Journal: bioRxiv

Article Title: Galectin-1 Modulates Hepatocellular Carcinoma Response to Thermal Ablation Through Regulating Glycolysis

doi: 10.1101/2024.12.12.628238

Figure Lengend Snippet: A-B) SNU449 survival rates (n=3 each) (A) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell growth (B) . C) Western blot of Gal-1 in SNU449, SNU423, and HepG2/C3a (positive control). β-Tubulin was used to as loading control. D-E) SNU449 cell growth (n=3 each) (D) post-thermal exposure at 37°C and 47°C with Gal-1 inhibitor OTX or DMSO at 24, 48, and 72 hours. Corresponding cell survival rates (E) . F) Western blot of Gal-1 in shGal-1-SNU449 (SNU449 with Gal-1 knockdown) and respective shControl-SNU449. G-H) shControl and shGal-1-SNU449 cell growth (n=3) (G) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell survival rates (H) . p-values were calculated using one-tailed-unpaired student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following day, cells were transduced with lentiviral shRNA particles carrying three to five expression constructs each encoding target-specific 19-25 nucleotides (plus hairpin) shRNA designed to silence the gene expression of galectin-1 (sc-35441-V, scbt) or respective shControl particles (sc-108080, scbt).

Techniques: Western Blot, Positive Control, Control, Knockdown, One-tailed Test

Journal: iScience

Article Title: IGFBP3 induced by the TGF-β/EGFRvIII transactivation contributes to the malignant phenotype of glioblastoma

doi: 10.1016/j.isci.2023.106639

Figure Lengend Snippet: Effect of IGFBP3 knockdown in vivo growth of U87-EGFRvIII cells using mice subcutaneous xenograft models. Nude mice were subcutaneously injected with pretreated U87-EGFRvIII- siIGFBP3 and U87-EGFRvIII- siControl cells (n = 5 for each group) (A) Tumor growth curves show tumor size measured every two days. (B) Tumor images and weights of the removed subcutaneous tumors after inoculation for 21 days. (C) The overall survival of mice in the U87-EGFRvIII-shControl and U87-EGFRvIII-shIGFBP3 cells implanted groups. Median survival of each group was 28 days for shControl, 38 days for shIGFBP3. n = 10, ∗∗p < 0.01 compared to the control, logrank test. (D) Representative images of IGFBP3, Ki-67 and tumor angiogenesis stained with CD31 in mouse subcutaneous tumors by IHC. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: Stable knockdown of IGFBP3 in U87-EGFRvIII cells: U87-EGFRvIII cells were transduced at 70%-80% density with purified shControl lentiviral particles (psi-LvRU6rLP-red firefly luciferase-shControl, GeneCopoeia) and purified shIGFBP3 lentiviral particles (psi-LvRU6rLP-red firefly luciferase-shIGFBP3, GeneCopoeia).

Techniques: In Vivo, Injection, Staining

Journal: iScience

Article Title: IGFBP3 induced by the TGF-β/EGFRvIII transactivation contributes to the malignant phenotype of glioblastoma

doi: 10.1016/j.isci.2023.106639

Figure Lengend Snippet:

Article Snippet: Stable knockdown of IGFBP3 in U87-EGFRvIII cells: U87-EGFRvIII cells were transduced at 70%-80% density with purified shControl lentiviral particles (psi-LvRU6rLP-red firefly luciferase-shControl, GeneCopoeia) and purified shIGFBP3 lentiviral particles (psi-LvRU6rLP-red firefly luciferase-shIGFBP3, GeneCopoeia).

Techniques: Luciferase, Microarray, Recombinant, Lysis, Transfection, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Reporter Gene Assay, BIA-KA, Cell Cycle Assay, Sequencing, shRNA, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software

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