Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: CD44s forms physical complexes with iASPP, both in mesenchymal and epithelial cells. ( A ) The expression levels of CD44, iASPP, and p53 were analysed in cell lysates from AG1523 fibroblasts, hTERT-BJ foreskin fibroblasts, breast cancer cells MCF-7, HEK293, lung carcinoma H1299, sarcoma U2OS, human dermal fibroblasts MTS64, and hepatoma HepG2 cells by immunoblotting (IB) with indicated antibodies, using anti-GAPDH as loading control. ( B ) Cell lysates from the selected cell lines were subjected to immunoprecipitation with an iASPP antibody or 3 µg/mL control rabbit IgG, followed by immunoblotting for CD44 and iASPP. ( C ) hTERT-BJ fibroblasts and MCF10A cells were cultured for 24 h in starvation medium prior stimulation, or not, with hyaluronan for 45 min. Cell lysates were then subjected to immunoprecipitation, using an iASPP antibody or control rabbit IgG, followed by immunoblotting with CD44, iASPP, and p53 antibodies. Note the absence of co-immunoprecipitated band at the expected position of CD44v just above the CD44s band. A representative experiment out of three performed is shown; lower panel in C indicates fold-change of mean values ± SD. of three independent experiments: * p < 0.05, Student’s t -test; significant difference compared with unstimulated cells. The uncropped blots are shown in .

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Cell Culture

Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: Schematic illustration of the structures of p53, iASPP, and CD44, and CD44 mutants. ( A ) An established interaction between domains in p53 and iASPP is indicated by a line, and the interaction found in the present study between iASPP and CD44s is indicated with a dashed line. ( B ) The cytosolic sequence of CD44 and the mutants used in this study are indicated.

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Sequencing

Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: p53 expression levels modulate formation of the complex between iASPP and CD44. hTERT-BJ cells, treated with p53 siRNA, or not, were stimulated with 10% FBS or 10 nM Nutlin. Lysates were subjected to immunoprecipitation (IP) with an iASPP antiserum, followed by immunoblotting with antibodies against iASPP and p53. Quantification of the data is shown below the blot. The data are plotted in bar graphs representing the mean ± SD of at least three independent experiments (* p < 0.05, Student’s t -test). The uncropped blots are shown in .

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: Depletion of CD44 promotes the translocation of p53 and iASPP to the cytoplasm. ( A ) hTERT-BJ cells treated with CD44 siRNA, or not, were stained for CD44 (blue), p53 (green), or iASPP (red). Images were taken using a Zeiss Axioplan 2 immunofluorescence microscope with a 63× objective. By ImageJ software, the localizations of p53 and iASPP in cytosol and nuclei were measured and are shown in the panels to the right. Scale bars, 10 µm. * p < 0.05, Student´s t -test. ( B ) Subcellular fractionation was performed on untreated or hyaluronan-stimulated hTERT-BJ cells transfected with siRNA for CD44 or scramble siRNA as control. Cell lysates were then immunoprecipitated with an iASPP antibody, followed by immunoblotting with iASPP, p53, and CD44 antibodies. The purity of the nuclear and cytoplasmic fractions was determined by immunoblotting for laminin and GAPDH, respectively. A representative experiment out of three independent experiments performed is shown. The data are plotted in bar graphs representing the mean ± SD of at least three independent experiments (* p < 0.05, Student’s t -test). The uncropped blots are shown in .

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Translocation Assay, Staining, Immunofluorescence, Microscopy, Software, Fractionation, Transfection, Control, Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: Knock-down of CD44 in AG1523 fibroblast leads to loss of contact inhibition. ( A , B ) AG1523 fibroblasts, transfected with CD44 siRNA or scramble siRNA, were seeded in 6-well plates and grown to a density of 10 5 cells per well (regular density; RD) or >10 6 cells per well (high density; HD), counted, and observed under microscopy. Scale bars, 20 µm. ( C ) Cell lysates from RD and HD cell cultures were subjected to immunoblotting using indicated antibodies. ( D ) AG1523 cells, treated with CD44 siRNA, iASPP siRNA, or scramble siRNA, were stimulated, or not, with hyaluronan, PDGF-BB, and 10% FBS, and then subjected to a 3 H-thymidine incorporation assay. Knock-down efficiencies are shown in immunoblots. ( E ) Cell lysates from RD and HD cell cultures, incubated in Nutlin or 10% FBS, were subjected to immunoprecipitation using an iASPP antibody, followed by immunoblotting using indicated antibodies. The data are plotted in bar graphs representing the mean ± SD of at least three independent experiments (* p < 0.05 and ** p < 0.01, Student’s t -test). The uncropped blots are shown in .

