, colored by cell types. (E) The expression of FAK in various cells. (F) T-SNE plots showing the various subpopulations of epithelial cells. (G) The expression levels of FAK various subpopulations of epithelial cells. (H) Cell-cell communication from FAK high cells and FAK low cells to T cells. (I) Representative images and quantitation of the expression of FAK and CD8 in PDAC. Scale bars, 50 µm.

Article Snippet: Human PDAC cell line BXPC3 was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 medium (Pricella, PM150110) supplemented with 10% fetal bovine serum (Gibco 10,100,147) under standard culture conditions (37°C, 5% CO 2 ).

Techniques: Clinical Proteomics, Expressing, Quantitation Assay

Inhibition of FAK can enhance the cytotoxic effect of PBMCs on pancreatic cancer PDOs. (A) Representative images and quantitation of the expression of FAK in pancreatic cancer PDOs. Scale bars, 50 µm. (B) Representative images of organoids treated with different concentrations of FAK inhibitors. Scale bars, 100 µm. (C) Dose-response curve of FAK inhibitors VS-6063 on human pancreatic cancer PANC1 cell viability. (D) Representative images of organoids treated with different concentrations of FAK inhibitors. Scale bars, 100 µm. (E) Western blotting analysis of FAK protein levels in control (NC) and FAK-knockdown (KD) BXPC3 cells. (F) FACS analysis PBMCs of CD69 + of anti-CD3/CD28-activated after co-culturing BXPC3-NC or BXPC3-FAK-KD ( n = 3). (G) FACS analysis CD8 of IFN-γ of anti-CD3/CD28-activated after co-culturing BXPC3-NC or BXPC3-FAK-KD ( n = 3). P-KD: BXPC3-FAK-KD; P-NC: BXPC3-NC. Error bars indicate standard error (SE). p-values were calculated by the Student’s t-test and Kruskal-Wallis test. ** p < 0.01; *** p < 0.001.

Journal: Oncoimmunology

Article Title: Inhibition of FAK promotes pancreatic cancer immunotherapy by mediating CXCL10 secretion to enhance CD8 + T cell infiltration

doi: 10.1080/2162402X.2025.2539442

Figure Lengend Snippet: Inhibition of FAK can enhance the cytotoxic effect of PBMCs on pancreatic cancer PDOs. (A) Representative images and quantitation of the expression of FAK in pancreatic cancer PDOs. Scale bars, 50 µm. (B) Representative images of organoids treated with different concentrations of FAK inhibitors. Scale bars, 100 µm. (C) Dose-response curve of FAK inhibitors VS-6063 on human pancreatic cancer PANC1 cell viability. (D) Representative images of organoids treated with different concentrations of FAK inhibitors. Scale bars, 100 µm. (E) Western blotting analysis of FAK protein levels in control (NC) and FAK-knockdown (KD) BXPC3 cells. (F) FACS analysis PBMCs of CD69 + of anti-CD3/CD28-activated after co-culturing BXPC3-NC or BXPC3-FAK-KD ( n = 3). (G) FACS analysis CD8 of IFN-γ of anti-CD3/CD28-activated after co-culturing BXPC3-NC or BXPC3-FAK-KD ( n = 3). P-KD: BXPC3-FAK-KD; P-NC: BXPC3-NC. Error bars indicate standard error (SE). p-values were calculated by the Student’s t-test and Kruskal-Wallis test. ** p < 0.01; *** p < 0.001.

Techniques: Inhibition, Quantitation Assay, Expressing, Western Blot, Control, Knockdown

FAK can mediate the immune response in pancreatic cancer through the secretion of CXCL10. (A) Communication status in different cells of CXCL signaling pathway network. (B) RT-PCR detection of the expression of CXCL-related genes after knockdown of FAK in BXPC3 cells. The data are presented as the means ± SD. p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. **** p < 0.0001; ns, non-significant. (C) ELISA validation of CXCL10 expression after knockdown of FAK in BXPC3 cells (FAK-KD) ( n = 3). p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. *** p < 0.001. (D) RT-PCR detection of the expression of CXCL10 after knockdown of CXCL10 (si-CXCL10) in BXPC3-FAK-KD cells. The data are presented as the means ± SD. p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. *** p < 0.001. (E) BXPC3 cells co-culture schematic diagram with PBMC. (F) FACS analysis PBMCs of CD69 + of anti-CD3/CD28-activated after co-culturing BXPC3 cells ( n = 3). p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. ** p < 0.01; *** p < 0.001. (G) Schematic diagram of the construction of a subcutaneous tumor graft model (KPC tumor) in C57BL/6J mice and the drug administration strategy. (H) Tumor growth curves of mice and tumor weight in different treatment groups ( n = 5/group). p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. * p < 0.5. (I) Representative images and quantification results of CD8 + T cell in different treatment groups of mice ( n = 5/group), scale bars, 50 µm. p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. ** p < 0.01.

