scramble shrna shcontrol  (Addgene inc)

Journal: bioRxiv

Article Title: Inhibition of Mitochondrial Fission and iNOS in the Dorsal Vagal Complex Protects from Overeating and Weight Gain

doi: 10.1101/2020.06.26.173641

Figure Lengend Snippet: ( A ) Experimental design and feeding study protocol. ( B ) Western blot analysis of the iNOS knockdown in the DVC. iNOS levels of n=8 for shControl and n=8 shiNOS are shown in the bar graph. Representative western blot image is shown at the bottom. ( C ) Representative confocal image of iNOS labelling in animals expressing ShRNA for iNOS or the shControl in the NTS of the DVC. Bar=20μm ( D ) NO levels in the DVC of RC-fed rats compared with HFD-fed rats expressing the control virus and HFD-fed rats expressing shiNOS. Data are shown as mean ± SEM, with each single point highlighted of n=9 RC rats and n=6 HFD-fed rats expressing either shControl or shiNOS. ( E ) Acute feeding study: total food intake at 4 hours comparing animals treated with insulin or a vehicle in the DVC. Data are shown as min ± SEM, with each single point highlighted of n=10 rats for control vehicle, n=7 control insulin, n=11 for shiNOS vehicle, n=7 for shiNOS insulin. ( F ) Chronic cumulative food intake, from day 1 (see schematic in A). ( G ) Chronic data showing body weight increase from day 1. ( H ) White adipose tissue: epididymal, retroperitoneal and visceral fat collected on the day of sacrifice. Data are shown as min ± SEM, with each single point highlighted. Data from F to H are representative of n=10 for shControl and n=8 for shiNOS. *p < 0.05, **p <0.01, *** p<0.001, ****p<0.0001

Article Snippet: On day 0 a lentiviral system was used to deliver ShRNA to knockdown of iNOS (shiNOS) or a control scramble ShRNA (shControl) (Santa Cruz Biotechnology, sc-29417-V and sc-108080 respectively) or on day 1 an adenoviral system was used to deliver either a constitutively active form of Drp1 (Drp1-S637A); a catalytically inactive form of Drp1 (Drp1-K38A) or a control of GFP expressed under CMV ( Filippi et al , 2017 ) or GFAP promoters.

Techniques: Western Blot, Knockdown, Expressing, shRNA, Control, Virus

Journal: bioRxiv

Article Title: Inhibition of Mitochondrial Fission and iNOS in the Dorsal Vagal Complex Protects from Overeating and Weight Gain

doi: 10.1101/2020.06.26.173641

Figure Lengend Snippet: Rats were fed for 28 days with HFD or control RC diet. On day 28 rats received DVC surgery. ShControl (ShC) and ShiNOS (ShI) virus were injected on surgery day while the GFP and Drp1-K38A viruses where injected on day 29. Acute feeding study was preformed 8 and 14 days after surgery (see Fig S3). (A) Body weight increase over 4-weeks in HFD-fed compared to RC fed animals (B) Cumulative food intake over the 4-weeks pre-surgery. Values are multiplied by calories of diet: 3.93Kcal/g RC, 5.51Kcal/g HFD. (A-B) Data are expressed as mean ± SEM, n=10 RC, n=24 HFD (C) Blood glucose pre-surgery. Bar charts represent mean ± SEM of individual rats shown as single points. (D and E) Acute feeding study in GFP and Drp1-K38A expressing animals (D) and ShC and ShI expressing animals (E). Graph shows the total food intake at 4 hours. Bar charts represent mean ± SEM of individual rats shown as single points (n=5 for RC GFP vehicle, n=5 RC GFP insulin, n=9 for HFD GFP vehicle, n=6 for HFD GFP insulin, n=8 for HFD Drp1-K38A vehicle, n=6 for HFD Drp1-K38A insulin; n=6 RC ShC insulin, n=6 for HFD ShCl vehicle, n=5 for HFD ShC insulin, n=7 for HFD ShI vehicle, n=5 for HFD ShI insulin). (F and H) Cumulative food intake starting from the day after surgery. (G and I) Body weight increase starting from the day after surgery (G). (J and K) Total white adipose tissue (sum of epididymal, retroperitoneal and visceral fat) collected on the day of sacrifice. Data are expressed as mean ± SEM, of n=5 GFP RC, n=6 GFP HFD, n=10 Drp1-K38A HFD (F, G and L) and of n=8 ShC RC, n=8 ShC HFD, n=7 ShI HFD (H, I, K). (L and M) Average blood glucose over 14 days of the study, data is an average of sampled readings taken before feeding studies and day of sacrifice. Data are expressed as mean ± SEM, n=6 GFP RC, n=15 GFP HFD, n=12 Drp1-K38A HFD and n=9 for ShC RC, ShC HFD, ShI HFD. *p <0.05, **p <0.01, *** p<0.001.