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Knockdown, Inhibition, Transfection, Microscopy, Western Blot, Thymidine Incorporation Assay, Incubation, Immunoprecipitation

Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: iASPP is required for hyaluronan-induced fibroblast migration. Confluent cultures of hTERT-BJ fibroblasts, transfected with iASPP siRNA or scrambled siRNA, were scratched and then stimulated, or not, with hyaluronan or PDGF-BB in DMEM medium containing 0.1% FBS. The wound size was examined after 24 h of culturing pictures were taken and analyzed using TScratch program (a software tool for automated analysis of wound-healing assays). The effect of hyaluronan stimulation and knock-down of iASPP on the migration of fibroblasts, are shown in bar graphs. The iASPP knock-down efficiency was determined by immunoblotting using an iASPP antibody; equal loading was determined by immunoblotting for GAPDH. The data are plotted in bar graphs representing the mean ± SD of at least three independent experiments (** p < 0.01 and *** p <0.001 Student’s t -test). The uncropped blots are shown in .

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Migration, Transfection, Software, Knockdown, Western Blot

Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: iASPP suppresses but p53 increases CD44-mediated adhesion of fibroblasts. ( A , B ) AG1523 fibroblasts incubated with iASPP siRNA ( A ), p53 siRNA ( B ), or scramble siRNA, were pre-treated, or not, with hyaluronan and blocking CD44 antibody or PMA and then seeded (30,000 cells per well) and incubated for 30 min. Adhered cells were then visualized by staining with crystal violet and quantified by measurement of the absorbance at 570 nm. The data are plotted in bar graphs representing the mean ± SD of at least three independent experiments (* p < 0.05, Student’s t -test). The uncropped blots are shown in .

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Incubation, Blocking Assay, Staining

Journal: Cancers

Article Title: Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival

doi: 10.3390/cancers15041082

Figure Lengend Snippet: iASPP-CD44 complexes suppress but CD44 increases p53-mediated ROS production. V5-tagged wt iASPP (iASPP wt-v5) or CD44 were transfected individually or in combination in HEK293 cells expressing high level of endogenous p53wt, and the amount of ROS was determined. The data are plotted in bar graphs representing the mean ± SD of at least three independent experiments (* p < 0.05, Student’s t -test).

Article Snippet: AG1523 cells depleted of iASPP or p53 by siRNA (100,000 cells/350 μL DMEM, 0.1% BSA) were collected in Eppendorf tubes and incubated with hyaluronan (100 μg/mL for 60 min), anti-CD44 Hermes1 antibodies (20 μg/mL for 30 min), PMA (100 nM; 10 min), or combinations thereof.

Techniques: Transfection, Expressing

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: Tenascin C contributes to NRP-1 associated migration. a. Dual immunofluorescence staining (40× magnification scale bar 10 μm) of NRP-1 and TNC on BT-474 and BT-474 NRP-1 cells indicates their colocalization in the cytoplasm. Treatment of BT-474 NRP-1 cells with TNC targeted siRNA molecules, b , reduced TNC gene expression, c , reduced migratory capacity and d , downregulated NRP-1 and vimentin expression. (TNC protein was not detected on western blot due to the lack of specific antibody for this application.) The gene expression fold change was measured by comparing the basal levels detected in the empty plasmid transfected BT-474 or in the case of the siRNA experiment, to the control siRNA treated BT-474 and normalized to β-Actin and GUSB reference gene expression. Wound healing assay images (panel d, 5× magnification, scale bar 500 μm) taken on day 0 and day 2 after siRNA transfection. Graphs represent the mean ± SEM of three independent experiments. Statistical analysis using independent samples t-test, p-value < 0.05 considered as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Staining, Gene Expression, Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Wound Healing Assay

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: NRP-1 overexpression activates integrin β3 and TNFR2 pathways. Representative western blot images of protein lysates from untransfected BT-474, BT-474 NRP-1 and empty vector control cells blotted with indicated antibodies involved in a. Integrin signaling, and downstream signaling targets FAK, Akt, GSK3-β and NF-kB b. siRNA-mediated TNC downregulation decreased phosphorylation of FAK and Akt-473. c. Blots show levels of tumor necrosis factor receptors (TNFRs). GAPDH protein expression is indicated as a loading control. (The prefix P beside the antibody names indicates the phosphorylated form)

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Over Expression, Western Blot, Plasmid Preparation, Control, Phospho-proteomics, Expressing

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