Journal: Oncoimmunology

Article Title: Inhibition of FAK promotes pancreatic cancer immunotherapy by mediating CXCL10 secretion to enhance CD8 + T cell infiltration

doi: 10.1080/2162402X.2025.2539442

Figure Lengend Snippet: FAK can mediate the immune response in pancreatic cancer through the secretion of CXCL10. (A) Communication status in different cells of CXCL signaling pathway network. (B) RT-PCR detection of the expression of CXCL-related genes after knockdown of FAK in BXPC3 cells. The data are presented as the means ± SD. p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. **** p < 0.0001; ns, non-significant. (C) ELISA validation of CXCL10 expression after knockdown of FAK in BXPC3 cells (FAK-KD) ( n = 3). p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. *** p < 0.001. (D) RT-PCR detection of the expression of CXCL10 after knockdown of CXCL10 (si-CXCL10) in BXPC3-FAK-KD cells. The data are presented as the means ± SD. p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. *** p < 0.001. (E) BXPC3 cells co-culture schematic diagram with PBMC. (F) FACS analysis PBMCs of CD69 + of anti-CD3/CD28-activated after co-culturing BXPC3 cells ( n = 3). p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. ** p < 0.01; *** p < 0.001. (G) Schematic diagram of the construction of a subcutaneous tumor graft model (KPC tumor) in C57BL/6J mice and the drug administration strategy. (H) Tumor growth curves of mice and tumor weight in different treatment groups ( n = 5/group). p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. * p < 0.5. (I) Representative images and quantification results of CD8 + T cell in different treatment groups of mice ( n = 5/group), scale bars, 50 µm. p-values were calculated by two-way ANOVA with Tukey’s multiple comparison test. ** p < 0.01.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Knockdown, Comparison, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Co-Culture Assay

(A) Representative immunoblots showing the effect of PI3K inhibition (GDC-0941, 2 μM) over a 6-hour time course on phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) in KRAS -deficient PDAC clones (8988T-KO, KP4-KO). HSP90 is loading control. Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01 *** p < 0.001, repeated measures ANOVA (rmANOVA) with Dunnett’s post-hoc test. (B) Representative immunoblots showing the effect of PI3K inhibition (GDC-0941, 2 μM) over a 6-hour time course on pERK1/2 and pAKT in non-malignant cells (293HEK, KRAS WT MEF cells). Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01, repeated measures ANOVA (rmANOVA) with Dunnett’s post-hoc test. (C) Immunoblots showing pERK1/2 and pAKT levels in designated cell lines following 4-hour treatment with isoform-selective PI3K inhibitors (2 μM each): BYL719 (BYL, p110α), GDC-0941 (GDC, pan-class IA), idelalisib (IDE, p110δ), TGX221 (TGX, p110β), and ZSTK474 (ZSTK, pan-class IA). HSP90 is loading control. (D) Correlation between pAKT/AKT and pERK/ERK (normalized to DMSO) across inhibitor treatments was assessed in each cell line in ( C ), indicating the extent to which PI3K signaling activity (pAKT) is associated with MAPK output (pERK). Pearson correlation coefficients (r) and associated p-values are shown. (E-H) Representative immunoblots and quantification of pERK1/2 and pAKT following treatment with DMSO (solvent), 2 μM GDC-0941 (GDC), 1 μM PI3K activator 1938, or their combination for 0.5 hour in 8988T-KO ( E ), KRAS WT MEF ( F ), NRAS WT MEF ( G ), and HRAS WT MEF ( H ) cells. Bar graphs represent mean ± SD normalized to DMSO from n = 3 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, repeated measures ANOVA (rmANOVA) with Šidák’s post-hoc test.

Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) Representative immunoblots showing the effect of PI3K inhibition (GDC-0941, 2 μM) over a 6-hour time course on phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) in KRAS -deficient PDAC clones (8988T-KO, KP4-KO). HSP90 is loading control. Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01 *** p < 0.001, repeated measures ANOVA (rmANOVA) with Dunnett’s post-hoc test. (B) Representative immunoblots showing the effect of PI3K inhibition (GDC-0941, 2 μM) over a 6-hour time course on pERK1/2 and pAKT in non-malignant cells (293HEK, KRAS WT MEF cells). Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01, repeated measures ANOVA (rmANOVA) with Dunnett’s post-hoc test. (C) Immunoblots showing pERK1/2 and pAKT levels in designated cell lines following 4-hour treatment with isoform-selective PI3K inhibitors (2 μM each): BYL719 (BYL, p110α), GDC-0941 (GDC, pan-class IA), idelalisib (IDE, p110δ), TGX221 (TGX, p110β), and ZSTK474 (ZSTK, pan-class IA). HSP90 is loading control. (D) Correlation between pAKT/AKT and pERK/ERK (normalized to DMSO) across inhibitor treatments was assessed in each cell line in ( C ), indicating the extent to which PI3K signaling activity (pAKT) is associated with MAPK output (pERK). Pearson correlation coefficients (r) and associated p-values are shown. (E-H) Representative immunoblots and quantification of pERK1/2 and pAKT following treatment with DMSO (solvent), 2 μM GDC-0941 (GDC), 1 μM PI3K activator 1938, or their combination for 0.5 hour in 8988T-KO ( E ), KRAS WT MEF ( F ), NRAS WT MEF ( G ), and HRAS WT MEF ( H ) cells. Bar graphs represent mean ± SD normalized to DMSO from n = 3 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, repeated measures ANOVA (rmANOVA) with Šidák’s post-hoc test.

Article Snippet: Established human PDAC cell lines (8988T, BxPC3, KP-4, AsPC1, and L3.3) and 293T cells (293HEK) were sourced from DSMZ-Germany, American Type Culture Collection (ATCC), and RIKEN.

