Fig. 4.
Quantitative assessment of Cd73 gene upregulation in myofibroblastic HSC and PF. A: upregulation of Cd73 mRNA determined by real-time RT-PCR. Total RNA was extracted from day 1 (quiescent) and day 7 (myofibroblastic) HSC. Cd73 gene expression was determined by real-time RT-PCR and calculated relative to housekeeping Gapdh gene expression, by the 2−ΔΔCT method. [Cd73]/[Gapdh] mRNA ratios from day 1 and day 7 HSC preparation samples (n ≥ 3 for each group, 3 separate experiments) were calculated and compared. As can be seen, day 7 HSC express roughly 3× the mRNA of day 1 HSC. ***P < 0.001, HSC day 1 vs. HSC day 7. B: semiquantitative RT-PCR to determine relative expression of Cd73 mRNA in human cells. Total RNA from human immortalized LX-1, LX-2 myofibroblastic HSC, primary isolated human myofibroblastic HSC (FH11 and FH13), HepG2 hepatocellular carcinoma cells, and human brain tissue extract (positive control) was reverse transcribed, and the resulting cDNAs (lanes 2-7) were used as templates for semiquantitative PCR to detect expression of CD73 (171-bp product) and GAPDH (loading control, 228-bp product) genes. All samples are positive for expression of CD73 mRNA, except for the water control (lane 1), in which no template was added. C: upregulation of CD73 protein in myofibroblastic HSC determined by immunoblot. Total protein fractions were isolated from rat day 1 and day 7 HSC preparations and used for immunoblot to study the expression of ecto-5′-nucleotidase/CD73 and β-tubulin (loading control) proteins. A representative experiment shows a faint band (∼70 kDa) indicating low basal expression of ecto-5′-nucleotidase/CD73 that is seen in day 1 HSC, that is of greater intensity in day 7 HSC. Bottom: ratios of signal intensity [ecto-5′-nucleotidase/CD73]/[β-tubulin] protein immunoblots from day 1 and day 7 HSC (n ≥ 3 for each group) samples were calculated and compared, **P < 0.01, HSC day 1 vs. HSC day 7. D: upregulation of CD73 protein in myofibroblastic PF determined by immunoblot. Total protein fractions isolated from day 1 and day 7 PF preparations were analyzed as in C to study the expression of ecto-5′-nucleotidase/CD73 and β-actin (loading control) proteins. A representative experiment shows that expression of ecto-5′-nucleotidase/CD73 is absent in day 1 PF whereas a strong band indicative of protein expression is seen in day 7 PF. Bottom: ratios of signal intensity [ecto-5′-nucleotidase/CD73]/[β-actin] protein immunoblots from day 1 and day 7 PF (n ≥ 3 for each group) samples were calculated and compared, **P < 0.01, PF day 1 vs. PF day 7.