Journal: Oncogenesis

Article Title: ACTL6A suppresses p21 Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

doi: 10.1038/s41389-021-00362-7

Figure Lengend Snippet: A ACTL6A was successfully knocked down using ACTL6A-siRNA which leads to an increase in p21 Cip1 protein level. B – D ACTL6A knockdown decreases spheroid number and size, invasion and migration. For spheroid assay, 40,000 cells were seeded on ultra-low attachment plates and counted at Day 4. Spheroid diameters were measured using ImageJ. E – H ACTL6A knockout cell clones, which lack ACTL6A, display elevated p21 Cip1 expression and this is associated with reduced cell proliferation, spheroid formation and invasion. I , J ACTL6A overexpression in ACTL6A knockout cell clones reduces p21 Cip1 protein and mRNA levels. Asterisks indicate a significant decrease ( n = 3, p ≤ 0.001).

Article Snippet: The pCMV3-ACTL6A-FLAG expression vector (HG10963-CF) was obtained from Sino Biologicals (Wayne State, PA).

Techniques: Migration, Knock-Out, Clone Assay, Expressing, Over Expression

Journal: Oncogenesis

Article Title: ACTL6A suppresses p21 Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

doi: 10.1038/s41389-021-00362-7

Figure Lengend Snippet: A – C p21 Cip1 knockdown enhances spheroid formation and invasion. D , E ACTL6A knockdown increases p21 Cip1 protein level and reduces invasion, and these actions are partially reversed following simultaneous p21 Cip1 -siRNA and ACTL6A knockdown. F – I ACTL6A knockout lines display enhanced p21 Cip1 levels and reduced spheroid formation. p21 Cip1 knockdown in these lines increases spheroid formation, invasion, and migration. This suggests that p21 Cip1 is a mediator of ACTL6A suppression of the cancer phenotype. Single asterisks indicate a significant decrease ( n = 3, p ≤ 0.001) and double asterisks indicate a significant increase ( n = 3, p ≤ 0.001).

Article Snippet: The pCMV3-ACTL6A-FLAG expression vector (HG10963-CF) was obtained from Sino Biologicals (Wayne State, PA).

Techniques: Knock-Out, Migration

Journal: Oncogenesis

Article Title: ACTL6A suppresses p21 Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

doi: 10.1038/s41389-021-00362-7

Figure Lengend Snippet: A – D ACTL6A knockdown increases p21 Cip1 protein level and is associated with reduced spheroid formation, invasion, and migration. E – H ACTL6A knockdown increases p21 Cip1 levels and reduces spheroid formation, invasion, and migration and p21 Cip1 knockdown partially reverses the ACTL6A-knockdown dependent reduction in spheroid formation, invasion, and migration. These findings suggest that ACTL6A suppresses p21 Cip1 to maintain the aggressive NCI-Meso-17 cell cancer phenotype. Single asterisks indicate a significant decrease ( n = 3, p ≤ 0.001) and double asterisks indicate a significant increase ( n = 3, p ≤ 0.005).

Article Snippet: The pCMV3-ACTL6A-FLAG expression vector (HG10963-CF) was obtained from Sino Biologicals (Wayne State, PA).

Techniques: Migration

Journal: Oncogenesis

Article Title: ACTL6A suppresses p21 Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

doi: 10.1038/s41389-021-00362-7

Figure Lengend Snippet: A , B ACTL6A knockdown increases Meso-1 cell p21 Cip1 mRNA expression and protein level. C ACTL6A knockout Meso-1 cells express increased p21 Cip1 mRNA. D Map of the full-length p21 Cip1 promoter showing the p53-1 and p53-2 sites in the distal promoter and six Sp1 site in the proximal promoter. The top sequence indicates the authentic p53 site sequences. Our previous report indicates the mutations that inactivate these sites . E ACTL6A knockout Meso-1 cells display enhanced p21 Cip1 promoter activity for the full-length (p21-2326) and proximal (p21-124) promoter, and mutation of the six Sp1 sites eliminates the response. F Chromatin IP, using Meso-1 extracts, shows ACTL6A binding to the p21 Cip1 promoter p53 binding sites and the proximal promoter Sp1 sites. G – I ACTL6A knockdown in NCI-Meso-17 cells increases p21 Cip1 mRNA level and p21 Cip1 promoter activity and this is associated with ATL6A binding to the p21 Cip1 promoter p53 and Sp1 sites. As observed for Meso-1 cells, mutation of the proximal Sp1 sites eliminates transcriptional activity. Double asterisks indicate a significant increase ( n = 3, p ≤ 0.001).