Techniques: Western Blot, Inhibition, Clone Assay, Control, Activity Assay, Solvent

(A) Representative immunoblots showing the effect of PI3K inhibition (ZSTK474, 2 μM) over a 6-hour time course on phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) in KRAS deficient PDAC clones (8988T-KO, KP4-KO). HSP90 serves as loading controls. Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01 *** p < 0.001, repeated measures ANOVA (rmANOVA) with Dunnett’s post-hoc test. (B) Representative immunoblots showing the effect of PI3K inhibition (ZSTK474, 2 μM) over a 6-hour time course on pERK1/2 and pAKT in non-malignant cells (293HEK, KRAS WT MEF cells). Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01, rmANOVA with Dunnett’s post-hoc test. (C) Immunoblots showing the effects of PI3K inhibition (GDC-0941, 2 μM) on pERK1/2 and pAKT in 293HEK cells transfected with real-time ERK kinase translocation reporter (ERK-KTR) and KRAS WT or KRAS G12V constructs. pERK is robustly increased with KRAS G12V , and is resistant to PI3K inhibition, unlike KRAS WT and control (not transfected with KRAS construct, thus representing endogenous signaling). HSP90 is loading control. (D) Representative images of ERK-KTR reporter in 293HEK: ERK-KTR cells in ( C ) showing cytoplasmic redistribution of ERK-KTR in KRAS G12V -expressing cells indicating elevated ERK activity. Bar graphs show quantification of ERK activity (cytoplasmic-to-nuclear fluorescence intensity ratio, mean ± SD, n = 6-7 biological replicates per group). ns = not significant, * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s post-hoc test. (E) Quantification of ERK activity (as in ( D )) normalized to average baseline intensity by time-lapse imaging of KP4-KO and 293HEK cells (untransfected or transfected with KRAS WT or KRAS G12V ) following treatment with GDC-0941 vs. DMSO solvent showed a rapid decrease in ERK activity in RAS wild-type cells (n=5-10 cells per group).

Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) Representative immunoblots showing the effect of PI3K inhibition (ZSTK474, 2 μM) over a 6-hour time course on phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) in KRAS deficient PDAC clones (8988T-KO, KP4-KO). HSP90 serves as loading controls. Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01 *** p < 0.001, repeated measures ANOVA (rmANOVA) with Dunnett’s post-hoc test. (B) Representative immunoblots showing the effect of PI3K inhibition (ZSTK474, 2 μM) over a 6-hour time course on pERK1/2 and pAKT in non-malignant cells (293HEK, KRAS WT MEF cells). Bar graphs quantify pERK/ERK and pAKT/AKT levels normalized to time 0 (mean ± SD, n = 3 biological replicates). ns = not significant, * p < 0.05, ** p < 0.01, rmANOVA with Dunnett’s post-hoc test. (C) Immunoblots showing the effects of PI3K inhibition (GDC-0941, 2 μM) on pERK1/2 and pAKT in 293HEK cells transfected with real-time ERK kinase translocation reporter (ERK-KTR) and KRAS WT or KRAS G12V constructs. pERK is robustly increased with KRAS G12V , and is resistant to PI3K inhibition, unlike KRAS WT and control (not transfected with KRAS construct, thus representing endogenous signaling). HSP90 is loading control. (D) Representative images of ERK-KTR reporter in 293HEK: ERK-KTR cells in ( C ) showing cytoplasmic redistribution of ERK-KTR in KRAS G12V -expressing cells indicating elevated ERK activity. Bar graphs show quantification of ERK activity (cytoplasmic-to-nuclear fluorescence intensity ratio, mean ± SD, n = 6-7 biological replicates per group). ns = not significant, * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s post-hoc test. (E) Quantification of ERK activity (as in ( D )) normalized to average baseline intensity by time-lapse imaging of KP4-KO and 293HEK cells (untransfected or transfected with KRAS WT or KRAS G12V ) following treatment with GDC-0941 vs. DMSO solvent showed a rapid decrease in ERK activity in RAS wild-type cells (n=5-10 cells per group).

Techniques: Western Blot, Inhibition, Clone Assay, Transfection, Translocation Assay, Construct, Control, Expressing, Activity Assay, Fluorescence, Imaging, Solvent

(A) G-LISA RAS activation assays in KRAS -deficient PDAC cells reconstituted with wild-type KRAS (8988T-KO: KRAS WT ) and treated with GDC-0941 (GDC, 2 μM), 1938 (1 μM), or solvent (DMSO) control for 30 minutes RAS-GTP levels are measured as absorbance (mean ± SD, n = 3 biological replicates per condition). Positive (active RAS control protein) and negative (buffer only) controls are shown. * p < 0.05, one-way ANOVA with Tukey’s post-hoc test. (B) G-LISA RAS activation assays in single RAS-expressing (KRAS WT , NRAS WT ) MEFs treated with GDC. RAS-GTP levels are measured as absorbance (mean ± SD, n = 3 biological replicates per condition). * p < 0.05, paired two-tailed t-test. (C) Representative immunoblots of RAS-GTP (pulldown assay using the RAF-RBD) and input lysate in 8988T-KO: KRAS WT cells upon PI3K inhibitors treatment (2 μM, 4 hours). (D) Representative immunoblots of GFP-tagged KRAS (GFP-KRAS) and associated effectors (BRAF and CRAF) following GFP nano-trap pulldown of KRAS assays upon PI3K inhibition (GDC-0941 or ZSTK474, 2 μM) in 8988T-KO cells reconstituted with GFP-KRAS (8988T-KO: GFP-KRAS WT cells. Densitometric quantification (mean ± SD, n = 3 biological replicates). *p < 0.05, one-way ANOVA with Tukey’s post hoc test. (E) G-LISA RAS activation assays in mutant KRAS-expressing cells (8988T parental cells, single RAS-expressing KRAS G12V MEFs, and 8988T-KO cells reconstituted with KRAS G12V (8988T-KO: KRAS G12V )) treated with GDC-0941 (GDC, 2 μM), ZSTK474 (2 μM), or solvent (DMSO) control for 4 hours. RAS-GTP levels are measured as absorbance (mean ± SD, n = 3 biological replicates per condition). ns = non-significant, one-way ANOVA with Tukey’s post-hoc test or paired two-tailed t-test. (F) Representative immunoblot of RAS-GTP pulldown assay using the RAF-RBD in 8988T-KO: KRAS G12V cells upon PI3K inhibitors treatment (2 μM, 4 hours). (G) Representative immunoblots and quantification of phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) following treatment with DMSO (solvent), 2 μM GDC-0941 (GDC), 1 μM PI3K activator 1938, or their combination for 0.5 hour in KRAS G12V MEFs. HSP90 is loading control. Bar graphs represent mean ± SD normalized to DMSO from n = 3 biological replicates. ns = non-significant, ** p < 0.01, repeated measures ANOVA (rmANOVA) with Šidák’s post-hoc test.

Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) G-LISA RAS activation assays in KRAS -deficient PDAC cells reconstituted with wild-type KRAS (8988T-KO: KRAS WT ) and treated with GDC-0941 (GDC, 2 μM), 1938 (1 μM), or solvent (DMSO) control for 30 minutes RAS-GTP levels are measured as absorbance (mean ± SD, n = 3 biological replicates per condition). Positive (active RAS control protein) and negative (buffer only) controls are shown. * p < 0.05, one-way ANOVA with Tukey’s post-hoc test. (B) G-LISA RAS activation assays in single RAS-expressing (KRAS WT , NRAS WT ) MEFs treated with GDC. RAS-GTP levels are measured as absorbance (mean ± SD, n = 3 biological replicates per condition). * p < 0.05, paired two-tailed t-test. (C) Representative immunoblots of RAS-GTP (pulldown assay using the RAF-RBD) and input lysate in 8988T-KO: KRAS WT cells upon PI3K inhibitors treatment (2 μM, 4 hours). (D) Representative immunoblots of GFP-tagged KRAS (GFP-KRAS) and associated effectors (BRAF and CRAF) following GFP nano-trap pulldown of KRAS assays upon PI3K inhibition (GDC-0941 or ZSTK474, 2 μM) in 8988T-KO cells reconstituted with GFP-KRAS (8988T-KO: GFP-KRAS WT cells. Densitometric quantification (mean ± SD, n = 3 biological replicates). *p < 0.05, one-way ANOVA with Tukey’s post hoc test. (E) G-LISA RAS activation assays in mutant KRAS-expressing cells (8988T parental cells, single RAS-expressing KRAS G12V MEFs, and 8988T-KO cells reconstituted with KRAS G12V (8988T-KO: KRAS G12V )) treated with GDC-0941 (GDC, 2 μM), ZSTK474 (2 μM), or solvent (DMSO) control for 4 hours. RAS-GTP levels are measured as absorbance (mean ± SD, n = 3 biological replicates per condition). ns = non-significant, one-way ANOVA with Tukey’s post-hoc test or paired two-tailed t-test. (F) Representative immunoblot of RAS-GTP pulldown assay using the RAF-RBD in 8988T-KO: KRAS G12V cells upon PI3K inhibitors treatment (2 μM, 4 hours). (G) Representative immunoblots and quantification of phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) following treatment with DMSO (solvent), 2 μM GDC-0941 (GDC), 1 μM PI3K activator 1938, or their combination for 0.5 hour in KRAS G12V MEFs. HSP90 is loading control. Bar graphs represent mean ± SD normalized to DMSO from n = 3 biological replicates. ns = non-significant, ** p < 0.01, repeated measures ANOVA (rmANOVA) with Šidák’s post-hoc test.

Techniques: Activation Assay, Solvent, Control, Expressing, Two Tailed Test, Western Blot, Inhibition, Mutagenesis

(A) Representative immunoblots of phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) in 8988T-KO cells reconstituted with various KRAS mutants (G12V, G13D, Q61R, G12C, G12D, G12R, Q61L) or KRAS WT treated with GDC-0941 or ZSTK474 (2 μM) for 4 hours. HSP90 is loading control. (B) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) for GDC-0941 and ZSTK474 of cell lines in (A) , demonstrating that mutant KRAS (but not KRAS WT ) decreased PI3K inhibitor sensitivity irrespective of mutant variant. (C) Representative immunoblots of pERK1/2 and pAKT in 8988T-KO KRAS WT cells and HEK293 cells stimulated with EGF (100 ng/mL) in the presence or absence of the PI3K inhibitor GDC-0941 (2 μM) for 30 minutes. (D) Representative immunoblots and quantification of pERK1/2 and pAKT following treatment with DMSO (solvent), 2 μM GDC-0941 (GDC), 1 μM PI3K activator 1938, or their combination for 0.5 hour in BRAF V600E MEFs. Bar graphs represent mean ± SD normalized to DMSO from n = 3 biological replicates. ns = non-significant, ** p < 0.01, *** p < 0.001, repeated measures ANOVA (rmANOVA) with Šidák’s post-hoc test. (E) Immunoblots showing phosphorylation levels of pERK1/2 and pAKT in BxPC3 PDAC cells (KRAS WT ; BRAF V600E ) following 4-hour treatment with isoform-selective PI3K inhibitors (2 μM each): BYL-719 (BYL, p110α), GDC-0941 (GDC, pan-class IA), idelalisib (IDE, p110δ), TGX-221 (TGX, p110β), and ZSTK474 (ZSTK, pan-class IA). Correlation between pAKT/AKT and pERK/ERK across inhibitor treatments was assessed, indicating that PI3K signaling activity (pAKT) is not associated with MAPK output (pERK). Pearson correlation coefficient (r) and associated p-value are shown,

Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) Representative immunoblots of phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) in 8988T-KO cells reconstituted with various KRAS mutants (G12V, G13D, Q61R, G12C, G12D, G12R, Q61L) or KRAS WT treated with GDC-0941 or ZSTK474 (2 μM) for 4 hours. HSP90 is loading control. (B) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) for GDC-0941 and ZSTK474 of cell lines in (A) , demonstrating that mutant KRAS (but not KRAS WT ) decreased PI3K inhibitor sensitivity irrespective of mutant variant. (C) Representative immunoblots of pERK1/2 and pAKT in 8988T-KO KRAS WT cells and HEK293 cells stimulated with EGF (100 ng/mL) in the presence or absence of the PI3K inhibitor GDC-0941 (2 μM) for 30 minutes. (D) Representative immunoblots and quantification of pERK1/2 and pAKT following treatment with DMSO (solvent), 2 μM GDC-0941 (GDC), 1 μM PI3K activator 1938, or their combination for 0.5 hour in BRAF V600E MEFs. Bar graphs represent mean ± SD normalized to DMSO from n = 3 biological replicates. ns = non-significant, ** p < 0.01, *** p < 0.001, repeated measures ANOVA (rmANOVA) with Šidák’s post-hoc test. (E) Immunoblots showing phosphorylation levels of pERK1/2 and pAKT in BxPC3 PDAC cells (KRAS WT ; BRAF V600E ) following 4-hour treatment with isoform-selective PI3K inhibitors (2 μM each): BYL-719 (BYL, p110α), GDC-0941 (GDC, pan-class IA), idelalisib (IDE, p110δ), TGX-221 (TGX, p110β), and ZSTK474 (ZSTK, pan-class IA). Correlation between pAKT/AKT and pERK/ERK across inhibitor treatments was assessed, indicating that PI3K signaling activity (pAKT) is not associated with MAPK output (pERK). Pearson correlation coefficient (r) and associated p-value are shown,

Techniques: Western Blot, Control, Mutagenesis, Variant Assay, Solvent, Phospho-proteomics, Activity Assay

(A) Immunoblots showing proximity-based biotinylation profiles from PDAC and 293HEK cells expressing BirA-tagged KRAS variants (WT, G12V, C185S, and HVR) following treatment with DMSO or GDC-0941 (2 μM). Biotin vs. no biotin confirms enrichment of specific proteins in cells harboring BirA constructs. (B) KEGG pathway enrichment analysis of decreased biotinylated proteins in 8988T-KO: KRAS WT cells treated with GDC-0941 (vs. DMSO control) reveals significant enrichment in RAS-MAPK, PI3K-AKT, focal adhesion, and actin cytoskeleton pathways. (C) Volcano plots (log2 fold-change (LFC) vs. −log10(adjusted p-value)) showing differential biotinylated protein enrichment between GDC-0941 and DMSO conditions 8988T-KO: KRAS HVR and 8988T-KO: KRAS C185S PDAC cells. (D) Volcano plot (LFC vs. −log10(adjusted p-value)) showing differential biotinylated protein enrichment between GDC-0941 and DMSO conditions 8988T-KO: KRAS G12V cells. (E) Volcano plot (LFC vs. −log10(adjusted p-value)) showing differential biotinylated protein enrichment between GDC-0941 and DMSO conditions 293HEK: KRAS WT cells, showing similar results in decreased proteins to 8988T-KO: KRAS WT cells.

Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) Immunoblots showing proximity-based biotinylation profiles from PDAC and 293HEK cells expressing BirA-tagged KRAS variants (WT, G12V, C185S, and HVR) following treatment with DMSO or GDC-0941 (2 μM). Biotin vs. no biotin confirms enrichment of specific proteins in cells harboring BirA constructs. (B) KEGG pathway enrichment analysis of decreased biotinylated proteins in 8988T-KO: KRAS WT cells treated with GDC-0941 (vs. DMSO control) reveals significant enrichment in RAS-MAPK, PI3K-AKT, focal adhesion, and actin cytoskeleton pathways. (C) Volcano plots (log2 fold-change (LFC) vs. −log10(adjusted p-value)) showing differential biotinylated protein enrichment between GDC-0941 and DMSO conditions 8988T-KO: KRAS HVR and 8988T-KO: KRAS C185S PDAC cells. (D) Volcano plot (LFC vs. −log10(adjusted p-value)) showing differential biotinylated protein enrichment between GDC-0941 and DMSO conditions 8988T-KO: KRAS G12V cells. (E) Volcano plot (LFC vs. −log10(adjusted p-value)) showing differential biotinylated protein enrichment between GDC-0941 and DMSO conditions 293HEK: KRAS WT cells, showing similar results in decreased proteins to 8988T-KO: KRAS WT cells.

Techniques: Western Blot, Expressing, Construct, Control, Protein Enrichment

(A) Schematic illustrating protein proximity labeling workflow. KRAS deficient PDAC cells reconstituted with various KRAS variants were treated with biotin and pharmacologic modulators of PI3K activity and protein lysates were subject to streptavidin pull-down prior to data-independent acquisition mass spectrometry (DIA-MS). (B) Heatmap showing row-normalized relative abundance of core RAS family proteins, effectors, scaffolds/adaptors, GAPs, and RTKs in detected in proximity to KRAS in reconstituted 8988T-KO cells in basal conditions. (C) Volcano plot (log2 fold-change (FC) vs. −log10(adjusted p-value) of 8988T-KO: KRAS WT cells treated with GDC-0941 vs DMSO showing reduced association of GAB1 and multiple scaffold/adaptor proteins upon PI3K inhibition. (D) Gene Ontology (GO) enrichment analysis of proteins significantly decreased following GDC-0941 treatment reveals enrichment of RAS-related signaling, actin cytoskeleton, and membrane-associated functions. (E) Schematic of genome-scale loss-of-function CRISPR screen (Brunello sgRNA library) performed in pools of 8988T KRAS intact and KO clones. Cells were grown for 14 population doublings following selection and genomic DNA was sequenced for sgRNA abundance at endpoint versus initial point. (F) Scatterplot showing the log2 fold-change (LFC) in abundance between end and initial time points of all genes in 8988T KRAS -intact and KO cells (calculated using MAGeCK). Blue dots indicate KRAS KO-specific dependencies (LFC < –1 and FDR < 0.25), while red dots indicate genes whose knockout confers a KRAS KO-specific growth advantage (LFC > 1 and FDR < 0.25). Dotted line separates genes associated with a >1 difference in LFC between KRAS -intact and KO cells with greater dependence in KO cells to the left of the line. RAS– MAPK signaling regulators identified in proximity biotinylation experiments in ( C ) are labelled. (G) KEGG pathway enrichment analysis was performed on genes that were significantly and specifically depleted in KRAS -KO cells (but not in KRAS -intact cells) in the genome-wide CRISPR screen. These KRAS KO-specific dependencies are enriched for pathways including PI3K signaling, MAPK signaling, and focal adhesion, highlighting key signaling and structural pathways required for cell survival in the absence of KRAS . (H) Dot plots showing relative changes in sgRNA abundance and FDR for key RAS family proteins, effectors, scaffolds/adaptors, GAPs, and RTKs in KRAS -intact and KO cells from the genome-wide CRISPR screen. Notably, positive regulators of MAPK signaling were more depleted in KRAS -KO cells while negative regulators ( e.g., GAPs) were more enriched.

Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) Schematic illustrating protein proximity labeling workflow. KRAS deficient PDAC cells reconstituted with various KRAS variants were treated with biotin and pharmacologic modulators of PI3K activity and protein lysates were subject to streptavidin pull-down prior to data-independent acquisition mass spectrometry (DIA-MS). (B) Heatmap showing row-normalized relative abundance of core RAS family proteins, effectors, scaffolds/adaptors, GAPs, and RTKs in detected in proximity to KRAS in reconstituted 8988T-KO cells in basal conditions. (C) Volcano plot (log2 fold-change (FC) vs. −log10(adjusted p-value) of 8988T-KO: KRAS WT cells treated with GDC-0941 vs DMSO showing reduced association of GAB1 and multiple scaffold/adaptor proteins upon PI3K inhibition. (D) Gene Ontology (GO) enrichment analysis of proteins significantly decreased following GDC-0941 treatment reveals enrichment of RAS-related signaling, actin cytoskeleton, and membrane-associated functions. (E) Schematic of genome-scale loss-of-function CRISPR screen (Brunello sgRNA library) performed in pools of 8988T KRAS intact and KO clones. Cells were grown for 14 population doublings following selection and genomic DNA was sequenced for sgRNA abundance at endpoint versus initial point. (F) Scatterplot showing the log2 fold-change (LFC) in abundance between end and initial time points of all genes in 8988T KRAS -intact and KO cells (calculated using MAGeCK). Blue dots indicate KRAS KO-specific dependencies (LFC < –1 and FDR < 0.25), while red dots indicate genes whose knockout confers a KRAS KO-specific growth advantage (LFC > 1 and FDR < 0.25). Dotted line separates genes associated with a >1 difference in LFC between KRAS -intact and KO cells with greater dependence in KO cells to the left of the line. RAS– MAPK signaling regulators identified in proximity biotinylation experiments in ( C ) are labelled. (G) KEGG pathway enrichment analysis was performed on genes that were significantly and specifically depleted in KRAS -KO cells (but not in KRAS -intact cells) in the genome-wide CRISPR screen. These KRAS KO-specific dependencies are enriched for pathways including PI3K signaling, MAPK signaling, and focal adhesion, highlighting key signaling and structural pathways required for cell survival in the absence of KRAS . (H) Dot plots showing relative changes in sgRNA abundance and FDR for key RAS family proteins, effectors, scaffolds/adaptors, GAPs, and RTKs in KRAS -intact and KO cells from the genome-wide CRISPR screen. Notably, positive regulators of MAPK signaling were more depleted in KRAS -KO cells while negative regulators ( e.g., GAPs) were more enriched.

Techniques: Labeling, Activity Assay, Data-independent acquisition, Mass Spectrometry, Inhibition, Membrane, CRISPR, Clone Assay, Selection, Knock-Out, Genome Wide

(A) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells treated with MRTX1133 +/- PI3K inhibitor (GDC-0941, 2 μM) or PI3K activator (1938, 1 μM). (B) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells treated with RMC-6236 +/- GDC-0941 (2 μM) or 1938 (1 μM). (C) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells transduced with PIK3CA WT or PIK3CA E545K and treated with MRTX1133. (D) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, of n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells transduced with PIK3CA E545K and treated with MRTX1133 +/- GDC-0941 (2 μM). (E) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells transduced with PIK3CA E545K and treated with RMC-6236 +/- GDC-0941 (2 μM).

Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells treated with MRTX1133 +/- PI3K inhibitor (GDC-0941, 2 μM) or PI3K activator (1938, 1 μM). (B) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells treated with RMC-6236 +/- GDC-0941 (2 μM) or 1938 (1 μM). (C) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells transduced with PIK3CA WT or PIK3CA E545K and treated with MRTX1133. (D) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, of n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells transduced with PIK3CA E545K and treated with MRTX1133 +/- GDC-0941 (2 μM). (E) Dose–response curves (three-parameter curve fit) of cell viability (normalized to DMSO control, mean ± SD, n = 3 technical replicates) of L3.3 and AsPC1 KRAS G12D mutant PDAC cells transduced with PIK3CA E545K and treated with RMC-6236 +/- GDC-0941 (2 μM).

