Journal: PLoS ONE

Article Title: Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2- b ]quinoline Tri-Alkylamine Derivatives

doi: 10.1371/journal.pone.0126891

Figure Lengend Snippet: A. p53 protein steady-state expression evaluated by immunoblot relative to DMSO (vehicle control), after 72 h exposure to equitoxic (IC 50 ) concentrations of 5-FU, TMPyP4 and IQ3A. B. Cell populations obtained by Guava ViaCount flow cytometry following 72 h incubation of 5-FU, TMPyP4 and IQ3A at equitoxic (IC 50 ) concentrations, or DMSO (vehicle control), in HCT116 p53 (+/+) and p53 (−/−) isogenic cell lines transfected with 40 nM siRNA KRAS or siRNA control. Results are expressed as mean ± SEM of at least three independent experiments. A. §p < 0.01 from DMSO (vehicle control); and ‡p < 0.01 from 5-FU. B. *p < 0.05 from siRNA control in P53 (+/+) cells; §p < 0.05 from siRNA control in p53 (-/-) cells; and †p <0.05 from siRNA KRAS in p53 (-/-) cells.

Article Snippet: Twenty four hours later, cells were transfected with 50 or 100 nM siRNA KRAS, siRNA control, siRNA HSP90 (HSP90α/β siRNA (h) (sc-35608 Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and co-transfected with 50 nM siRNA KRAS plus 50 nM siRNA HSP90.

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Incubation, Transfection

Journal: Journal of Neuroimmune Pharmacology

Article Title: Formyl Peptide Receptor-2-Suppressed Autophagy Promotes the Migration and Invasion of Human Glioblastoma Cells Through PI3K/Akt Signaling

doi: 10.1007/s11481-026-10284-z

Figure Lengend Snippet: The expression of FPR2 and SQSTM1 is associated with the invasiveness of glioma cells. ( A ) FPR2 positively modulates the protein expression of SQSTM. Compared with that in the shRNA or vector control groups, immunoblot analysis indicates that the protein expression level of SQSTM1 was clearly altered in FPR2-KD U87 cells, FPR2-OE LN-229 cells, and their corresponding controls; * p < 0.05, ** p < 0.01 indicate statistically significant differences in comparison with the shRNA or vector group.( C ) Correlation analysis of FPR2 and SQSTM1 in glioma patient tissues revealed a positive correlation between the mRNA levels of FPR2 and SQSTM1 (p<0.001, r=0.462) (http://gepia.cancer-pku.cn). ( D and F ) FPR2 regulates SQSTM1 expression via autophagy. Immunoblot analysis revealed that SQSTM1 expression levels in U87 cells with FPR2 knockdown and in scrambled shRNA control cells were influenced by treatment with BAF or CQ, as well as by transfection with ATG5 or BECN1 siRNA. ( F ). SQSTM1, BECN1, and ATG5 were quantified via densitometric analysis using ImageJ ( E and G ). * p < 0.05, ** p < 0.01 versus the vehicle or siNC group

Article Snippet: Glioma cell lines with stable knockdown or overexpression of FPR2, along with corresponding control cell lines, were established. siRNAs targeting BECN1 (sc-29797; Santa Cruz, USA), ATG5 (sc-41445; Santa Cruz, USA), control siRNA (sc-37007; Santa Cruz, USA) or SQSTM1 (sc-29679; Santa Cruz, USA) were transiently transfected into stable FPR2-knockdown and FPR2-overexpressing glioma cells using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s protocols.