Article Snippet: On day 0 a lentiviral system was used to deliver ShRNA to knockdown of iNOS (shiNOS) or a control scramble ShRNA (shControl) (Santa Cruz Biotechnology, sc-29417-V and sc-108080 respectively) or on day 1 an adenoviral system was used to deliver either a constitutively active form of Drp1 (Drp1-S637A); a catalytically inactive form of Drp1 (Drp1-K38A) or a control of GFP expressed under CMV ( Filippi et al , 2017 ) or GFAP promoters.

Techniques: Control, Virus, Injection, Expressing

Journal: Oncotarget

Article Title: Therapeutic inhibition of Mcl-1 blocks cell survival in estrogen receptor-positive breast cancers

doi: 10.18632/oncotarget.27070

Figure Lengend Snippet: (A) Western analysis of whole cell lysates harvested from cells treated with 1 µM ABT-263 and/or 1 µM VU661013 for 16 hrs. Antibodies used for western analysis are shown at left. (B) Caspase 3/7 activity was measured in cells treated with 1 µM VU661013 and/or 1 µM ABT-263 for 16 hrs. Data points are the average RLUs corrected for total protein in three technical replicates, midlines are the average RLUs corrected for total protein of 3-6 biological replicates (±S.E.). The average RLUs in control cells for each cell line was set at a value of 1, Two-way ANOVA . (C) Western analysis of cells expressing shControl or shMcl-1, treated with ABT-263 (1 µM) or with DMSO. Antibodies used are shown to left of each panel. Relative Mcl-1 expression was determined using densitometry analysis. (D) Caspase 3/7 activity was measured in cells expressing shControl or shMcl1 shRNA sequences, and treated with 1 µM ABT-263 for 4 hrs. Data points are the average RLUs corrected for total protein in three technical replicates, midlines are the average RLUs corrected for total protein of 6-9 biological replicates (±S.E.). The average RLUs in control cells for each cell line was set at a value of 1, Two-way ANOVA. (E) Cells were grown for 7 days with 1 µM VU661013 and/or 1 µM ABT-263. Average relative number of cells per well is shown, N = 6-10, Two-way ANOVA followed by Tukey's multiple comparison's test. (F–G) MCF7 tumor xenografts in athymic mice were treated with ABT-263 (20 mg/kg, once daily) and/or VU661013 (25 mg/kg, once weekly) for 12 days. TUNEL analysis was used to detect apoptotic cells in tumors collected on treatment day 12, one hour after final treatment (F). Each data point represents the percentage of TUNEL+ cells in 3 random fields per sample, N = 6-10 per group. Tumor volume of MCF7 xenografts were measured once every four days beginning on treatment day 0 (G). Average tumor volume (S.E.) is shown. N = 6-10.

Article Snippet: Mcl-1 expression was stably ablated used lentiviral particles containing two separate shRNA sequences against Mcl-1 (shMCL1), and a scramble control (shControl), according to the manufactures protocol (Santa Cruz Biotechnology, SC-35878-V) and as described previously.

Techniques: Western Blot, Activity Assay, Control, Expressing, shRNA, Comparison, TUNEL Assay

Journal: Molecular Medicine Reports

Article Title: HMGA1 participates in MHCC97H cell proliferation and invasion through the ILK/Akt/GSK3β signaling pathway

doi: 10.3892/mmr.2017.7820

Figure Lengend Snippet: Cell proliferation and apoptosis assay results.

Article Snippet: The lentiviral short hairpin (sh)RNA vector against HMGA1 (shHMGA1, 5′-GATCCAGCGAAGTGCCAACACCTATTCAAG-AGATAGGTGTTGGCACTTCGCTTTTTTTA-3′) and the scrambled control shRNA vector (shControl) were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Apoptosis Assay

Journal: Molecular Medicine Reports

Article Title: HMGA1 participates in MHCC97H cell proliferation and invasion through the ILK/Akt/GSK3β signaling pathway

doi: 10.3892/mmr.2017.7820

Figure Lengend Snippet: The effects on apoptosis in MHCC97H hepatocellular carcinoma cells treated with shHMGA1, ILK expression vector and/or MK2206. In each panel, B2 quadrant indicates only PI positive cells that were necrotic and the B4 quadrant indicates live cells. The B1 quadrant indicates Annexin and PI as late apoptosis cells and the B3 quadrant illustrates Annexin as early apoptotic cells. Apoptosis levels were also detected by flow cytometry in each of the seven experimental groups: (A) Blank control group; (B) shControl group; (C) shHMGA1 group; (D) shHMGA1 + EGFP group; (E) shHMGA1 + ILK group; (F) shHMGA1 + ILK + DMSO group; and (G) shHMGA1 + ILK + MK2206 group. DMSO, dimethylsulfoxide; EGFP, enhanced green fluorescent protein; HMGA1, high mobility group AT-hook 1; ILK, integrin-linked kinase; MK2206, an Akt-specific inhibitor; sh, short hairpin RNA.

Article Snippet: The lentiviral short hairpin (sh)RNA vector against HMGA1 (shHMGA1, 5′-GATCCAGCGAAGTGCCAACACCTATTCAAG-AGATAGGTGTTGGCACTTCGCTTTTTTTA-3′) and the scrambled control shRNA vector (shControl) were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, shRNA