Additional file 1 of The importin beta superfamily member RanBP17 exhibits a role in cell proliferation and is associated with improved survival of patients with HPV+ HNSCC
Creators: name: Mandic, Robert
name: Marquardt, André
name: Terhorst, Philip
name: Ali, Uzma
name: Nowak-Rossmann, Annette
name: Cai, Chengzhong
name: Rodepeter, Fiona R.
name: Stiewe, Thorsten
name: Wezorke, Bernadette
name: Wanzel, Michael
name: Neff, Andreas
name: Stuck, Boris A.
name: Bette, Michael
Publisher: name: figshare
Year: 2022
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Supplementary Table S1. Shown is the type and origin of the tissues used for Western blot analysis of RanBP17. Supplementary Table S2. Background information of cells and cell lines. Supplementary Figure S1. Uncropped Western blot images are shown for Supplementary Figure S2A (A), Supplementary Figure S2B (B), Figure 2A (C), Figure 4A (D), Figure 5A (E) and Figure 5B (F). Arrows point to the predicted size of the respective protein. Dashed rectangles refer to the region of the blot used in the final figure. Supplementary Figure S2. Western blot analysis, demonstrating protein expression of RanBP17 in tissues, cells and cell lines. A, A band at ~124 kDa (arrows) corresponding to the major RanBP17 isoform (UniProtKB/Swiss-Prot: Q9H2T7) was detected in all tested head and neck tumor tissues (HNSCC_1-15, Adeno CA_1-2) including tonsil (N1) but not muscle (N2) tissues (Supplementary Table S1). B, Similarly, a ~124 kDa band was also observed in all head and neck tumor derived cell lines and other cell lines derived from different tumors (Supplementary Table S2). Smaller sized immunoreactive bands are seen in most of the tested samples, especially cell lines. GAPDH was used as an input control. Short and long exposures are shown to visualize strong and weak bands. Indicated at the bottom in B is the histologic tumor/tissue type from which cells and cell lines were derived from. epi=epithelial, end=endothelial, mes=mesenchymal, neu=neural, mel=melanocytic, lym=lymphocytic. Supplementary Figure S3. Expression of RanBP17 exon-specific micro array probes in different HNSCC cell lines. To allow for a more precise statement about the differing RanBP17 expression levels between the tested HNSCC cell lines during micro array analysis, we also were looking at expression of the individual exons. In order to obtain this information from the microarray files, we first searched for the sample IDs given for RANBP17 on the Human Gene 1.0 ST Affymetrix chip used. Then, the .CEL files obtained from the microarray sequencing were imported in the program R (v. 4.0.4) using the tool oligo (v 1.54.1) and the function "read.celfiles". Afterwards, a background correction was performed using "backgroundCorrect" followed by normalization using "normalize". Finally, the obtained expression data for the previously searched RANBP17 Probe IDs were written into a table using write.csv2 and the results were analyzed afterwards. A, Background corrected normalized intensity of the respective Probe signal for each tested cell line. B, Graphical depiction of the exon position for the single RanBP17 specific probes according to the RanBP17 reference sequence NM_022897.4. Supplementary Figure S4. Shown on the left are the primer pairs used for the quantitative PCR analysis in Fig. 3 with their corresponding sequence information. A and B show comparable efficiencies of all primer pairs in A) untreated HCT116p53wt/wt cells and B) untreated UM-SCC-3 cells. In UM-SCC-3 cells, only primer pairs P3, P5, and P11 yielded amplicons. Supplementary Figure S5. Specificity of the RanBP17 antibody. The RanBP17 signal (left panel in A, B) disappears (right panel in A, B) after preincubating the RanBP17 antibody (HPA029568) with its corresponding antigen peptide sequence (APrEST73986) thereby confirming specificity of the antibody. TP53 mutation in UM-SCC-3: p.R248Q (nuclear p53); TP53 mutation in UT-SCC-26A: p.Y236* (cytoplasmic p53). Supplementary Figure S6. Timer2.0 (http://timer.cistrome.org/) was used for the analysis of A, the immune association for RANBP17 and CD8+ T-cells (left side) and B-cells (right side) showing the Spearman correlation coefficients. Color filled boxes indicate significantly higher values (p&lt;0.05) and B, for the correlation between the RANBP17 expression to CD8A/CD4/CD274 for HPV+ (upper part) and HPV- (lower part) samples of the TCGA-HNSC cohort (accessible at Gene_Corr of the Exploration tab), with the blue depicting the linear regression and the grey shaded area depicting the confidence interval. TPM: transcripts per kilobase million. Supplementary Figure S7. A, Boxplots for a scRNA-seq (single cell RNA-sequencing) HNSCC dataset (GEO103322) showing the expression of RANBP17, CD8a, CD4, and CD274 for the different cell types. Each dot represents the expression of one sequenced cell. B, Violin plots for the scRNA-seq immune cell HNSCC dataset (GEO139324), for which the expression details for each cell were extracted from the provided matrix files using Seurat for R. Plots were generated using the modules matplotlib and seaborn for python. Supplementary Figure S8. Boxplot analysis of the TP53 mutational status with regard to RUNX2 expression as well as correlation analysis of RUNX2 with RanBP17 for the A, HPV- and B, HPV+ TCGA HNSCC cohorts, showing no significant expression differences with regard to TP53 mutational status but a significant correlation in the HPV- cohort only. Analyses were performed using the TIMER2.0 Gene_Mutation module (http://timer.cistrome.org/). Left graphs: Wilcoxon test. Right graphs: linear regression with Spearman’s rho.