Journal: Oncology Letters

Article Title: PDGF-BB regulates the transformation of fibroblasts into cancer-associated fibroblasts via the lncRNA LURAP1L-AS1/LURAP1L/IKK/IκB/NF-κB signaling pathway

doi: 10.3892/ol.2021.12798

Figure Lengend Snippet: PDGF-BB induced the formation of the CAF phenotype in hOMF. (A) The cells were treated with PDGF-BB in a dose-dependent manner (10, 20, 30 ng/ml) in 10% fetal bovine serum for 72 h for three passages. The cells were subjected to western blot analysis with antibodies against CAF markers α-SMA, FAP-α, PDGFR-β and p-PDGFR-β. β-tubulin served as a loading control and sample loading was 20 µg. (B) Fluorescence microscopy of hOMF stained with α-SMA, FAP-α and PDGFR-β (probed with a primary and a secondary antibody). Cells were counterstained with DAPI. Data were expressed as means ± SEM (n=3). *P<0.05 vs. hOMF, # P<0.05 vs. 10 ng/ml PDGF-BB. PDGF, platelet-derived growth factor; CAF, cancer-associated fibroblast; hOMF, human oral mucosa (p3) 500K fibroblast; α-SMA, α-smooth muscle actin; FAP-α, fibroblast activation protein-α; PDGFR-β, platelet-derived growth factor receptor-β; p, phosphorylated.

Article Snippet: The following antibodies were used in the present study: α-SMA (cat. no. ab5694; 1:1,000), FAP-α (cat. no. ab53066; 1:500), IKKα (cat. no. ab32041; 1:1,000), IκBα (cat. no. ab32518; 1:1,000), NF-κB p65 (cat. no. ab16502; 1:1,000), p-NF-κBp65 (p-S536; cat. no. ab86299; 1:1,000), GAPDH (cat. no. ab181602; 1:10,000), β-tubulin (cat. no. ab179511; 1:1,000) and β-actin (cat. no. ab8227; 1:1,000) were obtained from Abcam; LURAP1L (cat. no. PA5-55072; 1:1,000) was obtained from Thermo Fisher Scientific, Inc., and PDGFR-β (cat. no. bs-0232R; 1:1,000) and p-PDGFR-β (Tyr740; cat. no. bs-3323R; 1:1,000) were obtained from BIOSS.

Techniques: Western Blot, Fluorescence, Microscopy, Staining, Derivative Assay, Activation Assay

Journal: Oncology Letters

Article Title: PDGF-BB regulates the transformation of fibroblasts into cancer-associated fibroblasts via the lncRNA LURAP1L-AS1/LURAP1L/IKK/IκB/NF-κB signaling pathway

doi: 10.3892/ol.2021.12798

Figure Lengend Snippet: Effects of the PDGFR-β inhibitor, CP-673451 on LURAP1L-AS1-LURAP1L/IKK/IκB/NF-κB signaling and CAF programming. (A) Reverse transcription-quantitative PCR analysis of LURAP1L and LURAP1L-AS1 expression levels. (B) Western blot analysis of FAP-α, α-SMA, IKKα, IκBα, NF-κB p65 and p-p65 expression following overexpression of LURAP1L-AS1 and treatment with PDGF-BB. *P<0.05 vs. PDGF-BB. PDGF, platelet-derived growth factor; LURAP1L, leucine-rich adaptor protein 1-like; LURAP1L-AS1, LURAP1L antisense RNA 1; IKKα, IκB kinase α; IKK, I-κB kinase; IκBα, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α; NF-κB, nuclear factor-κB; CAF, cancer-associated fibroblasts; p, phosphorylated; α-SMA, α-smooth muscle actin; FAP-α, fibroblast activation protein-α.

