Journal: International Journal of Oncology

Article Title: HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1

doi: 10.3892/ijo.2023.5493

Figure Lengend Snippet: HNF1A expression is associated with OXA resistance in PDAC tissue and cell lines (A) Reverse transcription-quantitative PCR analysis of HNF1A expression in OXA-S (n=35) and OXA-R (n=41) PDAC tissues. (B) Western blot analysis of HNF1A in PDAC tissue from ox OXA-S and OXA-R patients. (C) Immunohistochemistry analysis of HNF1A in OXA-S and OXA-R PDAC tissues. Scale bars, 50 µ m. (D) Survival of 76 patients with PDAC treated with platinum-based chemotherapy who had high or low HNF1A expression was analyzed based on Kaplan-Meier curves. (E) Dose-response curves were constructed for Panc-1 and MiaPaCa-2 cells to determine the IC 50 of OXA. (F) HNF1A mRNA expression levels in OXA-S and OXA-R Panc-1 and MiaPaCa-2 cells. (G) Western blot analysis of HNF1A protein expression in each group. Representative immunofluorescence images of HNF1A in OXA-S and OXA-R (H) Panc-1 and (I) MiaPaCa-2 cells. Scale bars, 50 µ m. The results are shown as the mean ± SD. *** P<0.001. HNF1A, hepatocyte nuclear factor 1 homeobox A; OXA, oxaliplatin; OXA-R, OXA-resistant; OXA-S, OXA-sensitive; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: First, 2 mg/ml oxaliplatin (cat. no. S1224; Selleck Chemicals, dissolved to storage concentration with DMSO and diluted to working concentrations with DMEM) solution was added to Panc-1 and MiaPaCa-2 cells and incubated at 37°C for 24 h; subsequently, the medium was replaced with oxaliplatin-free medium and cell proliferation was observed using a CCK-8 assay (cat. no. K1018; APExBIO Technology LLC).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Construct, Immunofluorescence

Journal: International Journal of Oncology

Article Title: HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1

doi: 10.3892/ijo.2023.5493

Figure Lengend Snippet: HNF1A mediates OXA resistance in pancreatic ductal adenocarcinoma cell lines in vitro . (A) Proliferation of Panc-1 and MiaPaCa-2 cells overexpressing HNF1A under OXA treatment (5 µ M), as determined using the Cell Counting Kit-8 assay. * P<0.05, ** P<0.01 and *** P<0.001 vs. Vector. (B) Proliferation of Panc-1 and MiaPaCa-2 cells following knockdown of HNF1A under OXA treatment (5 µ M), as determined using the Cell Counting Kit-8 assay. * P<0.05 and ** P<0.01 vs. si-NC. Representative images of the colony formation of Panc-1 and MiaPaCa-2 cells (C) post-HNF1A plasmid transfection or (D) post-si-HNF1A transfection and under OXA treatment (5 µ M). Sphere formation assays showing Panc-1 and MiaPaCa-2 cell proliferation (E) post-HNF1A plasmid transfection or (F) post-si-HNF1A transfection and under OXA treatment (5 µ M). Scale bars, 50 µ m. OXA-induced (5 µ M) apoptosis of Panc-1 and MiaPaCa-2 cells was examined through flow cytometry after (G) HNF1A plasmid or (H) si-HNF1A transfection. The results are shown as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001. HNF1A, hepatocyte nuclear factor 1 homeobox A; OXA, oxaliplatin; NC, negative control; si, small interfering.

Article Snippet: First, 2 mg/ml oxaliplatin (cat. no. S1224; Selleck Chemicals, dissolved to storage concentration with DMSO and diluted to working concentrations with DMEM) solution was added to Panc-1 and MiaPaCa-2 cells and incubated at 37°C for 24 h; subsequently, the medium was replaced with oxaliplatin-free medium and cell proliferation was observed using a CCK-8 assay (cat. no. K1018; APExBIO Technology LLC).

