automatic injector as 1555  (JASCO Inc)

Journal: STAR Protocols

Article Title: Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

doi: 10.1016/j.xpro.2025.104323

Figure Lengend Snippet: Schematic showing generation of isogenic A431-HA tagged cells engineered to express either KRAS WT or mutant KRAS G12V (A) Generation of retrovirus by transfecting HEK293T cells with vector expressing either KRAS-WT-HA or KRAS-G12V-HA and retroviral packaging vector. Retroviral particles are collected and filtered. (B) Generation of lentivirus by transfection HEK293T cells with vector expressing either KRAS-WT-Luc or KRAS-G12V-Luc and lentiviral packaging vectors. Lentiviral particles are collected and filtered. (C) Remove endogenous KRAS from A431 cells using CRISPR/Cas9 and identify clones by single cell cloning and PCR. (D) Transduce A431 KRAS knockout cells with retrovirus/lentivirus and select for successfully transduced cells using puromycin resulting in A431 cells expressing KRAS with HA tag or luciferase reporter.

Article Snippet: A431 , ATCC , Cat #CRL-1555; RRID: CVCL_0037.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Retroviral, Transfection, CRISPR, Clone Assay, Cloning, Knock-Out, Luciferase

Journal: STAR Protocols

Article Title: Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

doi: 10.1016/j.xpro.2025.104323

Figure Lengend Snippet: Schematic showing experimental workflow for in vitro evaluation of KRAS G12V selective siRNA (A) A431-KRAS(G12V/WT)-HA cells are transfected with siRNA using Lipofectamine RNAiMAX. After 48 hours, cells are collected and RNA is isolated and used for RT-qPCR for relevant mRNA targets. After 72 hours, cells are collected and protein is isolated and used for immunoblotting for relevant targets. (B) A431-KRAS(WT/G12V)-Luc cells are transfected with a serial dilution of siRNA using Lipofectamine RNAiMAX and plated into 96-well opaque plates. After 3 days, firefly and renilla luciferase readouts are completed.

Article Snippet: A431 , ATCC , Cat #CRL-1555; RRID: CVCL_0037.

Techniques: In Vitro, Transfection, Isolation, Quantitative RT-PCR, Western Blot, Serial Dilution, Luciferase

Journal: STAR Protocols

Article Title: Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

doi: 10.1016/j.xpro.2025.104323

Figure Lengend Snippet: Schematic showing experimental workflow for in vivo evaluations of KRAS G12V selective siRNA in tumor and mouse somatic tissues Mice are subcutaneously injected with A431-KRAS(G12V)-HA cancer cells and mice form tumors which are monitored by caliper measurement. After 1–2 weeks, mice are subcutaneously injected (away from the tumor) with the KRAS G12V siRNA. Tumor and mouse somatic tissues (kidney, bladder and skin) are collected at multiple timepoints from 24–96 hours after injection. Tissues are cut into smaller sections and processed with TRIzol for RNA isolation. RNA is used for RT-qPCR for relevant mRNA targets.

Article Snippet: A431 , ATCC , Cat #CRL-1555; RRID: CVCL_0037.

Techniques: In Vivo, Injection, Isolation, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

doi: 10.1016/j.xpro.2025.104323

Figure Lengend Snippet: Validation of mutant selectivity of KRAS G12V siRNA in vitro (A) UV melting curves and sequences of 23mer duplexes between the fully modified KRAS guide RNA and the targeted G12V mutant, WT, and G12D mutant RNA. (B) Western blot analysis in A431 cells stably expressing KRAS WT or G12V transiently transfected with siRNAs. Cells were analyzed at 48 hours and 72 hours. Blots were done separately, and densitometry quantification below is based on vinculin control for each individual blot. (C) RT-qPCR analysis in A431 cells transiently transfected with siRNAs at 20 nM. Cells were analyzed at 48 hours. Data shown as mean +/- SEM, experiments performed in duplicate. (D) Luciferase dose-response curve in A431-KRAS-WT or A431-KRAS-G12V cells stably expressing a luciferase reporter. Cells were analyzed at 72 hours. Data shown as mean +/- SEM, experiments performed in triplicate. (E) Volcano plots from RNA-sequencing in SKCO1 cells transiently transfected with siRNAs at 20 nM. Cells were analyzed at 24 hours. This figure includes data published in Stanland et al and has received permission to be shown in this figure.

Article Snippet: A431 , ATCC , Cat #CRL-1555; RRID: CVCL_0037.

Techniques: Biomarker Discovery, Mutagenesis, In Vitro, Modification, Western Blot, Stable Transfection, Expressing, Transfection, Control, Quantitative RT-PCR, Luciferase, RNA Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronic acid regulates cellular UDP-GlcNAc levels through CD44 to affect glycosylation and cell biological functions

doi: 10.1016/j.jbc.2025.111111

Figure Lengend Snippet: Establishment of HAS2 KO cells and confirmation of HA synthesis. A , comparison of HAS2 expression levels among different cell lines: HeLa (cervical adenocarcinoma cell line), A431 (epidermoid carcinoma cell line), PANC-1 (pancreatic ductal adenocarcinoma cell line), MIA PaCa-2 (pancreatic carcinoma cell line), and HepG2 (hepatocellular carcinoma cell line). The HAS2 mRNA level in HeLa was normalized to 1.0. All values are presented as mean ± SD from three independent experiments, based on one-way ANOVA with Tukey’s post hoc analysis. ∗∗∗ p < 0.001. B , comparison of HAS1 , HAS2 , and HAS3 expression levels in HeLa cells. The HAS1 mRNA level in HeLa was normalized to 1.0. All values are presented as mean ± SD from three independent experiments, based on one-way ANOVA with Tukey’s post hoc analysis. ∗∗∗ p < 0.001. C , Sanger sequencing was used to analyze exon 2 of the target sequence in HeLa WT and HAS2 KO cells with the canonical SpCas9 PAM sequence (TGG) highlighted in bold. D , WT and HAS2 KO cells were cultured in serum-free medium, and the conditioned medium was collected after 24 h to measure HA concentration. Statistical significance was determined from three independent experiments using an unpaired Student’s t test, with p values indicated as ∗∗∗ p < 0.001.

Article Snippet: A431 cells were obtained from ATCC.

Techniques: Comparison, Expressing, Sequencing, Cell Culture, Concentration Assay