(A) Quantitative Gli1 mRNA levels after treatment with CK2 subunit-specific siRNA detected by real-time RT-PCR. Silencing of CK2α significantly reduced Gli1 mRNA levels both in A549 and H1299 cell lines (by 50% and 45%, respectively). Silencing of CK2β also resulted in a significant decrease of Gli1 in both cell lines. Minimal Gli1 mRNA level was noticed in the normal lung (NL). * P<0.05, ** P<0.01, Student’s t-test. (B) Protein levels detected by Western blot. Silencing of CK2α led to 71% (A549) and 73% (H1299) reduction of Gli1, while silencing of CK2β led to 67% (A549) and 35% (H1299) decrease of Gli1. Treatment by CK2α’-targeting siRNA produced no obvious difference, rather than decrease of Gli1 in A549, and produced a 60% increase in H1299, both in mRNA and protein levels. (C) Localization of Gli1 detected by green fluorescence. The Gli1 protein mainly localizes in the nuclear compartment of the cells, and shows a great reduction after CK2α knockdown. Scale bar = 30 µm. (D) The transcriptional activity of Hh pathway in A549 detected by Gli reporter assay. Silencing of CK2α resulted in a significant decrease (more than 40% at 25 µM and 60% at 50 µM) of the transcriptional activity, compared with the control siRNA. * P<0.05, ** P<0.01, Student’s t-test. (E) Quantitative Gli1 mRNA levels after treatment with TBB and CX-4945 by real-time RT-PCR. Gli1 expression in A549 decreased noticeably at the dosage levels of 1 µM CX-4945 and 10 µM TBB, significantly at 10 µM CX-4945 and 50 µM TBB (50% and 60%). * P<0.05, ** P<0.01, Student’s t-test. (F) Protein levels detected by Western blot. In protein level, these decreases rose to 76% (10 µM CX-4945) and 89% (50 µM TBB) in A549, and 81% (10 µM CX-4945) and 93% (50 µM TBB) in H1299, respectively. (G) Protein expression of Gli1 detected by green fluorescence. Cells were treat with 30 µM TBB or vehicle DMSO, immunoflorescence staining with anti-Gli1 mAb on cultured cells showed a distinctly lower green florescence in the presence of 30 µM TBB. (Scale bar = 30 µm). (H) The transcriptional activity of Hh pathway in A549 detected by Gli reporter assay. A 50% decrease was detected in the presence of 10 µM CX-4945 or 10 µM TBB. * P<0.05, ** P<0.01, Student’s t-test.

(A) Hh downstream genes expression detected by real-time RT-PCR. The mRNA level of the four Hh target genes (Gli1, Ptc1, Ptc2 and Cyclin E1) decreased significantly after CK2α knockdown. * P<0.05, ** P<0.01, Student’s t-test. (B) The expression of ABCG2 decreased in CK2α-silenced A549 cells. (C) The proportion of SP cells dropped from 26.8% to 15.4%, and from 9.4% to 3.4% in presence of 50 µM verapamil, respectively. Cells were labeled with the Hoechst 33342 and then analyzed by flow cytometry. (right) Results when the cells were treated with 50 µM verapamil during the labeling procedure. The SP is outlined and shown as a percentage of the total cell population. These experiments were repeated at three times with similar results. (D) Bar chart for (C). (E) Model of Hh/Gli1 signaling regulated by CK2. Details are described in the text.

(A) RT-PCR. (B) Western blot. The CK2α gene was activated in the eight NSCLC cell lines examined (A549, A427, H460, H1299, H1650, H358, H838 and H322), and the Gli1 gene was expressed in all the cell lines except H358. (C) Linear correlation curve of CK2α and Gli1 mRNA levels. A mild correlation (r = 0.37, P<0.05) was shown in linear correlation analysis by SPSS. Primary NCSCL tissues from patients undergoing resection were collected at the time of surgery and immediately snap-frozen in liquid nitrogen. These tissue samples were kept at –170°C in a liquid nitrogen freezer before use.

(A)A549 cells were transfected either with a pcDNA3.1-CK2α or control pcDNA3.1-LacZ plasmid vectors, the over-expressed CK2α and up-regulated Gli1was detected by real-time RT-PCR. * P<0.05, ** P<0.01, Student’s t-test. (B)Western blot. The protein level of Gli1 was up-regulated in this system with a two-fold increase. (C)Moreover, the reporter assay also showed a significant (more than 2-fold) increase of the Gli1 transcriptional activity. * P<0.05, ** P<0.01, Student’s t-test. (D)In a protein degradation analysis, A549 cells showed reduced Gli1 protein at the time point of 2 hours after treated with CK2α siRNA.