Journal: Oncology Letters

Article Title: Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells

doi: 10.3892/ol.2020.12370

Figure Lengend Snippet: Downregulation of Beclin-1 enhances apoptosis and reduces cell viability induced by bortezomib in NB4 cells. Cells were infected with lentiviruses expressing shRNAs (non-targeting control or Beclin-1). Puromycin-resistant cells were pooled after each infection. (A) Cells transfected with the control shRNA and shBeclin-1 were treated with or without bortezomib (20 nM) for 24 h, and the expression levels of cleaved caspase-3, cleaved PARP, Bcl-2, p62 and Beclin-1, and LC3-I to LC3-II conversion were determined by western blotting. (B) Ratio of Bcl-2 and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. *P<0.05 and **P<0.01. (C) Ratio of LC3-II and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. **P<0.01. (D) Cells transfected with the control shRNA and shBeclin-1 were treated with bortezomib (20 nM) for 0 and 24 h, and cell viability was assessed using a water-soluble tetrazolium salts-8 assay. Data are presented as the mean ± standard deviation of three independent repeats. ***P<0.001. shRNA/sh, short hairpin RNA; CTRL, control; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Lentiviral particles containing short hairpin RNA (shRNA/sh) Beclin-1 (cat. no. 131209BZ; 5′-CCGACTTGTTCCTTACGGAAA-3′) and shRNA negative control (shCTRL; cat. no. 131127CZ; 5′-TTCTCCGAACGTGTCACGTTTC-3′) expression vectors were obtained from Shanghai GenePharma Co., Ltd., and were then cloned into pGLV3/H1/GFP-Puro vector (Shanghai GenePharma Co., Ltd.).

Techniques: Infection, Expressing, Transfection, shRNA, Western Blot, Standard Deviation

Journal: Journal of Clinical Laboratory Analysis

Article Title: A long non‐coding RNA OLBC15 promotes triple‐negative breast cancer progression via enhancing ZNF326 degradation

doi: 10.1002/jcla.23304

Figure Lengend Snippet: OLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01

Article Snippet: The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector.

Techniques: In Vitro, Over Expression, Transfection, shRNA, Plasmid Preparation

Journal: Journal of Clinical Laboratory Analysis

Article Title: A long non‐coding RNA OLBC15 promotes triple‐negative breast cancer progression via enhancing ZNF326 degradation

doi: 10.1002/jcla.23304

Figure Lengend Snippet: OLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01

Article Snippet: The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector.

Techniques: Expressing, Transfection, Construct, Ligation, Over Expression, Migration, shRNA

Journal: Biofactors (Oxford, England)

Article Title: ASNS Regulates H 2 O 2 ‐Induced Senescence, Oxidative Stress, and Glucose Metabolism in ARPE ‐19 Cells by Modulating USP13 Expression

doi: 10.1002/biof.70057

Figure Lengend Snippet: ASNS enhanced H 2 O 2 ‐induced retinal cell proliferation and attenuated senescence. ARPE‐19 cells were transfected with shASNS or ASNS‐OE. (A) The mRNA expression of ASNS was detected by RT‐qPCR. (B) The protein expression of ASNS was detected by WB. ARPE‐19 cells were divided into H 2 O 2 + shCtrl, H 2 O 2 + shASNS or H 2 O 2 + NC, H 2 O 2 + ASNS‐OE groups. (C) CCK8 assay was utilized to assess cell viability in H 2 O 2 ‐treated ARPE‐19 cells. (D) SA‐β‐gal staining experiments were conducted in the above groups. (E) Measurement of intracellular ROS in the above groups. (F) The protein expression of P53, P21, P16 was detected by WB. (G) The level of GSH and MDA detected by ELISA. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lentiviral vectors interfering with ASNS and USP13 expression (shASNS and shUSP13) and the negative control shCtrl were synthesized by GeneChem (Shanghai, China).

