Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Relative mRNA expression of UNC13B in multiple myeloma cell lines. ( A ) Quantitative real-time PCR (qPCR) analysis of UNC13B mRNA levels in U266, ARD, and RPMI 8226 cell lines. U266 was used as the reference control for relative quantification, and GAPDH served as the internal reference gene. Expression levels were calculated using the 2 -ΔΔCt method and are presented as fold-change relative to U266. The mean ± standard deviation of UNC13B expression was 1.00 ± 0.26 in U266, 3.07 ± 0.44 in ARD, and 1.41 ± 0.26 in RPMI 8226 cells. Overlaid symbols (●, ■, ▲) denote individual independent experiments ( n = 5 per group); different marker shapes are used only for visual distinction and carry no additional meaning. ( B ) Relative UNC13B mRNA expression in ARD cells following different treatments. The Scramble + 25 μM treatment group was used as the reference for comparison. **** indicate p < 0.0001; ns = not significant ( p ≥ 0.05). Data are presented as the mean ± standard deviation (SD, n = 5 independent experiments).

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Quantitative Proteomics, Gene Expression, Standard Deviation, Marker, Comparison

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on cell proliferation. The proliferation rate of ARD cells in the shUNC13B group was significantly inhibited ( p < 0.0001), suggesting that UNC13B expression correlated with the proliferative capacity of ARD cells. **** indicate p < 0.0001; Data are presented as mean ± SD ( n = 3 independent experiments).

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Expressing

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on the clonogenic ability of cells. ( A ) Representative images of colony formation in soft agar. ARD cells stably transduced with either shUNC13B or scrambled shRNA were seeded into 6-well plates (1000 cells per well) in soft agar consisting of a 0.35% agar upper layer over a 0.6% agar base layer. After 14 days of incubation at 37 °C in 5% CO 2 , visible colonies (>50 µm in diameter) were observed under a microscope. ( B ) Quantification of colony numbers. The total number of colonies per well was recorded.Overlaid symbols (●, ■) denote individual independent experiments; different marker shapes are used only for visual distinction and have no additional meaning. Data are presented as the mean ± SD. Statistical analysis was performed using a two-tailed unpaired t -test. ** indicates p < 0.01. Scale bar: 500 µm.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Stable Transfection, Transduction, shRNA, Incubation, Microscopy, Marker, Two Tailed Test

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on the cell cycle in ARD cells. Five days after transduction with shRNA lentiviral vectors, the shUNC13B group showed a reduced fraction of ARD cells in G1 ( p < 0.0001) and an increased fraction in S phase ( p < 0.0001) compared with the Scramble control, with no significant change in G2/M (ns, not significant). Histograms show DNA-content modeling: red areas denote fitted G1 and G2/M peaks, the blue hatched area denotes S phase, and the black line is the measured distribution; ▲ indicates software-generated peak positions (reference only, not used for quantification). **** indicate p < 0.0001.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Transduction, shRNA, Control, Software, Generated

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on apoptosis in ARD cells. ( A ) Flow cytometric analysis of apoptosis using Annexin V-APC staining. The shUNC13B group exhibited a significantly higher percentage of apoptotic ARD cells compared with the scrambled control group ( p < 0.01); Overlaid symbols (●, ■) denote individual independent experiments; different marker shapes are used only for visual distinction and have no additional meaning. ( B ) Western blot analysis of apoptosis- and signaling-related proteins in ARD cells stably transduced with scrambled shRNA or UNC13B shRNA (shUNC13B) for 72 h. Expression levels of PINK1, CDK2, and AKR7A3 were downregulated, while Bim was upregulated, and PKC levels changed only marginally. In the same samples, UNC13B protein expression was markedly reduced, accompanied by substantial increases in cleaved-PARP (~89 kDa), Bax (~21 kDa), and p21 (CDKN1A) levels. GAPDH served as the loading control. ** indicate p < 0.01.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Staining, Control, Marker, Western Blot, Stable Transfection, Transduction, shRNA, Expressing