Journal: Pharmaceuticals

Article Title: RNAi Screening in Tumor Cells Identifies Artificial microRNAs That Improve Oncolytic Virus Replication

doi: 10.3390/ph18050708

Figure Lengend Snippet: Enrichment of specific amiRNA sequences after serial passage of SINV library in tumor cells. ( A ) SINV titers obtained after 1 passage (P1) and 4 serial passages (P4) in CT26WT tumor cells infected at multiplicity of infection (MOI) of 0.1 for 48 h ( n = 5). Data are presented as means ± standard error of the mean (SEM). Student’s unpaired, one-tailed t -test was performed; **: p < 0.01. ( B ) Stacked area plot of percent frequency of each amiRNA in SINV–amiRNA library before passaging (P0) and after 4 rounds of serial passage (P4) in CT26WT cells. Cumulative percent frequencies of all 5 replicates are shown for P4. Each color represents an individual amiRNA. ( C ) Heat map showing percent frequency of reads of the 10 most enriched amiRNAs in each replicate at P0 (input) and after P4.

Article Snippet: The CT26WT (murine colon carcinoma), BHK-21 (baby hamster kidney fibroblast), U2OS (human osteosarcoma), HCT116 (human colorectal carcinoma), and Vero (African green monkey kidney epithelial cells) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, One-tailed Test, Passaging

Journal: Pharmaceuticals

Article Title: RNAi Screening in Tumor Cells Identifies Artificial microRNAs That Improve Oncolytic Virus Replication

doi: 10.3390/ph18050708

Figure Lengend Snippet: Enriched amiRNAs increase SINV replication. ( A ) Fluorescent images (4× magnification) and ( B ) virus titers from CT26WT cells transfected with siRNAs corresponding to amiRNA-1 and -2 targeting sequences and subsequently infected with SINV-GFP at MOI of 0.1. Fluorescent images were taken, and supernatants were collected 48 h post-infection ( n = 4). ( C ) Virus-induced cytotoxicity revealed by Coomassie Blue stain of CT26WT cells transfected with siRNAs corresponding to identified hits and subsequently infected with SINV-GFP. Cells were fixed and stained 72 h post-infection. ( D ) Enrichment of amiRNA-expressing SINV from competition experiments against parental SINV-GFP virus. The percentage of each virus is shown before competition (P0) and after 4 rounds of competition (P4) in CT26WT cells ( n = 3). Data are presented as means ± SEM. Student’s unpaired, one-tailed t -test was performed; **: p < 0.01; ***: p < 0.001.

Article Snippet: The CT26WT (murine colon carcinoma), BHK-21 (baby hamster kidney fibroblast), U2OS (human osteosarcoma), HCT116 (human colorectal carcinoma), and Vero (African green monkey kidney epithelial cells) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Virus, Transfection, Infection, Staining, Expressing, One-tailed Test

Journal: Pharmaceuticals

Article Title: RNAi Screening in Tumor Cells Identifies Artificial microRNAs That Improve Oncolytic Virus Replication

doi: 10.3390/ph18050708

Figure Lengend Snippet: The identified amiRNAs increase the replication of oncolytic VSVΔ51, MG1, and VVdd. ( A ) Fluorescent images (4× magnification) and virus titers from CT26WT cells transfected with siRNAs corresponding to the identified hits and subsequently infected with VSVΔ51-YFP (top panel, n = 6), MG1-GFP (middle panel, n = 3), or VVdd-mCherry (bottom panel, n = 5). Images were taken and supernatants were collected 48 h post-infection for VSVΔ51 and MG1 and at 72h for VVdd. ( B ) Virus-induced cytotoxicity as measured by AlamarBlue viability assay of CT26WT cells transfected with siRNAs corresponding to identified hits and subsequently infected with VSVΔ51-YFP. Cell viability was measured 72 h post-infection ( n = 3). ( C ) Dual-luciferase reporter assay. BHK cells were transfected with the indicated reporter plasmids possessing target sites for enriched amiRNAs in 3′ UTR of Renilla luciferase reporter and subsequently infected with the indicated VSV viruses. Luciferase activity was measured 24 h post-infection. ( n = 3). ( D ) Enrichment of amiRNA-expressing VSVΔ51 from competition experiments against parental virus VSVΔ51-YFP. The frequency of each virus is shown at P0 and P4 in CT26WT cells ( n = 3). Data are presented as means ± SEM. Student’s unpaired, one-tailed t -test was performed; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: The CT26WT (murine colon carcinoma), BHK-21 (baby hamster kidney fibroblast), U2OS (human osteosarcoma), HCT116 (human colorectal carcinoma), and Vero (African green monkey kidney epithelial cells) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Virus, Transfection, Infection, Viability Assay, Luciferase, Reporter Assay, Activity Assay, Expressing, One-tailed Test

