Fig. 4.

Knocking down mPINC_1.6 alters cell-cycle progression. (A) siRNA knockdown strategy. siRNAs were designed to target sequences in the unique 3′-terminal exon of mPINC_1.0 and mPINC_1.6 (illustrated diagrammatically) to allow transcript-specific knockdown of each mPINC isoform. (B) Immunofluorescent detection of BrdU incorporation in HC11s transfected with siRNAs against mPINC_1.0 (1.0 A), mPINC_1.6 (1.6 A), or an unrelated negative control oligo and mock-transfected cells. Transfected cells were growth-arrested for 72 h, and then normal growth medium was returned for 6 hours to reinitiate cell cycle. HC11s were labeled with BrdU for 45 min before fixation, and BrdU incorporation was measured by immunofluorescence by using a FITC-conjugated antibody against BrdU. (C) BrdU incorporation was measured by immunofluorescence (as described in B) and calculated as the percentage of BrdU-positive cells relative to total DAPI-stained nuclei averaged over six independent microscope fields. Knocking down mPINC_1.6 (1.6A) resulted in a 16-fold increase in the number of BrdU-positive cells compared with cells transfected with negative control siRNA. The percentage of BrdU-positive cells transfected with siRNA against mPINC_1.0 (1.0A) was similar to that of the control cells. ∗, P < 0.002.