Techniques: Expressing, shRNA, Plasmid Preparation, Control, Western Blot, Comparison, Knockdown, Transfection

Journal: Journal of Neuroimmune Pharmacology

Article Title: Formyl Peptide Receptor-2-Suppressed Autophagy Promotes the Migration and Invasion of Human Glioblastoma Cells Through PI3K/Akt Signaling

doi: 10.1007/s11481-026-10284-z

Figure Lengend Snippet: FPR2 suppresses autophagy and induces EMT-like transformation in glioma cells. ( A ) Immunoblot analysis demonstrating the expression of Snail, vimentin, N-cadherin and E-cadherin in FPR2-KD U87 cells, FPR2-OE LN-229 cells, and their respective control cells. ( B and C ) Quantitative graphs showing the protein expression levels of Snail, vimentin, N-cadherin and E-cadherin in FPR2-KD U87 cells and FPR2-OE LN-229 cells compared with those in the shRNA or vector control groups; ** p < 0.01, * p < 0.05. ( D and F ) Immunoblot analysis revealed the expression of Snail, vimentin, N-cadherin and E-cadherin in FPR2-KD U87 cells and control cells infected with shRNA and treated with ATG5 siRNA, BECN1-siRNA (D) or 3-MA ( F ). ( E and G ) Quantitative graphs depicting the relative protein expression levels of Snail, vimentin, N-cadherin and E-cadherin in FPR2-KD U87 cells and shRNA-infected control cells treated with ATG5 siRNA, BECN1-siRNA or 3-MA ( F ); * p < 0.05, ** p < 0.01 versus the Scr-siNC, FPR2 KD-siNC or FPR2 KD vehicle groups. ( H ) Immunoblot analysis revealed the protein levels of Snail in FPR2-KD U87 cells and scramble control cells following transfection with the SQSTM1 overexpression construct. ( I ) Quantitative graphs illustrating the protein levels of Snail in FPR2-KD U87 cells and scramble control cells after transfection with the SQSTM1 overexpression construct. ( J ) Western blot analysis showing the protein expression level of Snail in LN-229 cells overexpressing FPR2 and vector control cells after SQSTM1 siRNA transfection. ( K ) Quantitative images of Snail protein expression levels in FPR2-overexpressing LN-229 cells and vector control cells after transfection with SQSTM1 siRNA; * p < 0.05, ** p < 0.01 relative to the Scr-vector, FPR2 KD-vector, vector-siNC and FPR2 OE-siNC groups

Article Snippet: Glioma cell lines with stable knockdown or overexpression of FPR2, along with corresponding control cell lines, were established. siRNAs targeting BECN1 (sc-29797; Santa Cruz, USA), ATG5 (sc-41445; Santa Cruz, USA), control siRNA (sc-37007; Santa Cruz, USA) or SQSTM1 (sc-29679; Santa Cruz, USA) were transiently transfected into stable FPR2-knockdown and FPR2-overexpressing glioma cells using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s protocols.

Techniques: Transformation Assay, Western Blot, Expressing, Control, shRNA, Plasmid Preparation, Infection, Transfection, Over Expression, Construct

Journal: International journal of oncology

Article Title: ACK1 promotes hepatocellular carcinoma progression via downregulating WWOX and activating AKT signaling.

doi: 10.3892/ijo.2015.2910

Figure Lengend Snippet: Figure 6. The effect of ACK1 knockdown on MHCC-97H cells. (A) Representative images show the migration and invasion ability of MHCC-97H cells transfected with ACK1-shRNA or Control-shRNA (x200). (B) Data are presented as mean relative numbers of invaded or migrated cells from 5 fields (*P<0.01). (C) MHCC-97H cells transfected with ACK1-shRNA or Control-shRNA, respectively, were subjected to western blotting for ACK1, p-ACK1, WWOX, AKT, p-AKT, MMP2 and MMP9 (P<0.01).

Article Snippet: The ACK1 shRNA and scrambled shRNA vector pRS were purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Knockdown, Migration, Transfection, shRNA, Control, Western Blot

Journal: International journal of oncology

Article Title: ACK1 promotes hepatocellular carcinoma progression via downregulating WWOX and activating AKT signaling.

doi: 10.3892/ijo.2015.2910

Figure Lengend Snippet: Figure 5. ACK1 regulates apoptosis and proliferation in MHCC-97H cells. (A) Cell proliferation as measured by BrdU incorporation was inhibited by ACK1- shRNA in MHCC-97H cells (n=3, *P<0.01). (B) As assessed by MTT assay, ACK1 knockdown was found to reduce the viability of MHCC-97H cells (n=3, *P<0.01). (C) The activity of the pro-apoptotic caspases 3 and 7 was upregulated after ACK1 knockdown in MHCC-97H cells (n=3, *P<0.01). (D) Quantification of the apoptotic cell population by flow cytometry. ACK1 knockdown MHCC-97H cells were composed of a larger subset of apoptotic cells compared with the control group (n=3, *P<0.01). Values are depicted as mean ± SEM.