Journal: Pharmaceuticals

Article Title: RNAi Screening in Tumor Cells Identifies Artificial microRNAs That Improve Oncolytic Virus Replication

doi: 10.3390/ph18050708

Figure Lengend Snippet: siRNA-1 and -2 do not target the IFN pathway. ( A ) IFN-β levels in CT26WT cells transfected with the indicated siRNA and then infected with VSVΔ51 at an MOI of 0.001 for 24h, as measured by qPCR ( n = 2) and ( B ) ELISA ( n = 2). Data are presented as means ± SEM. Student’s unpaired, one-tailed t -test was performed; *: p < 0.05; **: p < 0.01. ( C ) Relative expression of various ISGs after siRNA transfection and infection of CT26WT cells with VSVΔ51 at an MOI of 0.001 for 24h, as measured by qPCR ( n = 3). Data are presented as means ± SEM. Student’s unpaired, one-tailed t -test was performed; *: p < 0.05; **: p < 0.01.

Article Snippet: The CT26WT (murine colon carcinoma), BHK-21 (baby hamster kidney fibroblast), U2OS (human osteosarcoma), HCT116 (human colorectal carcinoma), and Vero (African green monkey kidney epithelial cells) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Infection, Enzyme-linked Immunosorbent Assay, One-tailed Test, Expressing

Journal: Pharmaceuticals

Article Title: RNAi Screening in Tumor Cells Identifies Artificial microRNAs That Improve Oncolytic Virus Replication

doi: 10.3390/ph18050708

Figure Lengend Snippet: Gene expression changes induced by siRNA-1 and siRNA-2 reveals marked overlap of differentially expressed genes. ( A ) Microarray analysis of changes in gene expression 16h after VSVΔ51 infection of CT26WT transfected with siRNA-1 or siRNA-2. Heat map shows genes that were differentially expressed at least 3.5-fold compared with cells treated with control siRNA and infected with VSVΔ51. ( B ) Venn diagram showing number and overlap of differentially expressed genes (≥2-fold) in VSVΔ51-infected, siRNA-1- or siRNA-2-transfected cells, compared with control siRNA. Up arrows and down arrows indicate an increase and a decrease in expression, respectively. ( C ) qPCR analysis of selected genes identified as downregulated by microarray analysis of siRNA-treated CT26WT cells ( n = 3). Data are presented as means ± SEM. One-way ANOVA was performed with Dunnett’s multiple comparisons post hoc test. Compared with control siRNA, ##: p < 0.01; ###: p < 0.001. Compared with control siRNA + VSV, *: p < 0.05; **: p < 0.01; ***: p < 0.001. ( D ) Knockdown of target gene promotes VSV replication in CT26WT cells. CT26WT cells were transfected with the indicated siRNAs, and 48 h post-transfection, they were infected with VSVΔ51. Supernatants were collected 48 h post-infection, and virus was quantified ( n = 3). Data are presented as means ± SEM. Student’s unpaired, one-tailed t -test was performed; compared with control siRNA, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: The CT26WT (murine colon carcinoma), BHK-21 (baby hamster kidney fibroblast), U2OS (human osteosarcoma), HCT116 (human colorectal carcinoma), and Vero (African green monkey kidney epithelial cells) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Gene Expression, Microarray, Infection, Transfection, Control, Expressing, Knockdown, Virus, One-tailed Test

