Journal: Genes to Cells

Article Title: Functional Role of COP1 Gene in Hepatocellular Carcinoma Lipid Metabolism and Stemness

doi: 10.1111/gtc.70108

Figure Lengend Snippet: COP1 knockdown reduces motility, and stemness in HCC cells. (a‐b) Migration (a) or invasion (b) assay showing reduced motility in Huh7 and HepG2 cells treated with COP1‐siRNA, as quantified by the relative migration or invaded area. Statistical significance: *, p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. (c) Sphere formation assay images and quantitative data from Huh7 and HepG2 cells, demonstrating a decrease in both the number and size of spheres in COP1‐siRNA‐treated cells. (d) Sphere formation assay images and quantitative data from PLC/PRF/5 CD133+ cells. Compared to the control grpup, both the sphere number and size decreased upon COP1‐siRNA treatment. Scale bar, 100 μm Statistical significance: *, p < 0.05; *** p < 0.001 vs. control.

Article Snippet: The cells were treated with FcR Blocking Reagent and CD133 MicroBeads (130‐100‐857, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions and incubated for 15 min at 4°C in the dark.

Techniques: Knockdown, Migration, Control, Tube Formation Assay

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: Representative immunofluorescent staining of cultured mesothelioma cells using AX10 antibody (a), immunohistochemical staining (b), and secondary antibody‐drug conjugate assay in vitro (c). (a) AX10 immunoreactivity in MPM‐1, −2, and −3 cells, representing sarcomatoid, epithelioid, and biphasic type mesothelioma, respectively. All MPM‐1, −2, and −3 cells exhibited AX10 antibody immunoreactivity at the cell surface. The staining was analyzed using a Guava easyCyte cell analyzer and accompanying software to obtain a one‐parameter log histogram. (b) AX10 immunoreactivity in various mesothelioma tissue specimens. Weak or no AX10 immunoreactivity was detected in five out of 10 epithelioid mesothelioma tissues (a). One out of five biphasic mesotheliomas exhibited AX10 immunoreactivity in spindle sarcomatoid components (arrow) but weak immunoreactivity in epithelioid components (arrowhead) (b). Five out of six sarcomatoid mesothelioma tissues exhibited strong AX10 immunoreactivity (c). Little AX10 immunoreactivity was detected in normal human tissues. No significant AX10 immunoreactivity was detected in the lung (d) (pleural mesothelial cells; insert) tissue specimens. Weak AX10 immunoreactivity was detected in myofibrous cells in the uterus (e). We did not detect any significant AX10 immunoreactivity in the brain, liver, or kidney, whereas strong AX10 immunoreactivity was observed in a nonmelanocytic (hypomelanocytic) melanoma tissue sample that was supplementally included in the microarray (f) (staining without AX10 antibody; insert). (c) MPM‐1 sarcomatoid mesothelioma cells were incubated with AX10 at 10, 100, and 1000 ng/mL followed by incubation with anti‐murine IgG (Fc) antibody conjugated to duocarmycin. Representative staining with Annexin V‐PI is presented. Note the dose‐dependent Annexin V‐positive and PI‐negative apoptotic MPM‐1 cells in the presence of AX10 antibody

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Staining, Cell Culture, Immunohistochemical staining, In Vitro, Software, Microarray, Incubation

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: AX10 does not affect cell proliferation, but significantly decreases Matrigel invasion activity of MPM‐1 sarcomatoid mesothelioma cells in vitro. (a) Representative cell proliferation assay. At 24 h, the cell number was 1.80 ± 0.10 (mock) and 1.77 ± 0.06 (AX10). Respective numbers at 48 h were 2.40 ± 0.10 (mock) and 2.37 ± 0.12 (AX10), while at 72 h they were 3.90 ± 0.20 (mock) and 4.20 ± 0.61 (AX10). The data represent means ± SD from triplicate assays (Student's t ‐test, p > 0.5). (b) AX10 significantly reduced Matrigel invasion activity of MPM‐1 cells (Student's t ‐test, p < 0.01). The number of invading cells was 59.7 ± 7.02 (mock) and 10.3 ± 1.52 (AX10) at 24 h, and 210.7 ± 11.4 (mock) and 15.0 ± 3.00 (AX10) at 48 h. Data from triplicate assays are expressed as means ± SD ( n = 3). (c) Cells that migrated to the lower surface of the membrane are shown (48 h). Original magnification, ×100

