Journal: OncoTargets and Therapy

Article Title: Synergistic Effect of Ubiquitin-Specific Protease 14 and Poly(ADP-Ribose) Glycohydrolase Co-Inhibition in BRCA1-Mutant, Poly(ADP-Ribose) Polymerase Inhibitor-Resistant Triple-Negative Breast Cancer Cells

doi: 10.2147/ott.s463217

Figure Lengend Snippet: Figure 4 The NHEJ signaling is required for the combination effects of PARGi COH34 and USP14i IU1-248. (A and B) Long-term relative cell viability was measured by crystal violet assay for HCC1937 (A) and SUM149PT (B) cells. Cells were transfected with si-NC or si-Ku70 and treated with 2.5 μM COH34 and 5 μM IU1-248 as single- agents or in combination for 6 (SUM149PT) or 7 (HCC1937) days. Ku70 protein levels of Western blot analysis are shown. β-Actin was used as a loading control. (C) and (D) The synergistic effect of concomitant PARG and USP14 inhibition in HCC1937 (C) and SUM149PT (D) cells with or without Ku70-knockdown was measured by CCK8 assay after 72 h treatment. Notes: The data are shown as the mean ±S.D. *P< 0.05; **P< 0.01; ***P< 0.001 (Student’s t test).

Article Snippet: The primary antibodies used were PARG (27808-1-AP, Proteintech Group, Wuhan, China), USP14 (14,517- 1-AP, Proteintech Group), Ku70 (10,723-1-AP, Proteintech Group), BRCA1 (22,362-1-AP, Proteintech Group), c-Myc (5605, Cell Signaling Technology, MA, USA), and β-Actin (66009-1-Ig, Proteintech Group).

Techniques: Crystal Violet Assay, Transfection, Western Blot, Control, Inhibition, Knockdown, CCK-8 Assay

Journal: OncoTargets and Therapy

Article Title: Synergistic Effect of Ubiquitin-Specific Protease 14 and Poly(ADP-Ribose) Glycohydrolase Co-Inhibition in BRCA1-Mutant, Poly(ADP-Ribose) Polymerase Inhibitor-Resistant Triple-Negative Breast Cancer Cells

doi: 10.2147/ott.s463217

Figure Lengend Snippet: Figure 6 Schematic illustration to conclude the synergistic effect of USP14i and PARGi on BRCA1-mutant, PARP inhibitor-resistant TNBC cells. USP14 blockade increases the error-prone non-homologous end joining (NHEJ) through downregulation of c-Myc, and thus underlies PARG inhibitor sensitivity in BRCA-mutant, PARP inhibitor- resistant TNBC cells.

Article Snippet: The primary antibodies used were PARG (27808-1-AP, Proteintech Group, Wuhan, China), USP14 (14,517- 1-AP, Proteintech Group), Ku70 (10,723-1-AP, Proteintech Group), BRCA1 (22,362-1-AP, Proteintech Group), c-Myc (5605, Cell Signaling Technology, MA, USA), and β-Actin (66009-1-Ig, Proteintech Group).

Techniques: Mutagenesis, Non-Homologous End Joining

Journal: eLife

Article Title: Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-κB activation

doi: 10.7554/elife.15018

Figure Lengend Snippet: Figure 6. Sam68 plays a critical protective role for the survival of mouse and human colon cancers. (A) Methylene blue (MB) staining of the colons (with cecum, proximal colon [PC], distal colon [DC], and anus indicated) derived from 3-month old Apcmin716/+; Khdrbs1+/- and Apcmin716/+; Khdrbs1-/- mice. Red arrows indicate colon tumors. Scale bar, 1 cm. (B) Quantification of tumor number, tumor size, and tumor load in the colons from Apcmin716/+; Khdrbs1+/- (n = 6) and Apcmin716/+; Khdrbs1-/- mice (n = 3) following MB staining. (C) HCT8 and HCT116 cells were transfected with nonspecific control (si-NC) or Sam68-specific (si-Sam68) small interference RNAs. 72 hr later, whole cell lysates were derived and immunoblotted (IB) for indicated proteins, with b-actin as a loading control. (D) Immunofluorescence micrographs of Bcl-XL and PARylated (PAR) proteins in the si-NC and si-Sam68 transfected HCT116 cells, with nuclei counterstained by DAPI. Scale bar, 20 mm. (E) Percentage of HCT116 cells (>100 cells from 5–8 random fields) with Bcl-XL and PAR staining was quantified. (F) HCT116 cells transfected with indicated siRNAs as in (C) were stimulated with indicated doses of Camptothecin (CPT) for 6 hr. Whole cell lysates were derived and IB for indicated proteins, with b-actin as a loading control. c-Casp3, cleaved Caspase-3. The full-length and cleaved PARP1 are indicated by a black triangle and a red triangle, respectively; the two species of Bcl-XL proteins are labeled by open triangles. (G) HCT8 cells transfected with indicated siRNAs as in (C) were left untreated (UT) or stimulated with 10 mM of CPT for 12 hr, followed by propidium iodide (PI)/Annexin V staining and flow cytometry analysis. (H) Percentages of apoptotic (PI- Annexin V+) HCT8 cells treated as in (G) were quantified. Results in (B, E, and H) are expressed as mean and s.e.m. ns, non-significant difference; *p<0.05; **p<0.01; ***p<0.001 by Student’s t tests. Data in (A and C–H) are representative of at least three independent experiments. DOI: 10.7554/eLife.15018.018 The following figure supplements are available for figure 6:

Article Snippet: Human PARP1 and PARG siRNAs were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Derivative Assay, Transfection, Control, Immunofluorescence, Labeling, Flow Cytometry