Techniques: Control, Mutagenesis, Transduction

LDHA enhances the gemcitabine resistance and CSC properties of PDAC cells. ( A ) Left panel, the LDH enzyme activity in the PANC-1, PANC-1/GEM, MIA PaCa-2 and MIA PaCa-2/GEM cells. Right panel, the mRNA expression of LDHA and LDHB ( bottom ) and the protein expression of LDHA (top) were measured by RT-qPCR and Western blotting in these PDAC cell lines. α-tubulin was used as the internal protein loading control. ( B ) The LDHA and LDHB expression (left), and LDH enzyme activity (right) were determined for adherent and sphere PANC-1 cells. ( C ) Top, the protein expression of LDHA was analyzed by Western blotting in the indicated cell lines. Bottom , the viability of the indicated cell lines was measured by the MTT assay after treatment with 4 µM of gemcitabine for 48 h. ( D ) Representative images showed sphere formation (upper) and column graph presented number of spheres in the indicated PANC-1 cells (bottom). The total number of spheres was quantified across multiple replicate wells per condition, with the results shown as mean ± SEM. Scale bar: 20 μm. ( E ) The positive cells of CD133 expression and ALDH activity of the indicated PANC-1 cells were measured by flow cytometric analysis. ( F ) Kaplan-Meier plot of overall survival, relapse-free survival and recurrence-free survival in pancreatic cancer patients. Overall survival and relapse-free survival analyzed by the PROGgeneV2 online database. Recurrence-free survival in pancreatic cancer patients with gemcitabine treatment ( n = 20), stratified by LDHA expression. The LDHA expression in patient samples was classified according to the individual ΔCP values of LDHA relative to GAPDH , where the lower ΔCP values indicate higher expression of LDHA . The median of individual ΔCP values of patient samples was used as cut-off values to define high and low expression. P = 0.0009 (Log-rank test). ( G ) There was a positive correlation between LDHA and CSC marker expression, including CD44 , ALDHA1 and KLF4 , as determined by RT-qPCR in pancreatic cancer patients treated with gemcitabine ( n = 20). The Pearson’s correlation coefficient, r and P values, are shown in each panel. The results are presented as the means ± s.e.m. of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 (two-tailed Student’s t- test)

Journal: Cancer & Metabolism

Article Title: FOXO3a/miR-4259-driven LDHA expression as a key mechanism of gemcitabine sensitivity in pancreatic ductal adenocarcinoma

doi: 10.1186/s40170-025-00377-3

Figure Lengend Snippet: LDHA enhances the gemcitabine resistance and CSC properties of PDAC cells. ( A ) Left panel, the LDH enzyme activity in the PANC-1, PANC-1/GEM, MIA PaCa-2 and MIA PaCa-2/GEM cells. Right panel, the mRNA expression of LDHA and LDHB ( bottom ) and the protein expression of LDHA (top) were measured by RT-qPCR and Western blotting in these PDAC cell lines. α-tubulin was used as the internal protein loading control. ( B ) The LDHA and LDHB expression (left), and LDH enzyme activity (right) were determined for adherent and sphere PANC-1 cells. ( C ) Top, the protein expression of LDHA was analyzed by Western blotting in the indicated cell lines. Bottom , the viability of the indicated cell lines was measured by the MTT assay after treatment with 4 µM of gemcitabine for 48 h. ( D ) Representative images showed sphere formation (upper) and column graph presented number of spheres in the indicated PANC-1 cells (bottom). The total number of spheres was quantified across multiple replicate wells per condition, with the results shown as mean ± SEM. Scale bar: 20 μm. ( E ) The positive cells of CD133 expression and ALDH activity of the indicated PANC-1 cells were measured by flow cytometric analysis. ( F ) Kaplan-Meier plot of overall survival, relapse-free survival and recurrence-free survival in pancreatic cancer patients. Overall survival and relapse-free survival analyzed by the PROGgeneV2 online database. Recurrence-free survival in pancreatic cancer patients with gemcitabine treatment ( n = 20), stratified by LDHA expression. The LDHA expression in patient samples was classified according to the individual ΔCP values of LDHA relative to GAPDH , where the lower ΔCP values indicate higher expression of LDHA . The median of individual ΔCP values of patient samples was used as cut-off values to define high and low expression. P = 0.0009 (Log-rank test). ( G ) There was a positive correlation between LDHA and CSC marker expression, including CD44 , ALDHA1 and KLF4 , as determined by RT-qPCR in pancreatic cancer patients treated with gemcitabine ( n = 20). The Pearson’s correlation coefficient, r and P values, are shown in each panel. The results are presented as the means ± s.e.m. of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 (two-tailed Student’s t- test)

Article Snippet: Human pancreatic ductal adenocarcinoma (PDAC) cell lines (PANC-1, MIA PaCa-2, BxPC-3 and SUIT-2 cells) and human embryonic kidney 293 (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in culture medium according to their instructions.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Control, MTT Assay, Marker, Two Tailed Test

miR-4259 targets the LDHA 3’UTR and inhibits LDHA-mediated gemcitabine resistance in PDAC. ( A ) The miR-4259 expression (left) and LDHA -3’UTR luciferase activity (right) in PANC-1 and PANC-1/GEM cells was measured by RT-qPCR and a luciferase reporter assay, respectively. The RT-qPCR data were normalized to the level of U47 RNA in each individual sample. ( B ) A schematic diagram representing the predicted miR-4259-binding sequences or the mutated versions of the miRNA (left). The luciferase reporter activity (right) of the LDHA -3’UTR wild-type (+ 1 ~ + 937) and LDHA -3’UTR mutant reporters (mutant sites: 498, 498/518 and 498/518/818) were measured by a dual-luciferase reporter assay in HEK-293T cells transfected with miR-4259 and a reporter at different ratios. ( C ) The luciferase reporter activity of the LDHA -3’UTR wild-type and LDHA -3’UTR mutant reporters (triple-mutant sites, 498/518/818) in PANC-1/GEM and SUIT-2 cells and their expression of miR-4259. ( D ) The LDHA and miR-4259 expression (left) of PANC-1/GEM cells transfected with the indicated plasmids were analyzed by Western blotting and RT-qPCR, respectively. The cell viability (right) of these transfectants in the presence of gemcitabine treatment was measured by the MTT assay. ( E ) The LDHA and miR-4259 expression (left) of PANC-1 cells were analyzed by Western blotting and RT-qPCR, respectively. The cell viability ( right ) of these transfectants in the presence of gemcitabine treatment was measured by the MTT assay. The results are presented as the means ± s.e.m. of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 and n.s. not significant (two-tailed Student’s t -test)

