Journal: Life Science Alliance

Article Title: Rnd3 regulates lung cancer cell invasion and migration independently of ROCK1 signaling via alpha 5 integrin modulation

doi: 10.26508/lsa.202503494

Figure Lengend Snippet: (A, B, C) Representative Western blots in which decreased protein expression levels of Rnd3 (oligo A), ROCK1, and ROCK2 are observed, corresponding to siRNA treatment compared with non-silencing control (NSC) in (A) A549, (B) H460, and (C) A375 cells (as an experimental control). Tubulin was used as a loading control. (D, E, F) Invasion assay across a transwell membrane coated with Matrigel, percentage of invaded cells compared with NSC cells at (D) 24 h for A549, n = >4, (E) 48 h for H460, n = 6, and (F) 16 h for A375 cells, n = 3 (as an experimental control). (G, H) A549 wound healing assay. (G) Time course, percentage wound closure at each time-point compared with wound at 0 h, and data are presented as mean ± SEM. (H) Percentage wound closure at 24 h compared with wound at 0 h, n = 5. Data are presented as an individual mean for each experiment with bar representing overall mean ± SEM. Statistical comparisons were performed using one-way ANOVA with Dunnett’s multiple test correction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant.

Article Snippet: Authenticated A549 (CCL185) and H460 (HTB-177) lung adenocarcinoma cells and A375 (CRL1619) melanoma cells were obtained from the ATCC.

Techniques: Western Blot, Expressing, Control, Invasion Assay, Membrane, Wound Healing Assay

Journal: Life Science Alliance

Article Title: Rnd3 regulates lung cancer cell invasion and migration independently of ROCK1 signaling via alpha 5 integrin modulation

doi: 10.26508/lsa.202503494

Figure Lengend Snippet: (A, B, C, D, E, F, G, H, I) Quantification of representative Western blots with samples used in cell migration and cell invasion assays in . Densitometric analysis was performed for each sample set over multiple biological replicates for Rnd3 (A, D, G), ROCK1 (B, E, H), and ROCK2 (C, F, I) expression after transient transfection with siRNA oligo compared with expression levels in non-silencing control cells in (A, B, C) A549, n = 5, (D, E, F) H460, n = 4, and (G, H, I) A375 cells, n = 3. Expression levels of each protein of interest were normalized to own tubulin loading control, then normalized to the expression level of the protein of interest in the NSC cells. Data are presented as an individual mean for each experiment with bar representing overall mean ± SEM. Statistical comparisons were performed using one-way ANOVA with Dunnett’s multiple test correction. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Authenticated A549 (CCL185) and H460 (HTB-177) lung adenocarcinoma cells and A375 (CRL1619) melanoma cells were obtained from the ATCC.

Techniques: Western Blot, Migration, Expressing, Transfection, Control

Journal: Cancers

Article Title: Mechanisms of the Antiproliferative Effects of SIRT6 Inhibition in Melanoma: A Multi-Omics Analysis

doi: 10.3390/cancers18040590

Figure Lengend Snippet: Characterization of SIRT6 expression in G361 knockdown (KD) cells and proteogenomics workflow in melanoma cells. ( A ) CRISPR/Cas9-mediated SIRT6 KD G361 cells were created, and protein expression was determined by Protein Simple automated Western blotting. SIRT6 KD reduces the ( B ) proliferation and ( C ) colony forming potential of G361 melanoma cells. Data is representative of at least 2 biological and 3 technical replicates per group, with statistical significance determined using one-way ANOVA and data shown as mean ± SEM (* p ≤ 0.05, **** p ≤ 0.0001). ( D ) Schematic representation of the experimental workflow for integrative proteogenomic analysis in A375 and G361 melanoma cells, outlining the steps from sample preparation to data integration. For RNA-seq and proteomics, data was generated from three independent biological replicates per group. For all datasets, control cell line was matched for each knockdown strategy.

