Journal: Frontiers in Oncology

Article Title: Overexpression of miRNA-3613-3p Enhances the Sensitivity of Triple Negative Breast Cancer to CDK4/6 Inhibitor Palbociclib

doi: 10.3389/fonc.2020.590813

Figure Lengend Snippet: Overexpression of miR-3613-3p suppresses the proliferation, migration, and clonogenic ability induces G0/G1 cell-cycle arrest in TNBC cells. Effects of miR-3613-3p on TNBC cell growth and migration in vitro . After generation of miR-3613-3p stable overexpression in MDA-MB-231. (A) Cell vitality was evaluated by MTT assay, stably transfected with miR-3613-3p or NC lentivirus. (B, C) Colonigenic ability of different MDA-MB-231 cells were tested; generation of miR-3613-3p stable overexpression, the numbers of colony were counted and compared in the diagrams. (D, E) Cell migration of different MDA-MB-231 cells was tested using monolayer wound healing assay, generation of miR-3613-3p stable overexpression; the percent of wound closure was counted and compared in the diagrams. (F, G) Cell cycle of MDA-MB-231 cells stably transfected with miR-3613-3p or NC lentivirus was analyzed by flow cytometry assay; the percentage of cells in G0/G1, S, and G2/M phase is annotated in each column. (H, I) EdU assay of relative DAPI stained cells and EdU add-in cells. MDA-MB-231 cells were stably transfected with miR-3613-3p or NC lentivirus. At least 200 cells were counted per well. (J) Western blot analysis of positive cell cycle regulators CCND1, CCND2, CCND3, pRb, p-pRb, c-MYC, CDK4, and CDK6 protein in MDA-MB-231 cells transfected with transfected with miR-3613-3p lentivirus or vector control. P values were determined by two-tailed t-test, **P < 0.01; ****p < 0.0001.

Article Snippet: Primary antibodies were used as follows: anti-c-MYC (1:1,000, Proteintech), anti-EZH2, anti-SMAD2, anti-RB, anti-CDK4, anti-CDK6, anti-CCND1, anti-CCND2 and anti-CCND3 (1:1,000, Cell Signaling Technology) and anti-Actin (1:3,000, Sigma).

Techniques: Over Expression, Migration, In Vitro, MTT Assay, Stable Transfection, Transfection, Wound Healing Assay, Cytometry, EdU Assay, Staining, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: Translational Lung Cancer Research

Article Title: Deubiquitinase USP5 promotes non-small cell lung cancer cell proliferation by stabilizing cyclin D1

doi: 10.21037/tlcr-21-767

Figure Lengend Snippet: USP5 promotes cell cycle progression and proliferation in non-small cell lung cancer (NSCLC) cells. (A,B) A549 and H1299 cells were transfected with siUSP5 or siNC (150 nM) for 48 hours. Cell cycle phases were then analyzed by flow cytometry. Representative images of flow cytometric cell cycle analysis (A) and the percentage of cells in each cell cycle phase distribution (B) are shown. G1 or S in A549: G1, **, P<0.01 or S, ***, P<0.001; G1 or S in H1299: *, P<0.05, Student’s t-test. (C) In A549 and H1299 cells, the levels of proteins involved in the CCND1-CDK4-Rb signaling pathway after knockdown of USP5 were assessed by immunoblotting (IB) assay using the indicated antibodies (Abs) [primary Ab: anti-CCND1, 1:1,000, anti-CCND2, 1:1,000, anti-CCND3, 1:1,000, anti-CDK4, 1:1,000, anti-CDK6, 1:1,000, anti-pRb (Ser780), 1:1,000, or anti-Rb, 1:1,000; secondary Ab: horseradish peroxidase-labeled goat anti-rabbit or anti-mouse IgG, 1:2,000]. (D,E) Myc-USP5- and siUSP5-transfected A549 and H1299 cells along with the respective control cells were cultured for different time periods (24–72 hours), and cell viability was then measured by Cell Counting Kit-8 assay. **, P<0.01 or ***, P<0.001 compared with their respective control at the same time point, Student’s t-test. (F,G) Colony formation assay was performed using siUSP5-tranfected A549 and H1299 cells and their respective control cells. Representative images of colony formation assay (F) and the number of colonies (G) are shown. *, P<0.05, **, P<0.01, or ***, P<0.001 compared with the respective control, Student’s t-test.

Article Snippet: Antibodies and chemicals The following antibodies (Abs) were used: rabbit anti-CCND1 [Cat. No. 2922 for IP/immunoblotting (IB) and 2978 for immunohistochemistry (IHC), CST, Danvers, MA, USA], rabbit anti-USP5 (Cat. No. 10473-1-AP, Proteintech, Wuhan, Hubei, China), rabbit anti-CCND2 (Cat. No. 2924, CST), rabbit anti-CCND3 (Cat. No. 26775-1-AP, Proteintech), mouse anti-CDK4 (Cat. No. 12790, CST), mouse anti-CDK6 (Cat. No. 3136, CST), mouse anti-Rb (Cat. No. 9309, CST), rabbit anti-Phospho-Rb (Ser780) (Cat. No. 3590, CST), rabbit anti-β-tubulin (Cat. No. 10068-1-AP, Proteintech), mouse anti-Ub (Cat. No. sc-271289, Santa Cruz, Dallas, TX, USA), mouse anti-Myc (Cat. No. M192-3, MBL, Tokyo, Japan), mouse anti-Flag (Cat. No. M185-3, MBL), mouse anti-HA (Cat. No. M180-3, MBL), and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H+L) (Cat. No. A0208, Beyotime) and anti-mouse IgG (H+L) (Cat. No. A0216, Beyotime).

Techniques: Transfection, Flow Cytometry, Cell Cycle Assay, Knockdown, Western Blot, Labeling, Control, Cell Culture, Cell Counting, Colony Assay