quantification of the number of rearranged long filament actin following the siRNA treatment described as in = 3). PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100C184 or 100C200 strongly induces an F-actin structure called the actin comet tail, Obatoclax mesylate (GX15-070) not observed with WT PRR11. Our findings show that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells. is usually previously identified as a cancer-related gene that is implicated in lung malignancy development (32, 37); however, the mechanism behind PRR11-mediated cellular function remains unclear. To explore how PRR11 regulates cellular functions and lung tumorigenesis, we first performed PRR11 protein co-immunoprecipitation to identify its interacting partners. Multiple components of the seven-subunit actin-related protein 2/3 (Arp2/3) complex were recognized in the PRR11 immunoprecipitates by mass spectrometry (MS), including Arp2, Arp3, ARPC1A, ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, and ARPC5L (Fig. 1and and mass spectrometry analysis of PRR11-associated proteins. Lysates from H1299 cells transfected with Flag-WT PRR11 or pcDNA3.0 (vacant) were immunoprecipitated with anti-Flag and then analyzed by MS. Subunits of Arp2/3 complex (Arp2, Arp3, ARPC1A, ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, and ARPC5L) were recognized in the PRR11 immunoprecipitates. and conversation between PRR11 and Arp2 was confirmed by coimmunoprecipitation. cells were transfected with plasmids encoding Flag-WT PRR11 or pcDNA3.0. Cell lysates were immunoprecipitated with Flag antibody and blotted with different antibodies as indicated. to verify the conversation of endogenous proteins, the lysate of cell was immunoprecipitated with Arp2 antibody. Co-immunoprecipitation (endogenous PRR11 co-localizes with Arp2. Cells were fixed and stained for PRR11 and Arp2 antibody. indicates that Arp2 localizes at the lamellipodia, and zoomed images of the are shown at the (Western blot analysis for the indicated proteins in the pvN173- and WT PRR11-transfected cells. Cells were lysed 24 h after transfection Obatoclax mesylate (GX15-070) and analyzed for the indicated proteins. PRR11 overexpression stimulates F-actin assembly and reorganization. H1299 and A549 cells were fixed at 24 h after transfection with pvN173 FlagCcontrol or Flag-WT PRR11 and stained with Flag antibody and F-actinCbinding phalloidinCTRITC. indicates WT PRR11-expressing cells, and zoomed images of are shown at the (relative phalloidin fluorescence (Flag PRR11Cpositive cells/pvN173Cpositive cells) was quantitatively analyzed for = 3). Greater than 20 cells were counted per condition in every repeat. quantitative analysis of F-actin rearrangement of cells is usually exhibited in = 3). Greater than 20 cells were counted per condition in every repeat. siRNA-mediated silencing of ARPC1A. H1299 cells were transfected with unfavorable control siRNA (H1299 cells were transfected with siNC or siARPC1 for 24 h, and then the cells were transfected with FlagCPRR11 or pvN173 FlagCcontrol for another 24 h, respectively. The cells were fixed and Gdf2 stained for F-actin and Flag. Representative images are shown. relative fluorescence of F-actin (Flag PRR11-positive cells/pvN173-positive cells) was quantitatively analyzed for = 3). Greater than 20 cells were counted per condition in every repeat. quantitative analysis of F-actin rearrangement of cells is usually exhibited in = 3). Greater than 20 cells were counted per condition in every repeat. CK-666, the Arp2/3 complex inhibitor, represses Obatoclax mesylate (GX15-070) F-actin assembly driven by PRR11. H1299 cells transfected with FlagCPRR11 or pvN173 FlagCcontrol were treated with CK-666 (84 m), SMIFH2 (15 m), or CK-689 (100 m) for 24 h and then were fixed and stained with Flag Obatoclax mesylate (GX15-070) antibody and the F-actinCbinding phalloidinCTRITC. Representative images are shown. relative phalloidin fluorescence (FlagCPRR11-positive cells/pvN173Cpositive cells) was quantitatively analyzed for = 3). Greater than 50 cells were counted per condition in every repeat. quantitative analysis.