Article Snippet: A375 cells were grown per recommendation from ATCC in DMEM medium (Corning, Corning, NY, USA, #10-013-CV) supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA, #F2442) and G361 cells were grown in McCoy’s 5A medium (Corning, #10-050-CV) supplemented with 10% FBS. shRNA-mediated SIRT6 KD A375 cells and shNS control cells used for proteomics work were made as previously described [ ], while CRISPR/Cas9-mediated SIRT6 KD A375 cell lines used for RNA-seq work were purchased from Synthego (Redwood City, CA, USA) and clones selected as described previously [ ].

Techniques: Expressing, Knockdown, CRISPR, Western Blot, Sample Prep, RNA Sequencing, Generated, Control

Journal: Cancers

Article Title: Mechanisms of the Antiproliferative Effects of SIRT6 Inhibition in Melanoma: A Multi-Omics Analysis

doi: 10.3390/cancers18040590

Figure Lengend Snippet: Transcriptomic profiling and functional enrichment analysis of SIRT6 knockdown (KD) A375 cells. ( A ) The top 20 up/down regulated differentially expressed genes (DEGs) identified by RNA-seq in SIRT6 KD A375 cells denoted via volcano plot. Dashed line indicates significant genes with Log2 FC ≥ |1.5|. Subsequent Gene Ontology (GO) enrichment by PANTHER of top 200 significant DEGs found significant changes in several biological processes ( B ) and molecular functions ( C ) in SIRT6 KD A375 cells.

Article Snippet: A375 cells were grown per recommendation from ATCC in DMEM medium (Corning, Corning, NY, USA, #10-013-CV) supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA, #F2442) and G361 cells were grown in McCoy’s 5A medium (Corning, #10-050-CV) supplemented with 10% FBS. shRNA-mediated SIRT6 KD A375 cells and shNS control cells used for proteomics work were made as previously described [ ], while CRISPR/Cas9-mediated SIRT6 KD A375 cell lines used for RNA-seq work were purchased from Synthego (Redwood City, CA, USA) and clones selected as described previously [ ].

Techniques: Functional Assay, Knockdown, RNA Sequencing

Journal: Cancers

Article Title: Mechanisms of the Antiproliferative Effects of SIRT6 Inhibition in Melanoma: A Multi-Omics Analysis

doi: 10.3390/cancers18040590

Figure Lengend Snippet: Functional insights from Ingenuity Pathway Analysis (IPA). ( A ) Graphical summary generated by Ingenuity Pathway Analysis highlighting predicted target regulation activity based on differentially expressed genes (DEGs) in SIRT6 knockdown (KD) G361 cells. ( B ) Top canonical pathways affected in SIRT6 KD G361 cells as predicted by IPA based on the transcriptomic data. IPA analysis of differentially expressed genes shows regulation of cancer-related biological functions in SIRT6 KD G361 ( C ) and A375 cells ( D ).

Article Snippet: A375 cells were grown per recommendation from ATCC in DMEM medium (Corning, Corning, NY, USA, #10-013-CV) supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA, #F2442) and G361 cells were grown in McCoy’s 5A medium (Corning, #10-050-CV) supplemented with 10% FBS. shRNA-mediated SIRT6 KD A375 cells and shNS control cells used for proteomics work were made as previously described [ ], while CRISPR/Cas9-mediated SIRT6 KD A375 cell lines used for RNA-seq work were purchased from Synthego (Redwood City, CA, USA) and clones selected as described previously [ ].

Techniques: Functional Assay, Generated, Activity Assay, Knockdown

Journal: Cancers

Article Title: Mechanisms of the Antiproliferative Effects of SIRT6 Inhibition in Melanoma: A Multi-Omics Analysis

doi: 10.3390/cancers18040590

Figure Lengend Snippet: Proteomics analysis of SIRT6 knockdown (KD) A375 melanoma cells. Proteomic analysis of SIRT6 KD A375 cells showing peptide identifications ( A ) and abundance data ( B ) in respective proteins. Gene Ontology (GO) enrichment based on PANTHER analysis reveals regulation of multiple biological ( C ) and molecular ( D ) functions in SIRT6 KD A375 cells.