Journal: Pharmaceuticals

Article Title: RNAi Screening in Tumor Cells Identifies Artificial microRNAs That Improve Oncolytic Virus Replication

doi: 10.3390/ph18050708

Figure Lengend Snippet: The amiRNA-expressing viruses show enhanced replication and efficacy in vivo. ( A ) Quantification of SINV in tumors ( n = 6) and spleens ( n = 3) 48 h post-intratumoral treatment of CT26WT tumor-bearing mice with 1 × 10 8 PFU. Data are presented as means ± SEM. Student’s unpaired, one-tailed t -test was performed; *: p < 0.05. ( B ) Quantification of VSVΔ51 in tumors ( n = 14) and spleens ( n = 3) 48 h post-intravenous treatment of CT26WT tumor-bearing mice with 5 × 10 8 PFU. Data are presented as means ± SEM. Student’s unpaired, one-tailed t -test was performed; *: p < 0.05. ( C ) Tumor volumes of mice ( n = 6) bearing CT26WT subcutaneous tumors treated intravenously on day 1 and intratumorally on days 2 and 3 with 5 × 10 8 PFU of the indicated viruses. Values represent means ± SEM. Two-way ANOVA test was performed with Dunnett’s multiple comparisons post hoc test; ***: p < 0.001.

Article Snippet: The CT26WT (murine colon carcinoma), BHK-21 (baby hamster kidney fibroblast), U2OS (human osteosarcoma), HCT116 (human colorectal carcinoma), and Vero (African green monkey kidney epithelial cells) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, In Vivo, One-tailed Test

Journal: Molecular Oncology

Article Title: In‐depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

doi: 10.1002/1878-0261.13228

Figure Lengend Snippet: The overexpression of ∆Np73 in CRC cells produced the dysregulation of cytokines, growth factors, cell signaling proteins, and proteins related to tumorigenesis, metastasis and cell–cell adhesion as determined by antibody microarrays and SILAC quantitative proteomic analyses. (A) Confirmation by qPCR of the overexpression of ∆Np73 in stably transfected HCT116 CRC cells in comparison to mock control cells. Triplicates were analyzed ( n = 3). (B) The secretome of two different biological replicates of HCT116‐∆Np73 CRC cells and mock control cells was analyzed using L‐series antibody microarrays. (C) Microarrays were incubated with biotin‐labeled samples followed with Cy3‐streptavidin, and differential expression for proteins due to ∆Np73 overexpression was found in both biological replicates. a.u., arbitrary units. (D) For the SILAC approach, protein extracts from the conditioned media from metabolically labeled HCT116‐∆Np73 and mock cells were mixed 1 : 1 to perform forward and reverse experiments and run on SDS‐polyacrylamide gels. A total of 8730 and 6862 peptides in forward and reverse experiments were found, resulting in 1586 and 1338 identified proteins, respectively, with 1051 proteins in common in both experiments. (E, F) After data normalization using the 5% trimmed mean (E), 1590 proteins were quantified in both SILAC experiments (F), with 793 proteins quantified in common. (G) A total of 37 dysregulated proteins by ∆Np73 identified in the secretome of HCT116 cells with variability ≤ 20%, and fold‐change ≥ 1.5. At least two peptides in both biological replicates, with 19 and 18 up‐ and down‐regulated proteins, respectively, were observed. (H) The top 10 dysregulated proteins observed by SILAC were represented by a bar‐graph.

Article Snippet: The colon cancer cell line HCT116 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Produced, Multiplex sample analysis, Stable Transfection, Transfection, Comparison, Control, Incubation, Labeling, Quantitative Proteomics, Metabolic Labelling

Journal: Molecular Oncology

Article Title: In‐depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

doi: 10.1002/1878-0261.13228

Figure Lengend Snippet: Proteins differentially regulated by ∆Np73 identified by antibody microarrays and SILAC in the secretome of HCT116 CRC cells with at least 1.5‐fold change variation.