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Activity Assay, In Vitro, Proliferation Assay, Membrane

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: Inhibitory effect of AX10 on MPM‐1 xenotransplanted sarcomatoid mesothelioma cell proliferation. (a) Inoculation of AX10 antibody delayed the growth of xenotransplanted MPM‐1 sarcomatoid mesothelioma tumors. On day 0, SCID‐NOD mice were subcutaneously implanted with MPM‐1 cells. The following day, day 3, the mice were administered AX10 antibody or vehicle only by intraperitoneal injection and weekly thereafter as indicated by arrows. Values are represented as means ± standard error for n = 5 mice. Statistical significance was measured by a two‐sided unpaired Student's t ‐test (* p < 0.01). (b) On day 42, the xenotransplanted tumors were excised to determine their weight. Total tumor weights are represented as means ± standard error for n = 5 mice. Statistical significance was measured by a two‐sided unpaired Student's t ‐test ( p < 0.01). (c) Gross and histological appearance of a representative xenotransplanted tumor. Arrowhead indicates the tumor without AX10 antibody, while the arrow indicates the small tumor remaining following weekly AX10 injection. Note the elimination of tumor cells, which were histologically replaced by regenerative muscle in mice inoculated with AX10 antibody. Scale bar indicates 100 μm

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Injection

Journal: Human Molecular Genetics

Article Title: PD-linked CHCHD2 mutations impair CHCHD10 and MICOS complex leading to mitochondria dysfunction

doi: 10.1093/hmg/ddy413

Figure Lengend Snippet: PD-linked CHCHD2 mutants showed reduced binding to CHCHD10. ( A ) Co-immunoprecipitation of endogenous CHCHD2 by antibody against CHCHD2 in SK-N-SH cells. WCL: whole cell lysate, 5% total protein used in co-IP experiment. ( B ) Co-immunoprecipitation of endogenous CHCHD10 by antibody against CHCHD10 in SK-N-SH cells. WCL: whole cell lysate, 5% total protein used in co-IP experiment. ( C ) Co-immunoprecipitation of endogenous CHCHD2 by antibody against CHCHD2 in human brain tissue lysates. ( D ) Co-immunoprecipitation of CHCHD2 by a CHCHD2 antibody against middle region of CHCHD2 (labeled as CHCHD2-TF) in SK-N-SH cells transfected with non-tagged CHCHD2 WT/T61I/R145Q/Q126X. WCL: whole cell lysate, 5% total protein used in co-IP experiment. Lower arrow pointed to CHCHD2 Q126X and higher arrow pointed to full length CHCHD2. ( E ) Co-immunoprecipitation of CHCHD2 by a CHCHD2 antibody against middle region of CHCHD2 (labeled as CHCHD2-TF) in hESCs of H9 and isogenic lines harboring homozygous R145Q (−/−).WCL: whole cell lysate, 5% total protein used in co-IP experiment. Arrow pointed to CHCHD10. Representative results from R10 and R17 (R145Q−/−) were shown. ( F ) Quantification of protein abundance of CHCHD10 normalized with CHCHD2 on the co-immunoprecipitation complex from isogenic hESC lysates.

Article Snippet: Human brain tissue lysates were from Novus Centennial, CO. Elamipretide was from MedChemExpress Monmouth Junction, NJ.

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Labeling, Transfection, Quantitative Proteomics