Article Snippet: The ACK1 shRNA and scrambled shRNA vector pRS were purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: BrdU Incorporation Assay, shRNA, MTT Assay, Knockdown, Activity Assay, Flow Cytometry, Control

Journal: Oncogene

Article Title: Ras association domain family member 10 suppresses gastric cancer growth by cooperating with GSTP1 to regulate JNK/c-Jun/AP-1 pathway.

doi: 10.1038/onc.2015.300

Figure Lengend Snippet: Figure 3. Effect of RASSF10 on cell growth and cell migration/invasion. (a) Overexpression of RASSF10 in AGS and MKN45 cells after stable transfection with RASSF10 expression vectors was confirmed by western blotting. (b) MTS viability assays showed RASSF10 expression significantly suppressed growth of AGS and MKN45 cells. (c) Colony-formation ability of AGS and MKN45 cells was significantly inhibited by RASSF10 expression. (d1) RASSF10 expression was knocked down in GES-1 cells by transfection of shRNA against RASSF10. (d2) Cell growth of GES-1 was significantly increased after knockdown of RASSF10. (e) Cell migration ability was significantly inhibited by RASSF10 expression in AGS cells as indicated by wound-healing assay. (f) Cell invasiveness was significantly reduced by RASSF10 expression in AGS and MKN45 cells as indicated by matrigel invasion assay.

Article Snippet: Knockdown of gene expression by shRNA or siRNA Vectors carrying shRNA specifically targeting RASSF10 (shRASSF10) or scrambled control shRNA (shCtrl) were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Migration, Over Expression, Stable Transfection, Expressing, Western Blot, Transfection, shRNA, Knockdown, Wound Healing Assay, Invasion Assay

Journal: Oncotarget

Article Title: Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients

doi: 10.18632/oncotarget.17715

Figure Lengend Snippet: (A) mRNA expression analysis was performed for the A427 and A549 lung adenocarcinoma cell lines in the presence or absence of cisplatinum-based treatment, and the effects of negative control scramble siRNAs and specific anti-MEOX2 siRNAs were assessed. The results are shown for two representative biological experiments performed in triplicate, with * p ≤0.05, and ** p ≤0.01. (B) A427 and A549 lung adenocarcinoma cells exhibited a cisplatinum-inducible GLI-1 protein expression pattern at IC:12.5 (8 μM), while reduced inducible GLI-1 expression was observed following transfection with anti-MEOX2 siRNA in the presence of 8 μM cisplatinum. Western blot statistical analyses, assessed via quantitative densitometry, were performed to determine * p ≤0.05 and ** p ≤0.005 using Student's t -test as well as one-way ANOVA with Dunnett's and Tukey's multiple comparison tests. Western blot bands were quantified as the pixel total intensity rate and expressed as a Change Index normalized to GAPDH. Images are representative of 3 biological replicates. Quantification analyses were performed using cisplatinum (0 μM) treatment as a negative control reference. Images were obtained using a C-DIGIT device (LICOR). Pixel quantification and data analyses were carried out using Image Studio software; the total pixel intensity for each specific protein product was normalized to GAPDH.

Article Snippet: Two non-human related siRNAs obtained from Santa Cruz Biotechnology (Dallas, TX, USA) were used as negative controls (sc-37007 and sc-44230).