Article Snippet: The colon cancer cell line HCT116 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Multiplex sample analysis, Microarray, Variant Assay

Journal: Molecular Oncology

Article Title: In‐depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

doi: 10.1002/1878-0261.13228

Figure Lengend Snippet: Validation of ∆Np73‐dysregulated proteins. Validation of the dysregulated proteins found was carried out using different methodologies. (A) Dysregulation of selected genes due to the stably overexpression of ∆Np73 was firstly confirmed by PCR in comparison to stably transfected mock control cells. The image is representative of four different experiments where 30 or 35 PCR cycles were performed. GAPDH was used as control of the assay. bp, base pair. (B) At protein level, the dysregulation of BDNF, and EMAP‐II was verified in the conditioned media of HCT116 mock control cells by dot‐blot. Red Ponceau staining was used as loading control. Two independent biological replicates were measured for each condition assessed. (C) Quantification of BDNF, VEGFC, and VEGFR3 in the conditioned medium or EMAP‐II in cell extracts was performed by ELISA. One‐tailed Student's t ‐test was performed. All values are expressed as median ± standard deviation. Significant data were determined for VEGFR3 and BDNF, and almost significant for EMAP‐II ( P = 0.06635). (D) Dysregulation of EMAP‐II and BDNF was also verified by WB, using β‐actin as loading control. Quantification data is shown. L, low; M, medium; H, high are referred to the ∆Np73 levels after cell sorting of HCT‐116 CRC cells, where a lower (EMAP‐II) or higher (BDNF) expression is observed according to the expression of ∆Np73. All results either at mRNA or protein level showed consistency with the alterations observed by proteomics. Triplicates were analyzed with Mann–Whitney U ‐test. Error bars indicate standard deviation. (E) Left, confirmation by qPCR of the overexpression of ∆Np73 in stably transfected HCT116 p53 −/− CRC cells in comparison to mock control cells. Right, dysregulation of selected genes due to stably overexpression of ∆Np73 in HCT116 cells was confirmed by PCR in HCT116 p53 −/− cells in comparison to stably transfected mock control cells to demonstrate the presence of p53 was not affecting their dysregulation. The image is representative of four different experiments where 30 or 35 PCR cycles were performed. GAPDH was used as control of the assay. (F) The dysregulation of BDNF and EMAP‐II was also confirmed in the conditioned media of mock control and ∆Np73‐HCT116 p53 −/− cells by dot‐blot. Red Ponceau staining was used as loading control. Two independent biological replicates were measured for each condition assessed. (G) Dysregulation of the p53 target genes p21 and p16 in HCT116 and HCT116 p53 −/− ∆Np73 and mock control cells was analyzed by qPCR. Data were normalized with SDHA as reference gene. Triplicates were analyzed with Mann–Whitney U ‐test. Error bars indicate standard deviation.

Article Snippet: The colon cancer cell line HCT116 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Biomarker Discovery, Stable Transfection, Over Expression, Comparison, Transfection, Control, Dot Blot, Staining, Enzyme-linked Immunosorbent Assay, One-tailed Test, Standard Deviation, FACS, Expressing, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: In‐depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

doi: 10.1002/1878-0261.13228

Figure Lengend Snippet: ΔNp73 promotes angiogenesis and lymphangiogenesis in vitro . (A–C) HUVEC and HLEC primary cells were treated with conditioned media from HCT116‐Mock and HCT116‐ΔNp73 colon cancer cells. (A) Proliferation potential performed with the MTT assay. Five replicates of each experimental condition were measured and analyzed with Mann–Whitney U ‐test. Error bars indicate standard deviation. (B) Invasion capacity measured with a Transwell system using Matrigel as barrier. Five replicates of each experimental condition were measured and analyzed with Mann–Whitney U ‐test. Error bars indicate standard deviation. (C) Cell tube formation (scale bar = 500 μm). The parameters recorded are NS (number of segments); NMS (number of master segments); NJ (number of junctions); NMJ (number of master junctions); NMs (number of meshes); TL (total length); TSL (total segment length); TMSL (total master segment length). Five replicates of each experimental condition were measured and analyzed with Mann–Whitney U ‐test. Error bars indicate standard deviation. Statistical significance: * P < 0.05; ** P < 0.01; ¥ P = 0,06; RFU, fluorescence arbitrary units; Abs, absorbance.