Techniques: In Vitro, Cell Counting, Plasmid Preparation, Knockdown, Transfection, Flow Cytometry, Negative Control

Journal: International Journal of Oncology

Article Title: HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1

doi: 10.3892/ijo.2023.5493

Figure Lengend Snippet: HNF1A switches HR to NHEJ. Neutral comet assay was used to measure the DNA damage caused by OXA (5 µ M) in cells with (A) HNF1A overexpression or (B) HNF1A knockdown. Scale bar, 10 µ m. Representative images of γH2AX-positive foci in Panc-1 and MiaPaCa-2 cells with (C) HNF1A overexpression or (D) HNF1A knockdown and treated with OXA (5 µ M). Scale bar, 10 µ m. Panc-1 and MiaPaCa-2 cells HR-mediated DNA repair efficiency was detected by the (E) pDR-GFP reporter assay and (F) pimEJ5-GFP reporter assay in vector and HNF1A-overexpressing groups. (G) Panc-1 and MiaPaCa-2 cells HR-mediated DNA repair efficiency was detected by the pDR-GFP reporter assay si-NC and si-HNF1A groups. (H) Panc-1 and MiaPaCa-2 cells NHEJ-mediated DNA repair efficiency was detected by the pimEJ5-GFP reporter assay in si-NC and si-HNF1A groups. The technical replicates were performed three times. The results are shown as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001. HNF1A, hepatocyte nuclear factor 1 homeobox A; HR, homologous recombination; NC, negative control; NHEJ, nonhomologous end joining; OXA, oxaliplatin; si, small interfering.

Article Snippet: First, 2 mg/ml oxaliplatin (cat. no. S1224; Selleck Chemicals, dissolved to storage concentration with DMSO and diluted to working concentrations with DMEM) solution was added to Panc-1 and MiaPaCa-2 cells and incubated at 37°C for 24 h; subsequently, the medium was replaced with oxaliplatin-free medium and cell proliferation was observed using a CCK-8 assay (cat. no. K1018; APExBIO Technology LLC).

Techniques: Neutral Comet Assay, Over Expression, Knockdown, Reporter Assay, Plasmid Preparation, Homologous Recombination, Negative Control

Journal: International Journal of Oncology

Article Title: HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1

doi: 10.3892/ijo.2023.5493

Figure Lengend Snippet: 53BP1 reverses the effect of HNF1A knockdown. (A) Proliferation of Panc-1 and MiaPaCa-2 cells with the indicated treatment was measured by Cell Counting Kit-8 assay. (B) Representative images and semi-quantification of colony formation in Panc-1 and MiaPaCa-2 cells treated with OXA (5 µ M). (C) Representative images and semi-quantification of sphere formation in Panc-1 and MiaPaCa-2 cells with the indicated treatment under OXA incubation (5 µ M). Scale bars, 50 µ m. (D) Flow cytometry was performed to assess the apoptosis of Panc-1 and MiaPaCa-2 cells treated with OXA (5 µ M). (E) Representative images and semi-quantification of the neutral comet assay in Panc-1 and MiaPaCa-2 cells. In total, 50 cells per group were counted. Scale bar, 10 µ m. (F) Semi-quantification and representative images of γH2AX-positive foci in each group. More than 40 cells from each group were counted. (G and H) HR-mediated DNA repair efficiency was detected by the pimEJ5-GFP reporter assay, whereas NHEJ-mediated DNA repair efficiency was detected by the pDR-GFP reporter assay. Three independent replicates were performed. Data are expressed as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001. 53BP1, p53-binding protein 1; HNF1A, hepatocyte nuclear factor 1 homeobox A; HR, homologous recombination; NC, negative control; NHEJ, nonhomologous end joining; OXA, oxaliplatin; si, small interfering.

Article Snippet: First, 2 mg/ml oxaliplatin (cat. no. S1224; Selleck Chemicals, dissolved to storage concentration with DMSO and diluted to working concentrations with DMEM) solution was added to Panc-1 and MiaPaCa-2 cells and incubated at 37°C for 24 h; subsequently, the medium was replaced with oxaliplatin-free medium and cell proliferation was observed using a CCK-8 assay (cat. no. K1018; APExBIO Technology LLC).

