Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Immortal activated human hepatic stellate cells generated by ectopic telomerase expression.

doi: 10.1038/labinvest.3780426

Figure Lengend Snippet: Figure 2. Introduction of human telomerase reverse transcriptase (hTERT) in human HSCs leads to telomerase activity and permits maintenance of telomere length. Human HSCs were infected with a retrovirus encoding hTERT or a retroviral vector expressing only the neomycin resistance gene after 9 days in culture. Cells were selected in G418. A, The expression of hTERT was analyzed by semiquantitative RT-PCR using 1 g of total RNA. cDNA was analyzed from vector control–infected HSCs (vector) and from hTERT-infected HSCs (poly- clonal and single-cell clone 14). Telomerase-positive Hela cells and telomerase-negative primary human dermal fibroblasts (HDF) served as positive and negative control, respectively. Ubiquitin (below) was amplified to confirm equal amount of mRNA was present in each sample. B, Telomerase activity was measured in HSCs infected with a control vector (vector) or a vector expressing hTERT (polyclonal HSCs and HSCs from clone 14) by the telomeric repeat amplification protocol (TRAP) assay. For each sample 105

Article Snippet: RNA Isolation and RNase Protection Assay RNA was isolated from subconfluent HSCs using an RNA extraction kit (Qiagen) according to the manufacturer’s protocol.

Techniques: Reverse Transcription, Activity Assay, Infection, Retroviral, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Negative Control, Ubiquitin Proteomics, Amplification, TRAP Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Immortal activated human hepatic stellate cells generated by ectopic telomerase expression.

doi: 10.1038/labinvest.3780426

Figure Lengend Snippet: Figure 5. Gene expression patterns in activated human HSCs and telomerase-positive HSCs. A, Microarray analysis of gene expression in activated human HSCs and telomerase-positive HSCs was performed. The values of all genes in activated human HSCs (x axis) and telomerase-positive HSCs (y axis) were compared using scatter plot analysis. The upper and lower diagonals represent 2-fold higher gene expression in telomerase-positive and activated human HSCs, respectively. B, Uninfected and vector control–infected HSCs, telomerase- positive polyclonal HSCs, and HSCs from clone 14 were cultured on plastic. Total RNA (5 g) was subjected to RNase protection assay with riboprobes specific for human collagen 1(I) and human GAPDH mRNA. Migration of the protected bands is indicated. C, Western blot analysis was performed using whole cell extracts obtained from uninfected HSCs, vector control–infected HSCs, telomerase-positive polyclonal HSCs, and HSCs from clone 14. Proteins (10 g) were separated in a 12% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for SMA using monoclonal anti-smooth muscle -actin antibody.

Article Snippet: RNA Isolation and RNase Protection Assay RNA was isolated from subconfluent HSCs using an RNA extraction kit (Qiagen) according to the manufacturer’s protocol.

Techniques: Gene Expression, Microarray, Plasmid Preparation, Control, Infection, Cell Culture, Rnase Protection Assay, Migration, Western Blot, SDS Page, Membrane

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Immortal activated human hepatic stellate cells generated by ectopic telomerase expression.

doi: 10.1038/labinvest.3780426

Figure Lengend Snippet: Figure 6. Culturing telomerase-positive HSCs on a basement membrane-like matrix reverts them to a quiescent phenotype and down-regulates their collagen 1(I) mRNA. Telomerase-positive HSCs from clone 14 were plated in matrigel, a basement membrane-like matrix, at a density of 4 to 6105 per 60-mm dish. A, Phase microscopy demonstrates cluster formation without visible spreading of telomerase-positive HSCs in matrigel (original magnification, 100). B, HSCs were maintained in matrigel in hormonally defined medium (HDM) containing 2% FCS for 15 days. As controls telomerase-positive HSCs from clone 14 were cultured on plastic for 15 days after plating, either in HDM containing 2% FCS or in regular growth medium containing 10% FCS. RNase protection assay with collagen 1(I) and GAPDH gene-specific riboprobes and RNA (3.5 g) extracted from HSCs was performed. Migration of the protected bands is indicated. Expression of collagen 1(I) mRNA was normalized to GAPDH mRNA, and the ratios are indicated. The results are expressed relative to HSCs cultured on plastic in HDM containing 2% FCS.

Article Snippet: RNA Isolation and RNase Protection Assay RNA was isolated from subconfluent HSCs using an RNA extraction kit (Qiagen) according to the manufacturer’s protocol.

Techniques: Membrane, Microscopy, Cell Culture, Rnase Protection Assay, Migration, Expressing