Article Snippet: The following antibodies were used in the present study: α-SMA (cat. no. ab5694; 1:1,000), FAP-α (cat. no. ab53066; 1:500), IKKα (cat. no. ab32041; 1:1,000), IκBα (cat. no. ab32518; 1:1,000), NF-κB p65 (cat. no. ab16502; 1:1,000), p-NF-κBp65 (p-S536; cat. no. ab86299; 1:1,000), GAPDH (cat. no. ab181602; 1:10,000), β-tubulin (cat. no. ab179511; 1:1,000) and β-actin (cat. no. ab8227; 1:1,000) were obtained from Abcam; LURAP1L (cat. no. PA5-55072; 1:1,000) was obtained from Thermo Fisher Scientific, Inc., and PDGFR-β (cat. no. bs-0232R; 1:1,000) and p-PDGFR-β (Tyr740; cat. no. bs-3323R; 1:1,000) were obtained from BIOSS.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Over Expression, Derivative Assay, Activation Assay

Journal: Drug Design, Development and Therapy

Article Title:

miR-370-3p Alleviates Ulcerative Colitis-Related Colorectal Cancer in Mice Through Inhibiting the Inflammatory Response and Epithelial-Mesenchymal Transition

doi: 10.2147/dddt.s238124

Figure Lengend Snippet: Figure 3 Effect of miR-370-3p on inflammatory response and TLR4 signaling pathway in AOM/DSS-induced mice. (A) The expression of TNF-α, IL-1β, and IL-6 in colonic tissues of mice from the Sham group, UC-CRC group, Ad-control group, and Ad-miR-370-3p group was detected by qRT-PCR. (B) The production of TNF-α and PGE2 in the lysate of the tumor colon tissues were detected by ELISA. (C) The expression of TLR4 and COX-2 in the tumor colon tissues from each group was evaluated by using IHC staining. Scale bar=50 μm. (D) The expression levels of TLR4, COX-2, and pEGFR in the tumor colon tissues from each group were determined by Western blot, and the relative band intensity was analyzed.The comparison among groups was performed by One-way ANOVA. Data are presented as mean ± SD. ##p<0.01 compared with the Sham group, **p<0.01 compared with the Ad-control group.

Article Snippet: After blocking with 5% skim milk (Sangon Biotech, Shanghai China) at room temperature for 1 h, the membranes were incubated with corresponding primary antibodies incuding TLR4 (1:1000, proteintech, Wuhan, China), COX-2 (1:1000, proteintech, Wuhan, China), pEGFR (phosphorylated epidermal growth factor receptor, 1:1000, Affinity, Changzhou, Jiangsu, China), β-catenin (1:5000, proteintech, Wuhan, China), p53 (1:3000, proteintech, Wuhan, China), ki67 (1:1000, Affinity, Changzhou, Jiangsu, China), E-cadherin (1:10,000, proteintech, Wuhan, China), N-cadherin (1:5000, proteintech, Wuhan, China), Vimentin (1:5000, proteintech, Wuhan, China) and GAPDH (1:10,000, proteintech, Wuhan, China) overnight at 4° C. Then the membranes were washed by TBST and incubated with appropriate secondary HRPconjugated goat anti-rabbit or goat anti-mouse antibodies (1:3000, Solarbio, Beijing, China) for 1 h at 37 ° C. The proteins were visualized by using ECL Western Blotting Substrate (Solarbio, Beijing, China).

Techniques: Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot, Comparison

Journal: Biochemical and biophysical research communications

Article Title: Rebound pathway overactivation by cancer cells following discontinuation of PI3K or mTOR inhibition promotes cancer cell growth.