Article Snippet: The pCMV3-ACTL6A-FLAG expression vector (HG10963-CF) was obtained from Sino Biologicals (Wayne State, PA).

Techniques: Expressing, Knock-Out, Sequencing, Activity Assay, Mutagenesis, Chromatin Immunoprecipitation, Binding Assay

Journal: Oncogenesis

Article Title: ACTL6A suppresses p21 Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

doi: 10.1038/s41389-021-00362-7

Figure Lengend Snippet: A , B ACTL6A-knockdown increases p53 and p21 Cip1 mRNA levels and p21 Cip1 protein level, but p53 protein is not detected in ACTL6A competent or knockdown Meso-1 cells. C Chromatin IP assay shows ACTL6A binding to the distal p53 binding sites and the proximal promoter Sp1 binding sites in Meso-1 cell extracts, but no binding of p53 is detected. D p53 knockdown does not alter Meso-1 cell spheroid number or invasion. E , F p53 protein is absent in ACTL6A knockout cells even though p53 mRNA levels are increased. G ACTL6A knockdown increases p53 in NCI-Meso-17 cells. H ACTL6A binds to the p21Cip1 promoter Sp1 and p53 sites. p53 binds to the p53 binding sites (p21-p53 sites 1 and 2) but not the Sp1 binding sites (p21-proximal). I , J ACTL6A knockdown reduces spheroid formation and invasion, p53 knockdown increases spheroid formation and invasion, and combination treatment with ACTL6A- and p53-siRNA does not alter ACTL6A suppression of spheroid formation and invasion. This suggests that ACTL6A suppresses the ability of p53 to activate p21 Cip1 promoter transcription. K ACTL6A knockdown does not increase p53 mRNA level. Single asterisks indicate a significant decrease ( n = 3, p ≤ 0.001) and double asterisks indicate a significant increase ( n = 3, p ≤ 0.001). All statistical comparisons are to the control group. Single asterisks indicate a significant decrease ( n = 3, p ≤ 0.001) and double asterisks indicate a significant increase ( n = 3, p ≤ 0.001).

Article Snippet: The pCMV3-ACTL6A-FLAG expression vector (HG10963-CF) was obtained from Sino Biologicals (Wayne State, PA).

Techniques: Chromatin Immunoprecipitation, Binding Assay, Knock-Out

Journal: Oncogenesis

Article Title: ACTL6A suppresses p21 Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

doi: 10.1038/s41389-021-00362-7

Figure Lengend Snippet: A , B Wild-type (Meso-1) and ACTL6A knockout (Meso1-ACTL6A-KOc1-5-1) cells (3 million/site) were injected into each front flank in five NSG mice per treatment group (10 tumor/group) and tumor formation was monitored for 0–6 week. The images are two paired sets of tumors harvested at 6 weeks. Differences in tumor formation were analyzed using the Student’s t test and values are presented as mean ± SEM and p values are indicated. Single asterisks indicate a significant decrease ( n = 10 tumor, p ≤ 0.001). C Meso-1 cell tumors display low levels of p21 Cip1 while p21 Cip1 levels are elevated in Meso1-ACTL6A-KOc1-5-1 cell tumors. D Meso-1 and Meso1-ACTL6A-KOc1-5-1 tumors cells were dissociated and grown in culture for detection of ACTL6A and p21 Cip1 . These blots show that the Meso1-ACTL6A-KOc1-5-1 cells retained their ACTL6A-null status during tumor formation. E ACTL6A and p21 Cip1 levels were compared between NCI-Meso-17 cells and tumors isolated from three patients. F Schematic describing the role of ACTL6A in regulating cancer cell function. ACTL6A binds to the p21 Cip1 promoter p53-1 and 2 sites and the Sp1 sites to suppress p21 Cip1 transcription. The resulting reduction in p21 Cip1 level leads to enhanced cell proliferation, spheroid formation, Matrigel invasion, migration, and tumor formation.

Article Snippet: The pCMV3-ACTL6A-FLAG expression vector (HG10963-CF) was obtained from Sino Biologicals (Wayne State, PA).

Techniques: Knock-Out, Injection, Isolation, Cell Function Assay, Migration