Journal: Journal of Gastrointestinal Oncology

Article Title: Nestin overexpression reduces the sensitivity of gastric cancer cells to trastuzumab

doi: 10.21037/jgo-22-1048

Figure Lengend Snippet: Influence of Nestin on the activation of Nrf2/HO-1 and HER2/AKT/ERK1/2 of HER2-positive GC cells. The two cell lines received transfection by lentiviral-Nestin or lentiviral-empty, and then added to 10 μg/mL for 48 h. (A,B) Nestin mRNA level in MKN45 and NCI-N87 cells was detected by qRT-PCR. (C,D) WB was adopted to determine the protein level of Nestin, Nrf2, HO-1, NQO1, GST, AKT, ERK1/2, and HER2, in the MKN45 and NCI-N87 cells. (E,F) The cells were exposed to ARE luciferase reporter. 36 h post transfection, the luciferase activity was determined. Data are presented as the means ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. NTC, non-treated control; TRA, trastuzumab; TRA + NC, mice inoculated with cell infected with lentiviral-negative control were treated with TRA; TRA + Nestin, mice inoculated with cell infected with lentiviral-Nestin were treated with TRA; HO-1, hemeoxygenase-1; NQO1, NAD(P)H: quinone oxidoreductase-1; GST, glutathione S-transferase; AKT, protein kinase B; ERK1/2, extracellular regulated kinase 1/2; HER2, human epidermal growth factor receptor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, antioxidant response element; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting; SD, standard deviation.

Article Snippet: After a day of culture, the cells were treated with 200 ng of ARE luciferase reporter plasmids using OriGene transfection reagent (Company, City, Country).

Techniques: Activation Assay, Transfection, Quantitative RT-PCR, Luciferase, Activity Assay, Control, Infection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: BMC Cancer

Article Title: PKCα promotes the mesenchymal to amoeboid transition and increases cancer cell invasiveness

doi: 10.1186/s12885-015-1347-1

Figure Lengend Snippet: Expression of dominant-negative PKCα results in decreased invasiveness of cancer cells. Invasion analysis of A375m2 (A) , K2 (B) and MDA-MB-231 (C) strains overexpressing wt PKCα or expressing dominant-negative PKCα. Cells were transfected with pCMV6 vector expressing either wt PKCα or dominant-negative PKCα and let to invade into 3D collagen for 48 hours. 3D collagen cross-sections were analyzed using NIS Elements software (Nikon) and average invasion depth was calculated and normalized to that of untreated cells. The invasion depth in each individual field of view was assessed as sum of invasion depths of individual cells divided by the total number of cells in the respective field of view (including cells on the top of collagen). Statistical significance (marked by asterisks) was evaluated using ANOVA followed by Tukey’s honest significant difference test.

Article Snippet: Flag-tagged wt PKCα, constitutive active form (with myristoylation sequence from Src) and dominant-negative PKCα (K368R) in pCMV6 vector were all obtained from Addgene Inc. Silencer® Select Validated siRNA against PKCα was obtained from Applied Biosystems and a final concentration of 10 nM was used for transfections.

Techniques: Expressing, Dominant Negative Mutation, Transfection, Plasmid Preparation, Software

Journal: International Journal of Oral Science

Article Title: Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis

doi: 10.1038/ijos.2013.96

Figure Lengend Snippet: Genes identified as up-regulated or down-regulated by RNA-Seq and microarray analysis

Article Snippet: Human LOXL4 cDNA was purchased from Sino Biological and cloned in pCMV/hygro (CV004; Sino Biological, Beijng, China) for transient overexpression with a DNA Ligation Kit (2011A; TAKARA, Kyoto, Japan).

Techniques: Microarray

Journal: International Journal of Oral Science

Article Title: Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis

doi: 10.1038/ijos.2013.96

Figure Lengend Snippet: Candidate genes possibly involved in keratocystic odontogenic tumor progression

Article Snippet: Human LOXL4 cDNA was purchased from Sino Biological and cloned in pCMV/hygro (CV004; Sino Biological, Beijng, China) for transient overexpression with a DNA Ligation Kit (2011A; TAKARA, Kyoto, Japan).

Techniques: Microarray

Journal: International Journal of Oral Science

Article Title: Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis

doi: 10.1038/ijos.2013.96

Figure Lengend Snippet: Confirmation of RNA-Seq results. ( a ) QPCR analysis of TCTA, LARP6, RGS12 and LOXL4 mRNA expression in primary stromal fibroblasts of KCOT, DC and healthy GG. The data were consistent with the RNA-Seq and microarray results. ( b ) QPCR analysis of LOXL4 mRNA expression in primary stromal fibroblasts of KCOT, DC and GG. Each bar corresponds to the mean±s.d. of three independent experiments performed in triplicate with three technical repeats. * P <0.05, ** P <0.001. DC, dentigerous cysts; GG, gingival primary stromal fibroblast cells; KCOT, keratocystic odontogenic tumors; LARP6, La ribonucleoprotein domain family, member 6; LOXL4, lysyl oxidase-like 4; QPCR, quantitative PCR; RGS12, regulator of G-protein signaling 12; RNA-Seq, RNA sequencing; TCTA, T-cell leukemia translocation-associated gene.