doi: 10.1016/j.bbrc.2019.04.044

Figure Lengend Snippet: Fig. 4. Blocking IGF-1R prevents drug washout mediated cancer cell growth. (A) Washout of PI3K or mTOR inhibitors increases IGF-1Rb phosphorylation. HT-29 cells were treated for six hours with DMSO, BYL719 (10 mM) (BYL), BKM-120 (500 nM) (BKM), PP242 (5 mM) or Ku0063794 (5 mM) (Ku). Following washout, cells were incubated for six hours, and cell lysates were collected and analyzed for IGF-1Rb phosphorylation and actin. (B) IGF-1R inhibition with NVP-AEW541 blocks AKT phosphorylation mediated by washout of PP242 or BKM-120. HT-29 cells were treated with DMSO, PP242 (5 mM) or BKM-120 (500 mM) for 6 h. Following drug washout, cells were treated as indicated with sapitinib (2 mM), NVP- AEW541 (1 mM) (NVP-AEW) or erlotinib (1 mM) for six hours, and AKT phosphorylation was assessed by Western blot in cell lysates. (C) NVP-AEW541 inhibits cancer cell pro- liferation induced by washout of PP242 or BKM-120. HT-29 cells were treated with DMSO, PP242 (5 mM) or BKM-120 (500 nM) for six hours. Following drug washout, cells were incubated with DMSO or NVP-AEW541 (1 mM) for an additional 48 h. Cell proliferation was assessed with an MTS proliferation assay. Columns: Mean cell proliferation of three independent experiments expressed as percentage of DMSO treated cancer cells. Bars: SD. White columns: DMSO treated cells, grey columns: cells treated with PP242, and black columns: cells treated with BKM-120. *p < 0.05 compared to control cells.

Article Snippet: Anti-phospho-AKT (#4060), anti-AKT (#2920), and anti phospho-IGF-1Rb (#3024) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Blocking Assay, Phospho-proteomics, Incubation, Inhibition, Western Blot, Proliferation Assay, Control

Journal: Biology

Article Title: Chemical Composition, Antioxidant Capacity, and Anticancerous Effects against Human Lung Cancer Cells of a Terpenoid-Rich Fraction of Inula viscosa

doi: 10.3390/biology13090687

Figure Lengend Snippet: IVL DCM fraction inhibited the proliferation of A549 cells. A549 cells were treated with the indicated concentrations of IVL DCM for 48 h. ( A ) Ki67, P21, P27, p-P38, and P38 protein levels as detected by immunoblotting of A549 cell lysates. ( B ) Quantification of the bands in ( A ). Bar graphs of band intensity of target proteins normalized to the intensity of the loading control β-actin expressed as fold change of the vehicle-control and represented as the mean ± SEM of three independent experiments ( n = 3). p-P38 protein levels were normalized to P38 protein. * p < 0.05 and ** p < 0.01.

Article Snippet: Primary antibodies used were: anti-human poly-adenosine diphosphate (ADP) ribose polymerase (PARP) 46D11 rabbit mAb which can detect the full-length and cleaved forms of PARP (ref 9532 CST, dilution 1/1000), P53 rabbit mAb (ref 2527S CST, dilution 1/500), Ki67 rabbit polyclonal antibody (ref 28074-1-AP, Proteintech, Rosemont, IL, USA, dilution 1/1000), mouse anti-human B-cell lymphoma 2 (BCL2) (ref 15071S CST, dilution 1/1000), rabbit anti-Bcl-2 associated X protein (BAX) (D2E11 ref 5023 CST; dilution 1/1000), P38 MAPK polyclonal antibody (14064-1-AP, Proteintech, dilution 1/1000), phospho-p38 MAPK (Thr180/Tyr182) antibody(ref 9211 CST, dilution 1/1000), Caspase3 antibody (ref 9662 CST, 1/1000 dilution), cleaved Caspase3 (c-Caspase 3; Asp175) 5A1E rabbit mAb (ref 9664 CST, dilution 1/1000), β-actin 8H10D10 mouse mAb (ref 3760 CST, dilution 1/1000), P21 Waf1/Cip1 12D1 rabbit mAb (ref 2947 CST, 1/1000), P27 (CST, 1/1000), phospho-FAK (p-Fak Tyr 397) D20B1 rabbit mAb (ref 8556 CST, dilution 1/1000), FAK D2R2E rabbit mAb (ref 13009 CST, dilution 1/1000).

