Fig 2: RanBP17 does not localize to nucleoli. UM-SCC-3 and UT-SCC-26A cells were co-stained with the RanBP17-specific antibody HPA029568 and an antibody directed against Nucleolin (clone ZN004), exhibiting a distinct nuclear RanBP17 localization (arrowheads) that is different from nucleoli. DAPI was used to counterstain the nucleus. TP53 mutation in UM-SCC-3: p.R248Q (nuclear p53); TP53 mutation in UT-SCC-26A: p.Y236* (cytoplasmic p53)

Fig 3: CDDP treatment of normal human epidermal keratinocytes (NHEK) inhibits RanBP17 expression. A In one preliminary Western Blot experiment, a reduction of RanBP17 protein expression was observed in NHEK, treated with 5 and 50 µmol/L CDDP. B RT-qPCR analysis was subsequently performed to evaluate the response of RanBP17 RNA expression levels on CDDP treatment. Statistical tests in B One way ANOVA with Tukey´s correction for multiple comparisons (n=4) **p<0.01

Fig 4: Expression of RanBP17 during the cell cycle. A FACS sorting yields 4 cell populations, representing the G1/G0, S and G2/M phases of the cell cycle as well as a population labeled „All“, which contains all phases for comparison. Western blot analysis points to increased expression levels of RanBP17 (antibody orb226830) during the S-phase of the cell cycle, which is particularly prominent in UT-SCC-26A. Cell cycle associated proteins (PCNA, cyclin-B1, CDK1) show an expected increase of their expression during S- and into G2/M-phase, thereby helping to validate the accuracy of cell sorting to the respective phase of the cell cycle. B Total cellular protein was significantly elevated in S phase cells compared with cells in the G1/G0 phase of the cell cycle, which is an expected finding for cells in division. A similar observation was made for total RNA levels. C No significant differences in RanBP17 RNA levels could be noted between the different cell cycle phases for UM-SCC-3 (n=4) and UT-SCC-26A (n=3) cells. Statistical tests: B and C: One way ANOVA with Dunn´s correction for multiple comparisons. *p<0.05. (A: Protein bands shown in the upper two panels (RanBP17-orb226830 and GAPDH) and the lower six panels (RanBP17-GTX70420, PCNA, Cyclin-B1, GAPDH, CDK1, Beta-Tubulin) are derived from lysates of two independent experiments). TP53 mutation in UM-SCC-3: p.R248Q (nuclear p53); TP53 mutation in UT-SCC-26A: p.Y236* (cytoplasmic p53)

Fig 5: RNAi knockdown of RanBP17 inhibits cell proliferation independent of CDDP treatment. A XTT proliferation assay depicting the relative cell viability of the HNSCC cell line UM-SCC-3 after RNAi knockdown of RanBP17 using pool 1 siRNA (consists of si1, si2, si4, si5 as depicted in D) following incubation with CDDP (6.25, 12.5, 25, 50 and 100 µmol/L). Note the significant reduction in cell viability at 0 µmol/L CDDP in UM-SCC-3 cells after RanBP17 knockdown. B Effect of RanBP17 RNAi knockdown alone (using pool 1 siRNA) was tested in the indicated cell lines. Efficient RNAi knockdown of RanBP17 RNA using pool 1 siRNA is shown for HCT116p53 wt/wt cells. C The 3 most responsive cell lines from B were used to evaluate the effect of all 8 single RanBP17 specific siRNAs (si1-8). D Depicted are the siRNA (si1-8) binding sites as well as their location within the RanBP17 reference sequence NM_022897.4. Also included is the respective information for the updated RanBP17 reference sequence NM_022897.5, which differs from NM_022897.4 by lacking the first 136 nucleotides. Underlined nucleotides refer to the nucleotide sequence of the adjacent exon (shown in brackets). NT_non target RNA, siRanBP17=silencing RNA (pool 1) directed against RanBP17. Statistical tests; A, B, C: Unpaired Students t-test, two-sided (n=4 for all analyses) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001