1. Date of download: 7/3/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Short Hairpin RNA System to Inhibit Human p16 in Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2004;130(1):68-73. doi:10.1001/archotol.130.1.68 A, The chromosome 9p21 p16 Ink4a/p14 ARF locus. The p16 transcript is formed by exon 1α (green) and exons 2 and 3. The p14 ARF transcript has a distinct first exon, 1β, but shares sequence homology with p16 in its second exon. While there is sequence homology between the 2 transcripts, there is no amino acid homology. B, Overview of experimental design. Target sequences in exon 1α of p16 were selected and designated AB, CD, EF, GH, or IJ as indicated. pBluescript U6 (pBSU6)–short hairpin RNA (shRNA) cassettes (yellow) were created and screened for the strongest repressors. The most effective cassettes were introduced into viral vectors (pShuttle and pBabe) that were also screened for efficiency of inhibition. The strongest vectors were used to produce shRNA-producing adenovirus and retrovirus, which were tested in squamous cell carcinoma lines. Antibiotic selection vectors (modified pCDNA3) were also constructed for use in stable transfections. C, Sample shRNA sequence AB. Both the shRNA cassette and the resulting hairpin loop are illustrated. Figure Legend:

2. Date of download: 7/3/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Short Hairpin RNA System to Inhibit Human p16 in Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2004;130(1):68-73. doi:10.1001/archotol.130.1.68 A, The most effective pBluescript U6 (pBSU6)–short hairpin RNA (shRNA) cassettes targeting exon 1α of p16 were identified. The U-2 OS cells were cotransfected with the cytomegalovirus (CMV)-p16 expression vector and individual pBSU6-shRNA cassettes (AB, CD, and EF) vs control (U6). Western blot analysis shows high levels of p16 expression from the CMV-p16 expression vector in the presence of the control pBSU6 vector (first lane) and strong repression by all 3 shRNA vectors (second through fourth lane). Actin is used as a loading control. B, Short hairpin RNA directed again exon 1α did not repress p14 ARF expression. U-2 OS cells were cotransfected with the CMV-p14 ARF expression vector and the same p16-targeting pBSU6-shRNA cassettes (AB, CD, and EF) vs control (U6). Western blot analysis shows high levels of p14 ARF expression from the CMV p14 ARF expression vector in the presence of the control pBSU6 vector (first lane) and the absence of RNA interference in the presence of the shRNA vectors that repress p16 (second through fourth lane). Again, actin is used as a loading control. Figure Legend:

3. Date of download: 7/3/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Short Hairpin RNA System to Inhibit Human p16 in Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2004;130(1):68-73. doi:10.1001/archotol.130.1.68 Viral short hairpin RNA vectors effectively suppress p16 levels. The U-2 OS cells were cotransfected with the cytomegalovirus (CMV)-p16 expression vector and individual viral shRNA vectors. Western blot analysis shows high p16 expression in the presence of the control vectors (pShuttle and pBabeU6, left lanes). Viral vectors suppress p16 levels to variable degrees, depending on which target sequence (AB, CD, or EF) and orientations (forward [for] and reverse [rev]) are used. Actin is used as a loading control. A, Adenoviral pShuttle-shRNA vectors targeting sequences AB/reverse and CD/forward (third and fourth lanes) are the most effective repressors, with other sequences and orientations having an intermediate effect. B, Retroviral pBabe vectors carrying either the U6 promoter alone or shRNA are shown. Short hairpin RNA vectors targeting sequences AB/forward, CD/reverse, and EF/reverse are more effective than the opposite orientations for these sequences. Overall, the AB sequence resulted in the strongest inhibition. Figure Legend:

4. Date of download: 7/3/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Short Hairpin RNA System to Inhibit Human p16 in Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2004;130(1):68-73. doi:10.1001/archotol.130.1.68 Short hairpin RNA–producing adenovirus suppresses p16 expression. The U-2 OS cells with tetracycline-regulated expression of p16 were transduced (MOI 0.5) with adenovirus expressing AB/CD shRNA (fifth and sixth lanes). Control transductions were performed with non-shRNA producing adenovirus (U6, third and fourth lanes). Control cells with no adenoviral infection are also shown (first and second lanes). Western blot of lysates from noninduced (−) vs induced (+) cells were collected and compared 18 hours after induction. Actin is used as a loading control. Figure Legend:

5. Date of download: 7/3/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Short Hairpin RNA System to Inhibit Human p16 in Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2004;130(1):68-73. doi:10.1001/archotol.130.1.68 Short hairpin RNA–producing retrovirus inhibits p16 expression in squamous cells. The JHU-011-SCC cells with tetracycline- regulated expression of p16 were transduced with retrovirus either producing shRNA-AB/forward (fifth through tenth lanes) or containing only the U6 promoter (third and fourth lanes). Individual clones were isolated after hygromycin selection. Three stable shRNA clones and 1 control clone (U6) are shown. Lysates from JHU-011-SCC cells without retroviral infection are also shown for comparison (first and second lanes). Western blot of lysates from noninduced (−) vs induced (+) cells were collected and compared. Actin is used as a loading control. Figure Legend:

6. Date of download: 7/3/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Short Hairpin RNA System to Inhibit Human p16 in Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2004;130(1):68-73. doi:10.1001/archotol.130.1.68 Short hairpin RNA–producing retrovirus can block p16-associated cellular morphologic features in a laryngeal carcinoma line in vitro. View of JHU-011-SCC cells (original magnification ×40) with tetracycline-regulated expression of p16 that were transduced with retrovirus as described in Figure 5. Cells were induced with tetracycline to express high levels of p16 and photographed after 5 days. A, The JHU-011-SCC cells infected by control retrovirus show typical p16-associated perinuclear vacuoles, large cell volume, and relatively smooth cell membranes. B, The JHU-011-SCC cells infected by shRNA-producing retrovirus show only few vacuoles, smaller cell volume, and irregular cell membranes with multiple projections. Figure Legend: