Randa Kalaajieh

E-Cadherin Expression upon INHBA knockdown. Representative western blot (of n = 3 replicates) showing E-Cadherin protein expression in Mock KD and INHBA KD1/KD2 at t24 and t48.

Cell migration upon INHBA knockdown. A, Migration of native UDSCC1 cells, Mock KD, INHBA KD1/KD2 and native UDSCC2 cells as assessed by scratch assay. Results are shown as mean ± SD of the open wound area (n = 3) at baseline (t0), t24, and t48. Conditions were compared with native UDSCC1 using unpaired t test with ***, P ≤ 0.001; ****, P ≤ 0.0001; and ns, P > 0.05. B, Representative images of processed light microscopy pictures taken at 10-fold magnification of each condition at t0, t24, and t48.

Correlation analysis between miRNAs and INHBA expression output

Correlation analyses between INHBA expression, immune infiltrates and miRNAs. A, Waterfall plot showing Spearman correlation between INHBA expression level and infiltrating ratios (IR) of different immune cells, as computed by immune cell deconvolution. Rho > 0.4 was considered relevant (dashed line). *, P ≤ 0.05; ***, P ≤ 0.001; ns, not significant. B, Correlation matrix between INHBA and the pro-tumor M1 signature showing Spearman rho and P-value symbols (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) in the upper triangular matrix and scatter plots in the lower triangular matrix. TPM = transcripts per million. C, Waterfall plot showing Spearman correlation between INHBA expression level and miRNAs (TCGA cohort). miRNAs with Rho < −0.4 and P < 0.05 were considered relevant (dashed line). D, Venn diagram showing intersections between negatively correlating miRNAs from C and miRNAs predicted to interact with the 3′UTR of INHBA by miRDB and Targetscan resulting in three candidates of interest (hsa-mir-106a, hsa-mir-20b, hsa-mir-9-1/2/3). Their putative binding sites to the INHBA 3′UTR are highlighted. E–G, Kaplan–Meier plots with log-rank test for overall survival of patients with OPSCC (TCGA cohort) based on tumor miRNA expression of the outlined candidates (E: hsa-mir-106a, F: hsa-mir-20b, G: hsa-mir-9). Blue indicates low and red indicates high miRNA expression, as determined using median of raw counts as cutoff.

Gene set enrichment analysis output

Gene set enrichment analysis output

INHBA expression in OPSCC tumor and cell lines and its correlation with EMT transcription factors. Correlation of INHBA expression with the expression of SNAI1 (A), SNAI2 (B), and TWIST1 (C) in OPSCC tumors (Ulm cohort). TPM = transcripts per million. D, Relative expression of INHBA mRNA in native UDSCC1 (HPV-negative) and UDSCC2 (HPV-positive) cells as determined by qRT-PCR and normalized to RPL30 and UDSCC1. Results are shown as mean ± SD of n = 6 replicates. INHBA expression was not detected in n = 3 UDSCC2 samples. Expression level between UDSCC1 and UDSCC2 was compared by unpaired t test with **, P ≤ 0.01. E, Representative Western blot analysis (of n = 3 replicates) showing INHBA protein expression in native UDSCC1 and UDSCC2 cells. Numbers below blots indicate relative INHBA intensity normalized to GAPDH. F, Relative expression of INHBA, SNAI1, SNAI2, and TWIST1 mRNA in UDSCC1 cells upon transfection with Mock siRNA [Mock knockdown (KD)] and two INHBA siRNAs (INHBA KD1 and KD2) after 24 hours (t24) and 48 hours (t48). Target gene expression was determined by qRT-PCR and normalized to RPL30 and Mock KD. Results are shown as mean ± SD of n = 3 replicates. Expression level between Mock KD and INHBA KD1/2 was compared by unpaired t test with *, P ≤ 0.05; **, P ≤ 0.01; and ns, P > 0.05. G, Representative Western blot analysis (of n = 3 replicates) showing INHBA protein expression in Mock KD and INHBA KD1/KD2 at t24 and t48. Numbers below blots indicate relative INHBA intensity normalized to GAPDH.

GSEA identifying INHBA as a leading driver of EMT in HPV-negative tumors. A, Dot plot of pathways enriched and depleted in HPV-negative compared with HPV-positive OPSCC tumor as determined by GSEA of transcriptomes from n = 62 primary tumor samples (Ulm cohort). Results were ranked according to the NES, dots were colored according to FDR and size was determined by the number of genes. B, GSEA plot of the top pathway (EMT) showing enrichment score (green curve), position of genes (black lines) with the leading edge (p-EMT), NES, and FDR. C, Volcano plot showing the log2 fold change of genes upregulated in HPV-positive (red) and HPV-negative OPSCC (blue), with INHBA (red circle) as the highest upregulated leading edge in HPV-negative tumors.

INHBA correlates with stemness attributes. A, Correlation matrix between INHBA and stemness-related markers showing Spearman rho in scaled dots and P-value symbols (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) in the upper triangular matrix and Spearman rho values in the lower triangular matrix. B, Time series of ALDH enzyme activity for Mock KD and INHBA KD1/KD2. Results are shown as mean ± SD of ALDH activity (mU/mL; n = 3) and Δt (minutes). INHBA KD1/KD2 were compared with Mock KD using unpaired t test with *, P ≤ 0.05 and **, P ≤ 0.01.

E-Cadherin Expression upon INHBA knockdown. Representative western blot (of n = 3 replicates) showing E-Cadherin protein expression in Mock KD and INHBA KD1/KD2 at t24 and t48.