Article Snippet: A375 cells were grown per recommendation from ATCC in DMEM medium (Corning, Corning, NY, USA, #10-013-CV) supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA, #F2442) and G361 cells were grown in McCoy’s 5A medium (Corning, #10-050-CV) supplemented with 10% FBS. shRNA-mediated SIRT6 KD A375 cells and shNS control cells used for proteomics work were made as previously described [ ], while CRISPR/Cas9-mediated SIRT6 KD A375 cell lines used for RNA-seq work were purchased from Synthego (Redwood City, CA, USA) and clones selected as described previously [ ].

Techniques: Knockdown

Journal: Cancers

Article Title: Mechanisms of the Antiproliferative Effects of SIRT6 Inhibition in Melanoma: A Multi-Omics Analysis

doi: 10.3390/cancers18040590

Figure Lengend Snippet: Comparative analysis of multi-omics data. Comparison of SIRT6 knockdown (KD) G361 and A375 cell-based transcriptomics and proteomics data by Ingenuity Pathway Analysis (IPA) predicted regulation of multiple canonical pathways ( A ) and biological functions ( B ) that were predicted to be modulated and are related to cell proliferation, division, migration, invasion, and death at RNA and protein levels. By analyzing protein modulation ≥|1.2|-fold (red = increased levels; green = decreased levels), IPA predicted inhibition of cell proliferation ( C ) and activation of cell death of tumor cell lines ( D ) in our SIRT6 KD A375 cells as compared to the shNS control cells ( n = 3 per group).

Article Snippet: A375 cells were grown per recommendation from ATCC in DMEM medium (Corning, Corning, NY, USA, #10-013-CV) supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA, #F2442) and G361 cells were grown in McCoy’s 5A medium (Corning, #10-050-CV) supplemented with 10% FBS. shRNA-mediated SIRT6 KD A375 cells and shNS control cells used for proteomics work were made as previously described [ ], while CRISPR/Cas9-mediated SIRT6 KD A375 cell lines used for RNA-seq work were purchased from Synthego (Redwood City, CA, USA) and clones selected as described previously [ ].

Techniques: Biomarker Discovery, Comparison, Knockdown, Transcriptomics, Migration, Inhibition, Activation Assay, Control

Journal: Cancers

Article Title: Mechanisms of the Antiproliferative Effects of SIRT6 Inhibition in Melanoma: A Multi-Omics Analysis

doi: 10.3390/cancers18040590

Figure Lengend Snippet: Causal networks from multi-omics data. Comparative analysis of multi-omics data from SIRT6 knockdown (KD) G361 and A375 cells by Ingenuity Pathway Analysis (IPA) showed predicted regulation of multiple networks. ( A ) Common causal networks inferred from multi-omics data, integrating upstream regulators and downstream effects. ( B ) SIRT6 KD in A375 cells affects multiple proteins with ≥|1.2|-fold change ( n = 3 per group; shNS and shSIRT6) related to transcription factor family networks, highlighting top common pathways with activation z-scores ≥|2| in both transcriptomics and proteomics data.

Article Snippet: A375 cells were grown per recommendation from ATCC in DMEM medium (Corning, Corning, NY, USA, #10-013-CV) supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA, #F2442) and G361 cells were grown in McCoy’s 5A medium (Corning, #10-050-CV) supplemented with 10% FBS. shRNA-mediated SIRT6 KD A375 cells and shNS control cells used for proteomics work were made as previously described [ ], while CRISPR/Cas9-mediated SIRT6 KD A375 cell lines used for RNA-seq work were purchased from Synthego (Redwood City, CA, USA) and clones selected as described previously [ ].

Techniques: Biomarker Discovery, Knockdown, Activation Assay, Transcriptomics