Article Snippet: Human LOXL4 cDNA was purchased from Sino Biological and cloned in pCMV/hygro (CV004; Sino Biological, Beijng, China) for transient overexpression with a DNA Ligation Kit (2011A; TAKARA, Kyoto, Japan).

Techniques: RNA Sequencing Assay, Expressing, Microarray, Real-time Polymerase Chain Reaction, Translocation Assay

Journal: International Journal of Oral Science

Article Title: Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis

doi: 10.1038/ijos.2013.96

Figure Lengend Snippet: LOXL4 is overexpressed in the stroma of KCOT. ( a ) LOXL4 concentration in the supernatants of primary fibroblasts derived from KCOT, GG and DC ( n =3 per tissue) by ELISA. ( b ) LOXL4 concentration in the cell lysates of primary fibroblasts derived from KCOT, GG and DC ( n =3 per tissue) by ELISA. Data are the means of triplicates from a representative experiment±s.d.; * P <0.05, ** P <0.001. DC, dentigerous cysts; ELISA, enzyme-linked immunosorbent assay; GG, gingival primary stromal fibroblast cells; KCOT, keratocystic odontogenic tumors; LOXL4, lysyl oxidase-like 4.

Article Snippet: Human LOXL4 cDNA was purchased from Sino Biological and cloned in pCMV/hygro (CV004; Sino Biological, Beijng, China) for transient overexpression with a DNA Ligation Kit (2011A; TAKARA, Kyoto, Japan).

Techniques: Concentration Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oral Science

Article Title: Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis

doi: 10.1038/ijos.2013.96

Figure Lengend Snippet: Effects of LOXL4 on angiogenesis. ( a ) LOXL4 expression and microvessel density in DC, GG and KCOT stroma. Immunohistochemical analysis of LOXL4 ( a1 , a2 , a4 , a5 ). Few LOXL4-positive fibroblasts were observed in GG ( a1 , a2 ) or DC ( a4 , a5 ). ( a7 , a8 ) Most of the KCOT stromal fibroblasts were strongly LOXL4-positive. ( a3 , a6 ) Few CD105-positive microvessels were observed adjacent to the epithelium of normal GG ( a3 ) and dentigerous cysts ( a6 ). A high density of strongly CD105-positive microvessels was found in the stroma immediately beneath the epithelium lining KCOT ( a9 ). The intensity of LOXL4 protein expression was consistent with the density of CD105-positive microvessels in GG ( a3 ), DC ( a6 ) and KCOT ( a9 ). Scale bar=100 µm. ( b ) Effects of LOXL4 on the proliferation and migration of HUVECs. ( b1 , b2 ) There was a significant increase in the expression of LOXL4 both in intracellular and extracellular of HUVECs transfected with LOXL4 than transfected with empty plasmid. ( b3 ) Transient LOXL4 transfection increased cell growth, cell proliferation was measured with Cell Counting Kit-8 at 6, 12, 24, 72 and 96 h. ( b4 ) HUVECs migration ability was assessed by scratching assay with or without LOXL4 transfection. Representative images are shown (magnification: ×100). ( b5 ) HUVECs migration ability was also tested by Boyden Chamber assay with or without LOXL4 transfection. Representative images are shown (magnification: ×200). Each treatment was triplicate and six fields were randomly chosen from each well for counting mean number of migrated cells. Data are presented as mean±s.e.m., * P <0.05, ** P <0.01, Student's t -test. DC, dentigerous cysts; ELISA, enzyme-linked immunosorbent assay; GG, gingival primary stromal fibroblast cells; HUVEC, human umbilical vein endothelial cell; KCOT, keratocystic odontogenic tumors; LOXL4, lysyl oxidase-like 4; OD, optical density; Ctl, control.

Article Snippet: Human LOXL4 cDNA was purchased from Sino Biological and cloned in pCMV/hygro (CV004; Sino Biological, Beijng, China) for transient overexpression with a DNA Ligation Kit (2011A; TAKARA, Kyoto, Japan).

Techniques: Expressing, Immunohistochemical staining, Migration, Transfection, Plasmid Preparation, Cell Counting, Boyden Chamber Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oral Science

Article Title: Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis

doi: 10.1038/ijos.2013.96

Figure Lengend Snippet: Microvessel density and LOXL4 in keratocystic odontogenic tumors, dentigerous cysts and gingival tissues

Article Snippet: Human LOXL4 cDNA was purchased from Sino Biological and cloned in pCMV/hygro (CV004; Sino Biological, Beijng, China) for transient overexpression with a DNA Ligation Kit (2011A; TAKARA, Kyoto, Japan).

Techniques: Standard Deviation