Techniques: Expressing, Negative Control, Transfection, Western Blot, Comparison, Software

Journal: Oncotarget

Article Title: Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients

doi: 10.18632/oncotarget.17715

Figure Lengend Snippet: (A) A549 and H1975 lung cancer cells demonstrated an inducible GLI-1 protein expression pattern following treatment with 8 μM cisplatinum and reduced GLI-1 inducible expression following the application of specific anti-MEOX2 siRNA and/or anti-MEOX2 siRNA plus anti-GLI1 siRNA in the presence of 8 μM cisplatinum. Western blot statistical analyses, assessed via quantitative densitometry, were performed to determine * p ≤0.05 by one-way ANOVA and Dunnett's test for multiple comparisons to identify significant differences with respect to controls. Student's t -test was performed to identify significant differences between control and cisplatinum treatment. Quantification analyses were normalized to scrambled siRNA as a negative control for gene silencing. Images were obtained using a C-DIGIT device (LICOR), and pixel quantification and data analyses were carried out using Image Studio software. Total pixel intensity for each specific protein product was normalized to GAPDH. (B) Cell culture images and graphs showing the quantitative analysis of cellular migration as a percentage (transwell migration assays) indicated significant MEOX2 and GLI-1 protein-dependent functions following the individual and combined application of anti-MEOX2 and anti-GLI1 siRNAs in A549, NH2347 and H1975 lung adenocarcinoma cells; ** p ≤0.005 and *** p ≤0.0001 based on one-way ANOVA and Dunnett's multiple comparisons test. (C) Cell culture images and graphs showing the quantitative analysis of cellular proliferation (clonogenic assays) indicated significant MEOX2 and GLI-1 protein-dependent functions following the individual and combined application of anti-MEOX2 and anti-GLI1 siRNAs in A549, NH2347 and H1975 lung adenocarcinoma cells; ** p ≤0.005 and *** p ≤0.0001 based on one-way ANOVA and Dunnett's multiple comparisons test. Transwell migration index and colony growth (clonogenic assays) rates were normalized and quantified using the ImageJ Colony Number plugin tool (see Materials and Methods).

Article Snippet: Two non-human related siRNAs obtained from Santa Cruz Biotechnology (Dallas, TX, USA) were used as negative controls (sc-37007 and sc-44230).

Techniques: Expressing, Western Blot, Control, Negative Control, Software, Cell Culture, Migration

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Dual-specificity phosphatase 14 (DUSP14/MKP6) negatively regulates TCR signaling by inhibiting TAB1 activation.

doi: 10.4049/jimmunol.1300989

Figure Lengend Snippet: FIGURE 6. The enhanced JNK and IKK activa- tion of DUSP14-deficient T cells is attenuated by TAB1 knockdown. (A) Flow cytometry analysis of TAB1 expression in TAB1 shRNA-transfected cells. Murine primary T cells were transfected with GFP-tagged TAB1 shRNAs or a scrambled GFP- tagged shRNA for 36 h, followed by intracellular staining for TAB1. The GFP-tagged shRNA-trans- fected cells were GFP gated for flow cytometry analysis. Samples were stained with the secondary Ab only as negative controls (shaded graph). (B and C) WT and DUSP14-deficient (DUSP14-KO) T cells were transfected with GFP-tagged TAB1 shRNAs or a scrambled GFP-tagged shRNA for 36 h and then stimulated with anti-CD3 Ab for 15 min. The phosphorylation of JNK (B) and IKK (C) in shRNA-transfected cells (GFP gated) was ex- amined by intracellular staining. Data (mean 6 SEM) are representative of three independent ex- periments. *p , 0.05, two-tailed t test.

Article Snippet: Murine primary T cells were transfected with a mixture of four unique 29-mer GFP-tagged TAB1 short hairpin RNAs (shRNAs) or a scrambled GFP-tagged shRNA (both from OriGene Technologies) for 36 h. Cells were stimulated with biotinconjugated anti-CD3 (3 mg/ml; 500A2; eBioscience) Ab plus streptavidin (3 mg/ml; Sigma) for 15 min, followed by intracellular staining for p-JNK (Cell Signaling), p-IKK (Santa Cruz), or p-ERK (Cell Signaling).

Techniques: Knockdown, Flow Cytometry, Expressing, shRNA, Transfection, Staining, Cytometry, Phospho-proteomics, Two Tailed Test