Techniques: Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Biofactors (Oxford, England)

Article Title: ASNS Regulates H 2 O 2 ‐Induced Senescence, Oxidative Stress, and Glucose Metabolism in ARPE ‐19 Cells by Modulating USP13 Expression

doi: 10.1002/biof.70057

Figure Lengend Snippet: ASNS regulated glucose metabolism pathways in H 2 O 2 ‐induced retinal cells. ARPE‐19 cells were divided into H 2 O 2 + shCtrl, H 2 O 2 + shASNS or H 2 O 2 + NC, H 2 O 2 + ASNS‐OE groups. (A) Detection of glucose uptake using a kit in H 2 O 2 ‐treated ARPE‐19 cells. (B) Detection of lactate production using a kit in H 2 O 2 ‐treated ARPE‐19 cells. (C) Measuring ECAR in H 2 O 2 ‐treated ARPE‐19 cells using the XF‐96 extracellular flux analyzer. (D) Measuring OCR in H 2 O 2 ‐treated ARPE‐19 cells using the XF‐96 extracellular flux analyzer. (E) The mRNA expression of Glut1, Glut4, HK2, and PGK1was detected by RT‐qPCR. (F) The protein expression of Glut1, Glut4, HK2, and PGK1was detected by WB. (G) The protein expression of HIF‐1α was detected by WB. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lentiviral vectors interfering with ASNS and USP13 expression (shASNS and shUSP13) and the negative control shCtrl were synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Biofactors (Oxford, England)

Article Title: ASNS Regulates H 2 O 2 ‐Induced Senescence, Oxidative Stress, and Glucose Metabolism in ARPE ‐19 Cells by Modulating USP13 Expression

doi: 10.1002/biof.70057

Figure Lengend Snippet: Validation of ASNS's impact on the AMD disease process in vivo. Sprague–Dawley rats injected with sodium iodate (30 mg/kg body weight) and divided into MOCK group (No lentivirus injections, n = 10), shCtrl group (Injection of shCtrl lentivirus, n = 10), and shASNS (Injection of shASNS lentivirus, n = 10) group. (A) H&E staining analysis of rat retinal tissues in each group. (B) SA‐β‐gal staining of rat retinal tissues were conducted in the above groups. (C) Measuring ECAR and OCR in retinal tissues using the XF‐96 extracellular flux analyzer. (D) The mRNA expression of Glut1, Glut4, HK2, and PGK1 in retinal tissues was detected by RT‐qPCR. (E) The protein expression of Glut1, Glut4, HK2, and PGK1 in retinal tissues was detected by WB. (F) The protein expression of ASNS and USP13 in retinal tissues was detected by WB. (G) IF was used to analyze the expression of ASNS and USP1 in retinal tissues. (H) The protein expression of HIF‐1α in retinal tissues was detected by WB. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lentiviral vectors interfering with ASNS and USP13 expression (shASNS and shUSP13) and the negative control shCtrl were synthesized by GeneChem (Shanghai, China).

Techniques: Biomarker Discovery, In Vivo, Injection, Staining, Expressing, Quantitative RT-PCR

Journal: Biofactors (Oxford, England)

Article Title: ASNS Regulates H 2 O 2 ‐Induced Senescence, Oxidative Stress, and Glucose Metabolism in ARPE ‐19 Cells by Modulating USP13 Expression

doi: 10.1002/biof.70057

Figure Lengend Snippet: Validation of USP13's impact on the AMD disease process in vivo. Sprague–Dawley rats injected with sodium iodate (30 mg/kg body weigh) and divided into MOCK group (No lentivirus injections, n = 10), shCtrl group (Injection of shCtrl lentivirus, n = 10), and shUSP13 (Injection of shUSP13 lentivirus, n = 10) group. (A) H&E staining analysis of rat retinal tissues in each group. (B) SA‐β‐gal staining of rat retinal tissues were conducted in the above groups. (C) Measuring ECAR and OCR in retinal tissues using the XF‐96 extracellular flux analyzer. (D) The mRNA expression of Glut1, Glut4, HK2, and PGK1 in retinal tissues was detected by RT‐qPCR. (E) The protein expression of Glut1, Glut4, HK2, and PGK1 in retinal tissues was detected by WB. (F) The protein expression of USP13 in retinal tissues was detected by WB. (G) The protein expression of HIF‐1α in retinal tissues was detected by WB. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lentiviral vectors interfering with ASNS and USP13 expression (shASNS and shUSP13) and the negative control shCtrl were synthesized by GeneChem (Shanghai, China).

Techniques: Biomarker Discovery, In Vivo, Injection, Staining, Expressing, Quantitative RT-PCR