Article Snippet: The colon cancer cell line HCT116 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, MTT Assay, MANN-WHITNEY, Standard Deviation, Fluorescence

Journal: Molecular Oncology

Article Title: In‐depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

doi: 10.1002/1878-0261.13228

Figure Lengend Snippet: BDNF and EMAP‐II silencing effects on endothelial and lymphatic vasculature. (A) BDNF (left) and EMAP‐II (right) relative expression of HCT116‐ΔNp73 cells transiently transfected with a control siRNA (Scr) or a siRNA against BDNF (siBDNF) or EMAP‐II (siEMAP‐II). The efficiency of the siBDNF and siEMAP‐II was around 75% and 85%, respectively. (B, C) Tube formation assay (scale bar = 500 μm) in HUVEC and HLEC cells exposed to conditioned medium of HCT116‐∆Np73 treated with Scr or siBDNF or siEMAPII. The parameters recorded are NS (number of segments); NMS (number of master segments); NJ (number of junctions); NMJ (number of master junctions); NMs (number of meshes); TL (total length); TSL (total segment length); TMSL (total master segment length). Five replicates of each experimental condition were measured and analyzed with Mann–Whitney U test. Error bars indicate standard deviation. Statistical significance: * P < 0.05; ** P < 0.01.

Article Snippet: The colon cancer cell line HCT116 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transfection, Control, Tube Formation Assay, MANN-WHITNEY, Standard Deviation

Journal: Biomedical reports

Article Title: Potential prognostic and predictive value of UBE2N, IMPDH1, DYNC1LI1 and HRASLS2 in colorectal cancer stool specimens.

doi: 10.3892/br.2023.1604

Figure Lengend Snippet: Figure 8. Model validity of patients with CRC and non‑CRC controls. (A) Probability of CRC from pre‑ and postsurgical stool specimens. Data are presented as the mean ± standard error of the mean (n=2). (B) Boxplot showing the probability of CRC cDNA array. HCRT104 cDNA array (OriGene Technologies, Inc.) of 48 colon tissues was used. Data were presented as the mean ± standard error of the mean of non‑CRC status (n=8) and four CRC stages (n=40). Black spot, outliers. CRC, colorectal cancer.

Article Snippet: In addition, one cDNA array (HCRT104; OriGene Technologies, Inc.) of 48 colon tissues covering non‐CRC status (n=8) and four CRC stages (n=40) was purchased to examine the predictive model. LOOCV, discriminant analyses and predictive model for CRC risk.

Techniques:

Journal: Cell Death & Disease

Article Title: FADS1 promotes the progression of laryngeal squamous cell carcinoma through activating AKT/mTOR signaling

doi: 10.1038/s41419-020-2457-5

Figure Lengend Snippet: Hierarchical clustering ( a ) and volcano plots ( b ) for the expression profiles of mRNAs. c The top 10 significant terms by KEGG pathway analysis for differential expressed mRNAs in the microarray. d Verification of the differentially expressed transcripts by qRT-PCR in 30 paired LSCC tissues. * P < 0.05, ** P < 0.01, *** P < 0.001, two-tail paired t -test.

Article Snippet: The stable FADS1-OE and NC cells were used for transcriptome profile by the Agilent One-Color Gene Expression Microarray (KangChen Bio-Tech Inc.).

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays

doi: 10.1007/978-1-4939-6584-7_11

Figure Lengend Snippet: Overview of SMM strategy to identify RNA-binding small molecules. Small molecule libraries prepared in 384-well plates are utilized to print onto isocyanate-functionalized slides by the microarrayer. Prepared SMMs are next incubated with the labeled RNA of interest and scanned to detect hits

Article Snippet: Printing of Small Molecule Microarrays MicroGrid II Microarrayer (BioRobotics) or equivalent.

Techniques: RNA Binding Assay, Incubation, Labeling