Journal: Cancer & Metabolism

Article Title: FOXO3a/miR-4259-driven LDHA expression as a key mechanism of gemcitabine sensitivity in pancreatic ductal adenocarcinoma

doi: 10.1186/s40170-025-00377-3

Figure Lengend Snippet: miR-4259 targets the LDHA 3’UTR and inhibits LDHA-mediated gemcitabine resistance in PDAC. ( A ) The miR-4259 expression (left) and LDHA -3’UTR luciferase activity (right) in PANC-1 and PANC-1/GEM cells was measured by RT-qPCR and a luciferase reporter assay, respectively. The RT-qPCR data were normalized to the level of U47 RNA in each individual sample. ( B ) A schematic diagram representing the predicted miR-4259-binding sequences or the mutated versions of the miRNA (left). The luciferase reporter activity (right) of the LDHA -3’UTR wild-type (+ 1 ~ + 937) and LDHA -3’UTR mutant reporters (mutant sites: 498, 498/518 and 498/518/818) were measured by a dual-luciferase reporter assay in HEK-293T cells transfected with miR-4259 and a reporter at different ratios. ( C ) The luciferase reporter activity of the LDHA -3’UTR wild-type and LDHA -3’UTR mutant reporters (triple-mutant sites, 498/518/818) in PANC-1/GEM and SUIT-2 cells and their expression of miR-4259. ( D ) The LDHA and miR-4259 expression (left) of PANC-1/GEM cells transfected with the indicated plasmids were analyzed by Western blotting and RT-qPCR, respectively. The cell viability (right) of these transfectants in the presence of gemcitabine treatment was measured by the MTT assay. ( E ) The LDHA and miR-4259 expression (left) of PANC-1 cells were analyzed by Western blotting and RT-qPCR, respectively. The cell viability ( right ) of these transfectants in the presence of gemcitabine treatment was measured by the MTT assay. The results are presented as the means ± s.e.m. of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 and n.s. not significant (two-tailed Student’s t -test)

Techniques: Expressing, Luciferase, Activity Assay, Quantitative RT-PCR, Reporter Assay, Binding Assay, Mutagenesis, Transfection, Western Blot, MTT Assay, Two Tailed Test

FOXO3a/miR-4259 reduces the LDHA expression and enhances the gemcitabine sensitivity of PDAC in vivo. ( A ) Mice were subcutaneously implanted with the (PANC-1/shCtrl, PANC-1/shFOXO3a, PANC-1/shFOXO3a/pLemiR, PANC-1/shFOXO3a/pLemiR-4259) until the resulting tumours reached approximately 200 mm 3 . The mice were then intraperitoneally treated with vehicle or 50 mg/kg gemcitabine once a week. Each column represents the means ± s.e.m. of the tumour volumes of eight mice in each group. The tumour volume was calculated as described in the Methods section. ( B ) The indicated tumours from mice treated with the vehicle and gemcitabine for twenty-four days were dissected from the surrounding tissue. ( C ) Western blotting was performed to confirm the expression of FOXO3a and LDHA in the indicated groups of tumour samples. α-tubulin was used as a loading control. The relative expression of ( D ) miR-4259, ( E ) CD133 , CD44 and ALDHA1 were measured by RT-qPCR analysis. The results are shown as the means ± s.e.m. of three independent experiments. * P < 0.05, ** P < 0.01 and n.s. not significant (two-tailed Student’s t test)

Journal: Cancer & Metabolism

Article Title: FOXO3a/miR-4259-driven LDHA expression as a key mechanism of gemcitabine sensitivity in pancreatic ductal adenocarcinoma

doi: 10.1186/s40170-025-00377-3

Figure Lengend Snippet: FOXO3a/miR-4259 reduces the LDHA expression and enhances the gemcitabine sensitivity of PDAC in vivo. ( A ) Mice were subcutaneously implanted with the (PANC-1/shCtrl, PANC-1/shFOXO3a, PANC-1/shFOXO3a/pLemiR, PANC-1/shFOXO3a/pLemiR-4259) until the resulting tumours reached approximately 200 mm 3 . The mice were then intraperitoneally treated with vehicle or 50 mg/kg gemcitabine once a week. Each column represents the means ± s.e.m. of the tumour volumes of eight mice in each group. The tumour volume was calculated as described in the Methods section. ( B ) The indicated tumours from mice treated with the vehicle and gemcitabine for twenty-four days were dissected from the surrounding tissue. ( C ) Western blotting was performed to confirm the expression of FOXO3a and LDHA in the indicated groups of tumour samples. α-tubulin was used as a loading control. The relative expression of ( D ) miR-4259, ( E ) CD133 , CD44 and ALDHA1 were measured by RT-qPCR analysis. The results are shown as the means ± s.e.m. of three independent experiments. * P < 0.05, ** P < 0.01 and n.s. not significant (two-tailed Student’s t test)

Techniques: Expressing, In Vivo, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test

Review

Structured Review

Images

Similar Products

Image Search Results