Techniques: Western Blot, Control

Journal: Biology

Article Title: Chemical Composition, Antioxidant Capacity, and Anticancerous Effects against Human Lung Cancer Cells of a Terpenoid-Rich Fraction of Inula viscosa

doi: 10.3390/biology13090687

Figure Lengend Snippet: IVL DCM induces the apoptosis of A549 cells. A549 cells were treated with the indicated concentrations of IVL DCM for 48 h. ( A ) P53, BCL2, BAX, Caspase 3, c-Caspase 3, PARP, c-PARP protein levels as detected by immunoblotting of A549 cell lysates. β-actin was immunoblotted as a loading control ( B ). Quantification of the bands in ( A ). Bar graphs of band intensity of target proteins normalized to the intensity of the loading control β-actin expressed as fold change of the vehicle control and represented as the mean ± SEM of three independent experiments ( n = 3). The right panel of ( B ) shows the ratio of BAX/BCL2 ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Primary antibodies used were: anti-human poly-adenosine diphosphate (ADP) ribose polymerase (PARP) 46D11 rabbit mAb which can detect the full-length and cleaved forms of PARP (ref 9532 CST, dilution 1/1000), P53 rabbit mAb (ref 2527S CST, dilution 1/500), Ki67 rabbit polyclonal antibody (ref 28074-1-AP, Proteintech, Rosemont, IL, USA, dilution 1/1000), mouse anti-human B-cell lymphoma 2 (BCL2) (ref 15071S CST, dilution 1/1000), rabbit anti-Bcl-2 associated X protein (BAX) (D2E11 ref 5023 CST; dilution 1/1000), P38 MAPK polyclonal antibody (14064-1-AP, Proteintech, dilution 1/1000), phospho-p38 MAPK (Thr180/Tyr182) antibody(ref 9211 CST, dilution 1/1000), Caspase3 antibody (ref 9662 CST, 1/1000 dilution), cleaved Caspase3 (c-Caspase 3; Asp175) 5A1E rabbit mAb (ref 9664 CST, dilution 1/1000), β-actin 8H10D10 mouse mAb (ref 3760 CST, dilution 1/1000), P21 Waf1/Cip1 12D1 rabbit mAb (ref 2947 CST, 1/1000), P27 (CST, 1/1000), phospho-FAK (p-Fak Tyr 397) D20B1 rabbit mAb (ref 8556 CST, dilution 1/1000), FAK D2R2E rabbit mAb (ref 13009 CST, dilution 1/1000).

Techniques: Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: COX2 Enhances Neovascularization of Inflammatory Tenocytes Through the HIF-1α/VEGFA/PDGFB Pathway

doi: 10.3389/fcell.2021.670406

Figure Lengend Snippet: Knockdown of VEGFR2 and PDGFRβ inhibited the tube-forming ability of RAOECs of the conditioned medium from COX2-overexpressing tenocytes. (A,B) Western blot was performed to detect the protein expression level of VEGFR2 and PDGFRβ in RAOECs with VEGFR2-siRNA and PDGFRβ-siRNA transfection. (C,D) A three-dimensional Matrigel assay was conducted to assess the tube formation of RAOECs with VEGFR2-siRNA and PDGFRβ-siRNA transfection. The data were presented as the means ± SD; n = 3; ∗∗ p < 0.01; scale bar: 50 μm; the triangles represent the position of the tubes. Western blot was performed using tenocytes. The three-dimensional Matrigel assay was performed using RAOECs.

Article Snippet: After being blocked, the primary antibodies COX2 (Cell Signaling Technology, United States, #12282), HIF-1α (Cell Signaling Technology, United States, #14179), K-AC (Cell Signaling Technology, United States, #9441), VEGFR2 (Cell Signaling Technology, United States, #9698), PDGFRβ (Cell Signaling Technology, United States, #3161), and β-Actin (Cell Signaling Technology, United States, #8457) were added to membranes overnight at 4°C.

