Journal: Cancer Science

Article Title: GPAM mediated lysophosphatidic acid synthesis regulates mitochondrial dynamics in acute myeloid leukemia

doi: 10.1111/cas.15835

Figure Lengend Snippet: (A) Representative Western blot images showing the knockdown efficiency of shRNA targeting GPAM in THP‐1 cells. β‐actin was used as a loading control. (B) Cell growth curves of acute myeloid leukemia (AML) cell lines transfected with shRNA targeting GPAM or scrambled control. Data are represented as mean ± SD. (C) Frequency of green fluorescent protein positive (GFP + ) cells in the bone marrow (BM) of NSG mice 28 days after xenotransplantation of THP‐1 cells. (D) The Kaplan–Meier survival curves of NSG mice after xenotransplantation of THP‐1 cells. p ‐values were calculated using Student's t ‐test (B), Wilcoxon rank‐sum test (C), or log‐rank Mantel‐Cox test (D). * p < 0.05, *** p < 0.001.

Article Snippet: For GPAM knockdown, lentivirus vectors containing shRNA targeting human GPAM and scrambled control sequences were purchased from VectorBuilder Inc. (VB180824‐1060qzm, VB180824‐1062qxz, VB180824‐1063qtf, and VB151023‐10034).

Techniques: Western Blot, Knockdown, shRNA, Control, Transfection

Journal: Cancer Science

Article Title: GPAM mediated lysophosphatidic acid synthesis regulates mitochondrial dynamics in acute myeloid leukemia

doi: 10.1111/cas.15835

Figure Lengend Snippet: (A) Representative fluorescent images showing the mitochondria in THP‐1 cells transfected with shRNA targeting GPAM or scrambled control taken by Cellomics ArrayScan VTI HCS Reader. Nuclei were stained with Hoechst 33342 (blue), and mitochondria were stained with Mito‐Tracker Deep Red (red). (B) Quantification of Mito‐Tracker images by Cellomics ArrayScan VTI HCS Reader. The mean fluorescence intensity values were normalized to those of the untreated control. (C) Representative transmission electron microscopy (TEM) images showing the mitochondria in THP‐1 cells transfected with shRNA targeting GPAM or control. Red dotted lines outline the individual mitochondria. (D) TEM analysis of the lengths of 300–450 mitochondria derived from a random selection of THP‐1 cells transfected with shRNA targeting GPAM or control. (E) Cellular growth of THP‐1 cells transfected with shRNA targeting GPAM or control in the presence or absence of Mdivi‐1. (F) TEM analysis of the lengths of 300–450 mitochondria derived from a random selection of THP‐1 cells transfected with shRNA targeting GPAM or control treated with DMSO or Mdivi‐1 for 4 h. (G) Seahorse extracellular flux analysis of basal and stressed oxygen consumption rates (OCRs) in THP‐1 cells transfected with shRNA targeting GPAM or control. (H) Representative fluorescent images showing the reactive oxygen species (ROS) in THP‐1 cells transfected with shRNA targeting GPAM or control taken by Cellomics ArrayScan VTI HCS Reader. Nuclei were stained with Hoechst 33342 (blue), and ROS were stained with CellROX Deep Red (red). (I) Quantification of CellROX Deep Red by Cellomics ArrayScan VTI HCS Reader. Data are represented as mean ± SD. p ‐values were calculated using an unpaired Student's t ‐test (B, E, I) or Wilcoxon rank‐sum test (D, F). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For GPAM knockdown, lentivirus vectors containing shRNA targeting human GPAM and scrambled control sequences were purchased from VectorBuilder Inc. (VB180824‐1060qzm, VB180824‐1062qxz, VB180824‐1063qtf, and VB151023‐10034).

Techniques: Transfection, shRNA, Control, Staining, Fluorescence, Transmission Assay, Electron Microscopy, Derivative Assay, Selection

Journal: Cell Death & Disease

Article Title: Inhibition of HtrA2 alleviated dextran sulfate sodium (DSS)-induced colitis by preventing necroptosis of intestinal epithelial cells

doi: 10.1038/s41419-019-1580-7

Figure Lengend Snippet: a , b HT-29 cells were pretreated with Nec-1s (10 μM) or different doses of UCF-101 for 1 h, followed by stimulation with TNF-α (20 ng/mL)/Smac mimetic (2 μM)/Z-VAD (25 μM) for 8 h. PI positive cells were analyzed by PI/Hoechst staining ( a ), and cell viability was determined by CCK8 analysis ( b ). c – e HT-29 cells were stably infected with lentiviruses carrying scramble shRNA (shNC) or two different shRNAs targeting two individual sites of HtrA2 (shHtrA2-1 or shHtrA2-2). HtrA2 protein levels were detected by immunoblotting ( c ). Indicated cells were stimulated with T/S/Z for 8 h. PI positive cells were analyzed by PI/Hoechst staining ( d ), and cell viability was determined by CCK8 ( e ). In a , b , d , e , data are presented as means ± SEM. ** P < 0.01; *** P < 0.001 (two-tailed unpaired Student’s t test)

Article Snippet: HtrA2 shRNA (shHtrA2) and nontarget control shRNA (shNC) constructs were purchased from Cyagen Biosciences (China).

Techniques: Staining, Stable Transfection, Infection, shRNA, Western Blot, Two Tailed Test