Fig. 2.

Reduction of progranulin transcript and protein expression levels and cell proliferative effects of liver fluke granulin. Panels A and B, reduction of huPGRN transcription levels from ΔhuPGRN-H69 cell; red bar (∼70%) comparing with H69 reference (black bar). The huPGRN differential transcript after normalization with human GAPDH gene; mean ± SD, n = 3 (biological replicates); P < 0.0001 (****), unpaired t-test. Diminished level of huPGRN revealed by WB analysis using anti-PGRN antibody (64 kDa) compared with anti-GAPDH antibody (36 kDa). Progranulin levels in ΔhuPGRN-H69 cells (red bars) was reduced by ∼80%. The GAPDH levels were stable within three biological replicates. These decreased levels of progranulin were significantly different from H69 (black bars); n = 3 (biological replicates); P < 0.0001 (****), unpaired t-test. Panel C, real time monitoring of H69 vs ΔhuGRN-H69 cell proliferation before and after addition of liver fluke granulin. The lower normalized cell index (nCI) of ΔhuGRN-H69 cells (discontinuous red lines) compared with H69 cells (discontinuous gray lines) were monitored over 48 h. The nCI of ΔhuGRN-H69 cells was recovered as in H69 cells nCI value after addition of liver fluke granulin at 100 nM for 24 h, and higher than H69 from 24 to 48 h. The nCI signals are shown as mean ± SD, for ≥3 independent experiments, and assessed using two-way ANOVA with Dunnet’s multiple correction. Comparing 100 nM liver fluke granulin treatments on each cell type to untreated cells (either H69 vs 100 nM liver fluke granulin treated H69 or ΔhuPGRN-H69 vs 100 nM liver fluke granulin -treated ΔhuPGRN-H69): P < 0.05 (*); P < 0.01 (**); P < 0.001 (***). Comparing 100 nM liver fluke granulin-treated H69 to 100 nM liver fluke granulin-treated ΔhuPGRN-H69: P < 0.05 (#); P < 0.001 (###). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)