Techniques: Knockdown, Western Blot, Expressing, Transfection, Matrigel Assay

Journal: Journal of Biological Chemistry

Article Title: Silencing of Insulin-like Growth Factor-binding Protein-2 in Human Glioblastoma Cells Reduces Both Invasiveness and Expression of Progression-associated Gene CD24

doi: 10.1074/jbc.m609567200

Figure Lengend Snippet: FIGURE 2. Modulation of IGF/IGF-IR signaling caused by knockdown of IGFBP-2. The IGFBP-2-knockdown cells (IGFBP-2 KD) and control cells were treated with the indicated concentrations of IGF-I or IGF-II in serum-free con- dition, and extents of phosphorylation of IGF-IR (p-IGF-IR), Akt (p-Akt), and ERK1/2 (p-Erk1/2) were analyzed by immunoblot and a representative result of U251 was shown. The band intensity was measured and the ratio of phos- phorylated protein to the corresponding total protein was calculated.

Article Snippet: The following primary antibodies were used: anti-human IGFBP-2 mouse monoclonal antibody (C-10; Santa Cruz Biotechnology, Santa Cruz, CA), anti- -actinmousemonoclonal antibody (AC-74; Sigma), anti-phosphoERK1/2 rabbit monoclonal antibody (Thr185/Tyr187, clone AW39;Upstate Cell Signaling Solutions, Lake Placid, NY), antiphospho-IGF-IR rabbit polyclonal antibody (Tyr1131), antiIGF-IR rabbit polyclonal antibody, anti-phospho-Akt mouse monoclonal antibody (Ser473), anti-Akt rabbit polyclonal antibody, anti-phospho-ERK1/2 rabbit monoclonal antibody (Thr185/Tyr187, clone AW39; Upstate Cell Signaling Solutions, Lake Placid, NY), anti-ERK1/2 rabbit polyclonal antibody, anticaspase-3, and cleaved capase-3 (Asp175) rabbit polyclonal antibodies, and anti-heat shock protein 70 rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Knockdown, Control, Phospho-proteomics, Western Blot

Journal: Blood Advances

Article Title: Prevalence and impact of diabetes on survival of patients with multiple myeloma in different racial groups

doi: 10.1182/bloodadvances.2023010815

Figure Lengend Snippet: Progression of myeloma xenografts is faster in a mouse model of type 2 diabetes than without diabetes. (A) Representative MM1.S tumor xenograft growth trajectories from Rag1 −/− (Rag wild-type [WT]), and Rag1 −/− /MKR (Rag MKR) male mice; n = 3 to 5 per group. (B) Plasma insulin concentrations in Rag1 −/− and Rag1 −/− /MKR male mice; n = 3 to 5 per group. (C) Representative western blot analysis of MM1.S tumor xenograft protein lysates from Rag1 −/− and Rag1 −/− /MKR mice, as indicated. (D-E) Quantification of total insulin receptor expression corrected for β actin, and S6rp phosphorylation, relative to total S6rp protein levels. Results are expressed as relative difference to that of Rag1 −/− mice. (F) Representative western blot analysis of MM1.S tumor cell protein lysates from with and without insulin stimulation, as indicated. (G-I) Quantification of pIR/IGF-1R, pAkt, and pS6rp relative to total protein levels; n = 3 per group. ∗ P < .05 between groups; ∗∗∗ P < .001, as indicated.

Article Snippet: Primary antibodies and dilutions used were as follows: anti–phosphorylated IGF-1Rβ (Tyr1150/1151)/phosphorylated insulin receptor β (pIRβ) (Tyr1135/1136) (#3024, 1:1000, Cell Signaling Technology [CST], Danvers, MA), total IGF-1R (1:1000, #3027, CST), total IRβ (1:200, C-19, Santa Cruz Biotechnology, Santa Cruz, Dallas, TX), phosphorylated Akt (pAkt) (Ser473) (1:1000, #9271, CST), total Akt (1:2000, #2920, CST), phosphorylated S6 ribosomal protein (pS6rp) (Ser235/236) (1:1000, #2211, CST), total S6 ribosomal protein (1:1000, #2317, CST), and β-actin (1:10 000, A228, Sigma-Aldrich).

Techniques: Clinical Proteomics, Western Blot, Expressing, Phospho-proteomics