Journal: Cancer Science

Article Title: Pancreatic ductal adenocarcinoma cells regulated the gemcitabine‐resistance function of CAFs by LINC00460

doi: 10.1111/cas.15547

Figure Lengend Snippet: LINC00460 could directly bind with PDAP1. (A) FISH assay showed the sub‐cellular location of LINC00460 in CFPAC‐1. (U6: nucleus location positive control, 18S: cytoplasm control) The subcellular location of LINC00460 is mainly in cytoplasm, followed by the nucleus. (B) LINC00460 expression in the nucleus and cytoplasm fractions by qRT‐PCR were consistent with FISH results. Data from three independent experiments were expressed as mean ± SD. (C) RNA‐pull‐down‐MS results showed 291 proteins have the possibility of binding to LINC00460 in the intersection of three PDAC cell lines. (D) Volcano plot of RNA‐pull‐down‐MS revealed that PDAP1 (Log 10 [iBAQ] = 9.10) interacts with LINC00460. The red font indicates PDAP1. (E) RNA immunoprecipitation (RIP)‐qPCR assay detects the coupling of PDAP1 and LINC00460. (F) Predicted structure of the LINC00460 determined by RNAfold software and the prediction of LINC00460/PDAP1 interaction probabilities using RPIseq website. (G) Immunoblot of PDAP1 in RNA pull‐down extracts with different LINC00460 fragment sequences (1–237, 238–747, and 748–913).

Article Snippet: Thereafter, immunoblottings were performed with below primary antibodies against PDAP1 (#15081‐1‐AP, Proteintech), PDGFA (#203911, Abcam), PDGFR (#32570, Abcam), a PDGFR Activation Antibody Sampler Kit (#12651, CST), β‐actin (#4970, CST), and GAPDH (#60004‐1‐Ig, Proteintech) in TBST buffer containing 5% BSA overnight.

Techniques: Positive Control, Control, Expressing, Quantitative RT-PCR, Binding Assay, RNA Immunoprecipitation, Software, Western Blot

Journal: Cancer Science

Article Title: Pancreatic ductal adenocarcinoma cells regulated the gemcitabine‐resistance function of CAFs by LINC00460

doi: 10.1111/cas.15547

Figure Lengend Snippet: LINC00460 could regulate the proliferation of CAFs via the PDAP1/PDGFA/PDGFR pathway. (A) qRT‐PCR assay detected the mRNA level of PDAP1 and PDGFA after transduced LINC00460 shRNAs. (B) Western blot assay detected the protein level of PDAP1 and PDGFA after transduced LINC00460 shRNAs. (C) The concentration of PDGFA in the medium supernatant of CFAPC‐1 and PANC‐1 after silencing LINC00460 was measured by ELISA assay; values represent means ± SD of six replicate samples in each group. (D) Western blot assay validated KO‐PDAP1 cell lines. (E) Western blot assay showed the expression level of PDGFA in PANC‐1 cell line (KO‐PDAP1) after transduction of LINC00460 shRNAs. (F) ELISA assay showed the concentration of PDGFA in medium supernatant of PANC‐1 cell line (KO‐PDAP1) after transduction of LINC00460 shRNAs. Values represent means ± SD of six replicate samples in each group. (G) Western blot assay showed PDGFR‐β expression in eight pairs of PDAC CAFs and tumor cells. (H) Cell viability showed the different proliferation rate of CAFs after co‐cultured with the supernatant of CFPAC‐1 and PANC‐1. (I) Cell viability showed that crenolanib can reverse the proliferation of CAFs caused by silencing LINC00460. (J) Western blot analysis of the PDGFR‐β pathway activated genes in after transduced LIINC00460 shRNAs whose co‐cultured with CFPAC‐1 or PANC‐1 supernatant. (K) Western blot shows crenolanib can reverse PDGFR‐β pathway activation.

Article Snippet: Thereafter, immunoblottings were performed with below primary antibodies against PDAP1 (#15081‐1‐AP, Proteintech), PDGFA (#203911, Abcam), PDGFR (#32570, Abcam), a PDGFR Activation Antibody Sampler Kit (#12651, CST), β‐actin (#4970, CST), and GAPDH (#60004‐1‐Ig, Proteintech) in TBST buffer containing 5% BSA overnight.

Techniques: Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transduction, Cell Culture, Activation Assay

Journal: Cancer Science

Article Title: Pancreatic ductal adenocarcinoma cells regulated the gemcitabine‐resistance function of CAFs by LINC00460

doi: 10.1111/cas.15547

Figure Lengend Snippet: Proposed schematic model illustrating the interaction between LINC00460 and PDAP1, leading to the increase of CAF proliferation, which could cause chemotherapy resistance.

Article Snippet: Thereafter, immunoblottings were performed with below primary antibodies against PDAP1 (#15081‐1‐AP, Proteintech), PDGFA (#203911, Abcam), PDGFR (#32570, Abcam), a PDGFR Activation Antibody Sampler Kit (#12651, CST), β‐actin (#4970, CST), and GAPDH (#60004‐1‐Ig, Proteintech) in TBST buffer containing 5% BSA overnight.

Techniques:

Journal: International journal of oncology

Article Title: Synergistic effects of imatinib and carboplatin on VEGF, PDGF and PDGF-Rα/ß expression in squamous cell carcinoma of the head and neck in vitro.

doi: 10.3892/ijo.2011.912

Figure Lengend Snippet: Figure 1. Immunohistochemistry for PDGF-α expression; (A and B) Positive immunohistochemical reactivity against PDGF-α (x20) after incubation with carboplatin and imatinib (3+18 µmol) after 48 h. (C and D) Positive immunohistochemical reactivity against PDGF-α (x20-40) with carboplatin and imatinib (7.5+30 µmol) after 120 h. (E-F) Control group with strong immunoreactivity.

Article Snippet: Immunohistochemistry of PDGF-A/B ligand. immunohistochemical studies were performed using a monoclonal rabbit antibody directed against human PDGF-A/B (ACRIS Antibodies, Herford, Germany).

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining, Incubation, Control