human liver whole tissue lysate  (Novus Biologicals)

Journal: Cancers

Article Title: Mitochondrial Respiratory Defect Enhances Hepatoma Cell Invasiveness via STAT3/NFE2L1/STX12 Axis

doi: 10.3390/cancers12092632

Figure Lengend Snippet: Identification of downstream target genes commonly upregulated by mitochondrial defect and NFE2L1. Transcriptome data of four cell lines (Ch-L and SNU387 harboring active mitochondria, and SNU354 and SNU423 harboring defective mitochondria) were obtained from our previous report . Two independent sets of SNU387 cells transfected with pcDNA-NFE2L1 plasmid (NFE2L1_OE) for 48 h and of SNU423 cells with siNFE2L1 (NFE2L1_KD) for 72 h were applied to cDNA microarray for transcriptome profiling. ( A ) Venn diagram of commonly upregulated genes with mitochondrial defect (Mito defect, SNU354 and SNU423 vs. Ch-L and SNU387) and NFE2L1-dependent regulation. ( B ) Heatmaps show the expression of 10 commonly upregulated genes in four hepatoma cell lines (left), NFE2L1-depleted SNU423 cells (middle), and NFE2L1-overexpressed SNU387 cells (right). Each column and row represent independent samples and the indicated genes, respectively. Red and blue color indicates the high and low expression, respectively. ( C , E ) Validation of four target mRNA levels by qRT-PCR. ( D , F ) Validation of four target protein levels by Western blot analysis. #1 and #2 indicate two different siRNAs for NFE2L1. ** p < 0.01 vs. pcDNA or siNC by the Student t -test. ( G ) Associations of NFE2L1 with the 10 commonly upregulated genes were evaluated using the TCGA-LIHC cohort ( n = 371). Pearson’s product moment correlation test was performed. The correlation estimate and the p-value with statistical significance are marked. ( H ) Boxplot for STX12 expression level of primary HCC tissues from the TCGA-LIHC cohort. Bottom, middle, and top lines of each box indicate the first quartile, median, and third quartile values, respectively. Whiskers represent the minimum and maximum values. For comparison, primary tumors were divided into two groups, based on the median expression level of NFE2L1 and NDUFA9: a group with both above the median NFE2L1 level and below the median NDUFA9 level, a group with both below the median NFE2L1 level and above the median NDUFA9 level. p -value from Wilcoxon t-test is indicated.

Article Snippet: Briefly, paraffin-embedded tissue slides of human HCC tissue microarray (TMA) (NBP2-30221, Novus Biologicals) were deparaffinized and rehydrated.

Techniques: Transfection, Plasmid Preparation, Microarray, Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Comparison

Journal: Cancers

Article Title: Mitochondrial Respiratory Defect Enhances Hepatoma Cell Invasiveness via STAT3/NFE2L1/STX12 Axis

doi: 10.3390/cancers12092632

Figure Lengend Snippet: STX12 is a key regulator of hepatoma cell invasiveness. ( A , B ) SNU423 cell was transfected with STX12 siRNA for 72 h. ( A ) Cell invasion assay using a Matrigel-coated Transwell system. Representative invaded cell images are shown in the right panel. ** p < 0.01 vs. siNC by the Student t-test. ( B ) Western blot (left) and cell growth rate (right). #1 and #2 indicate two different siRNAs for STX12. ( C , D ) Ch-L was transfected with STX12 plasmid for 48 h. ( C ) Cell invasion assay. Representative invaded cell images are shown in the right panel. ** p < 0.01 vs. GFP by the Student t-test. ( D ) Western blot (left) and cell growth rate (right). ( E – G ) SNU423 clone, which was stably suppressing NFE2L1 with sgNFE2L1 RNA, was further infected with lentivirus harboring STX12 or GFP. ( E ) Cell invasion assay. ** p < 0.01 vs. sgNC; # < 0.05 vs. GFP by the Student t -test. ( F ) Cell growth. ( G ) Western blot. ( H ) Immunohistochemistry of HCC tissue microarray, as described in ‘Materials and Methods’. Lower panel shows representative images for NFE2L1 and STX12 immunostaining. ( I , J ) Boxplots of the normalized enrichment score (NES) for EMT signature of the primary HCC tissues from the TCGA_LIHC dataset. Bottom, middle, and top lines of each box indicate the first quartile, median, and third quartile values, respectively. Whiskers represent the minimum and maximum values. p -values from Welch two-sample t -test are indicated. ( I ) Tumor samples were divided into high and low groups, according to the expression level of NFE2L1 (left) or STX12 (middle), respectively, as described in ‘Materials and Methods’. To evaluate the effect of co-expression on the EMT signature, tumor samples were divided into two groups, based on the co-expression level of both NFE2L1 and STX12 (right). ( J ) The NFE2L1 and STX12 co-expressing group (right panel of I) was further subdivided into an H ( n = 11) and L group ( n = 23) according to the NDUFA9 expression level. ( K ) Overall survival (OS) time of the H and L group was compared based on the Kaplan–Meier survival analysis. Statistical significance for KM survival was estimated by the Cox–Mantel log-rank test.

Article Snippet: Briefly, paraffin-embedded tissue slides of human HCC tissue microarray (TMA) (NBP2-30221, Novus Biologicals) were deparaffinized and rehydrated.

Techniques: Transfection, Invasion Assay, Western Blot, Plasmid Preparation, Stable Transfection, Infection, Immunohistochemistry, Microarray, Immunostaining, Expressing