Techniques: Knockdown, Cell Counting, Incubation, Flow Cytometry, Neutral Comet Assay, Reporter Assay, Binding Assay, Homologous Recombination, Negative Control

Journal: International Journal of Oncology

Article Title: HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1

doi: 10.3892/ijo.2023.5493

Figure Lengend Snippet: HNF1A knockdown promotes OXA resistance in vivo . (A) Representative IVIS images of an orthotopic xenograft model, n=5/group. (B) Calculation of the luminescence intensity of the orthotopic xenograft model. (C) Representative pancreatic tumor images of the orthotopic xenograft model. (D) Representative IHC images for HNF1A, 53BP1, γH2AX and Ki-67 in the orthotopic xenograft model. Scale bars, 50 µ m. (E) Once the tumor volume reached 200 mm 3 , the xenograft subcutaneous model received oxaliplatin (5 mg/kg) once every 3 days, n=5/group. Changes in (F and G) tumor volume and (H) weight were monitored. (I) Representative IHC images for HNF1A, 53BP1 and γH2AX in the patient-derived xenograft model. The results are presented as the mean ± SD. *** P<0.001, 53BP1, p53-binding protein 1; HNF1A, hepatocyte nuclear factor 1 homeobox A; IHC, immunohistochemistry; NC, negative control; OXA, oxaliplatin.

Article Snippet: First, 2 mg/ml oxaliplatin (cat. no. S1224; Selleck Chemicals, dissolved to storage concentration with DMSO and diluted to working concentrations with DMEM) solution was added to Panc-1 and MiaPaCa-2 cells and incubated at 37°C for 24 h; subsequently, the medium was replaced with oxaliplatin-free medium and cell proliferation was observed using a CCK-8 assay (cat. no. K1018; APExBIO Technology LLC).

Techniques: Knockdown, In Vivo, Derivative Assay, Binding Assay, Immunohistochemistry, Negative Control

Journal: International Journal of Oncology

Article Title: HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1

doi: 10.3892/ijo.2023.5493

Figure Lengend Snippet: Clinical significance of the HNF1A/53BP1 axis in PDAC. (A) Immunohistochemistry analysis of 53BP1 in OXA-S and OXA-R PDAC tissues. Scale bars, 50 µ m. (B) Semi-quantification of the percentage of OXA-S and OXA-R PDAC specimens with low or high 53BP1 expression. (C) Correlation between HNF1A expression and 53BP1 protein levels in PDAC tissues. (D) Kaplan-Meier curves of progression-free survival associated with 53BP1 expression evaluated by IHC in patients with PDAC who received platinum-based chemotherapy. (E) Graphical illustration of the mechanism by which HNF1A mediates 53BP1 activation in PDAC cells to reduce oxaliplatin resistance. 53BP1, p53-binding protein 1; HNF1A, hepatocyte nuclear factor 1 homeobox A; HR, homologous recombination; IHC, immunohistochemistry; NHEJ, nonhomologous end joining; OXA, oxaliplatin; OXA-R, OXA-resistant; OXA-S, OXA-sensitive; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: First, 2 mg/ml oxaliplatin (cat. no. S1224; Selleck Chemicals, dissolved to storage concentration with DMSO and diluted to working concentrations with DMEM) solution was added to Panc-1 and MiaPaCa-2 cells and incubated at 37°C for 24 h; subsequently, the medium was replaced with oxaliplatin-free medium and cell proliferation was observed using a CCK-8 assay (cat. no. K1018; APExBIO Technology LLC).

Techniques: Immunohistochemistry, Expressing, Activation Assay, Binding Assay, Homologous Recombination

Journal: STAR Protocols

Article Title: Single-embryo RNA sequencing for continuous and sex-specific gene expression analysis on Drosophila

doi: 10.1016/j.xpro.2023.102535

Figure Lengend Snippet:

Article Snippet: Microseal 'F' PCR seal (for storage at −80°C) , Bio-Rad , Cat# MSF1001.

Techniques: Recombinant, Saline, Reverse Transcription, Sensitive Assay, SYBR Green Assay, Software, Transferring