Journal: The Journal of biological chemistry

Article Title: PP2A activation overcomes leptomeningeal dissemination in group 3 medulloblastoma.

doi: 10.1016/j.jbc.2024.107892

Figure Lengend Snippet: Figure 2. ATUX-1215 and ATUX-5800 activate PP2A in human group 3 MB PDX cells. A, D425 cells (1 × 106) were treated with ATUX-1215 or ATUX-5800 at increasing concentrations (0, 2.5, and 5 mM) for 4 h. PP2A activation was measured with a kit. PP2A activity was increased in the D425 cells with all compounds but did not reach statistical significance. B, D341 cells (1 × 106) were treated with ATUX-1215 or ATUX-5800 at increasing concentrations (0, 2, 4 mM of ATUX-1215 or 0, 4, 6 mM ATUX-5800) for 4 h and PP2A activity was measured. Both drugs increased PP2A activation significantly in the D341 cells. (C) immunoblotting of D425 and (D) D341 whole cell lysates demonstrate decreased CIP2A expression with ATUX-1215 and ATUX-5800. I2PP2A expression increased with ATUX-1215 or ATUX-5800 treatment. Vinculin and GAPDH served as internal loading controls. (CIP2A control GAPDH same blot as 3E, total Parp and cleaved caspase 9). Data are normalized to no treatment group (100) and presented as mean ± standard deviation (SD). Each dot represents an independent biologic replicate. Statistical comparison was completed with two-tailed Student’s t test. ***p = 0.001, ****p = 0.0001. CIP2A, cancerous in- hibitor of PP2A; I2PP2A, inhibitor 2 of protein phosphatase 2A; MB, medulloblastoma; PDX, patient-derived xenograft; PP2A, protein phosphatase 2A.

Article Snippet: Primary antibodies used for immunoblotting included: rabbit polyclonal anti-CIP2A (ab99518) from Abcam; rabbit polyclonal anti-SET (55201-AP) from Proteintech; rabbit monoclonal antibodies-anti-PARP (9532), anti-cleaved caspase-9 (20750S), anti-Bad (9239S), anti-pBad (5284S), antivinculin (13901S), anti-NF-kB (8242S), and anti-pNF-kB (3033S), and rabbit polyclonal antibodies-anti-total AKT (9272) and anti-phospho-AKT (S473; 9271) from Cell Signaling Technology; mouse monoclonal anti-b-actin from Sigma (A1978, Sigma-Aldrich).

Techniques: Activation Assay, Activity Assay, Western Blot, Expressing, Control, Standard Deviation, Comparison, Two Tailed Test, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: PP2A activation overcomes leptomeningeal dissemination in group 3 medulloblastoma.

doi: 10.1016/j.jbc.2024.107892

Figure Lengend Snippet: Figure 3. ATUX-1215 and ATUX-5800 impairs viability in human group 3 MB PDX cells. A–C, group 3 MB PDX cells were treated with increasing concentrations of PP2A activators (ATUX-1215 and ATUX-5800) for 24 h (D425) or 72 h (D341), and cell viability was measured using alamarBlue assay. A and B, decreased cell viability was dose responsive with increasing PP2A concentrations. Dose-response curves demonstrate variation in lethal dose 50% (LD50) ranging between 5.06 and 14.04 mM for ATUX-1215 and ATUX-5800 in D341 and D425 cells. C, LD50 data from three independent biologic replicates are presented in tabular form. D–G, D425 and D341 cells were treated with ATUX-1215 or ATUX-5800 at increasing concentrations (0–6 mM) for 24 h (D425) or 72 h (D341). Immunoblotting of whole cell lysates was completed. D and E, indication of apoptosis in treated D425 and D341 cells includes decreased total PARP and increased cleaved caspase 9. (Total Parp and cleaved caspase 9C control GAPDH same blot as 2D CIP2A). When investigating mechanisms for increased apoptosis we found that treatment with the ATUX compounds decreased phosphorylation of Bad (pBad) in D425 and D341 cells. (BAD control GAPDH same blot as 7B, NF-kB). F and G, phosphorylation of AKT is decreased with increasing concentrations of PP2A activating small molecules, indicating a potential mechanism for decreased cell viability and apoptosis. (BAD control GAPDH same blot as 7B, CCL2) b-actin, vinculin, and GAPDH provided an internal control for equal protein loading. Data are presented as mean ± standard error of the mean (SEM) and represent at least three independent biologic replicates. Statistical comparison was completed with two-tailed Student’s t test. **p = 0.01, ***p = 0.001, ****p = 0.0001. CIP2A, cancerous inhibitor of PP2A; MB, medulloblastoma; PDX, patient-derived xenograft; PP2A, protein phosphatase 2A.

Article Snippet: Primary antibodies used for immunoblotting included: rabbit polyclonal anti-CIP2A (ab99518) from Abcam; rabbit polyclonal anti-SET (55201-AP) from Proteintech; rabbit monoclonal antibodies-anti-PARP (9532), anti-cleaved caspase-9 (20750S), anti-Bad (9239S), anti-pBad (5284S), antivinculin (13901S), anti-NF-kB (8242S), and anti-pNF-kB (3033S), and rabbit polyclonal antibodies-anti-total AKT (9272) and anti-phospho-AKT (S473; 9271) from Cell Signaling Technology; mouse monoclonal anti-b-actin from Sigma (A1978, Sigma-Aldrich).

Techniques: Alamar Blue Assay, Western Blot, Control, Phospho-proteomics, Comparison, Two Tailed Test, Derivative Assay