MiR-9 modulates and predicts the response to radiotherapy and EGFR-inhibition in HNSCC (2021)
Figure 1

Figure 1. miR-9 expression sustains the tumor initiating properties of HNSCC cells.

A. qRT-PCR analyses of miR-9 expression in control (shCTR) and antimiR-9 FaDu cells. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance.

B. On the right, Western Blot (WB) analyses of the indicated protein expression in FaDu cells as in A. Actin was used as loading control. Right graph reports the quantification of protein expression normalized on Actin loading control. Data represent the mean (±SD) of three independent experiments. Unpaired t test was used to verify the statistical significance.

C. Cell viability analyses of cells described in A/B over a period of 5 days using the MTS assay. Data represent the mean (±SD) of three independent experiments performed in sextuplicate. Unpaired t-test was used to verify the statistical significance per each time point.

D. Colony formation assay of the cells described in A/B. Left graph reports the number of clones per well. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance. Right, representative images of clones are shown.

E. Soft agar assay of cells described in A/B. Left graph reports the number of clones per well. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance. Right, representative images of 10X field are shown.

F. Sphere forming assay of cells described in A/B. Left, typical images of 4X and 10X fields are shown. Middle graph reports the number of spheres formed in first- and second-generation. Right graph reports the area of second-generation spheres. Each dot represents one analyzed sphere. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance.

G. Graph reporting the tumor onset in NSG mice injected with different numbers of control (shCTR) and antimiR-9 FaDu cells, as described, and followed for up to 35 days (n=5 mice/group). Data represent the mean (±SD) and two-way ANOVA test was used to calculate the statistical significance among groups.

H. Left graph reports the tumor volume in NSG mice injected with control (shCTR) and antimiR-9 FaDu cells followed for up to 35 days (n=5 mice/group). Data represent the mean (±SD) and unpaired t-test was used to verify the statistical significance at each time point. On the right, typical images of explanted tumors formed by control (shCTR) and antimiR-9 FaDu cells at necropsy.

I. Typical images of Hematoxylin&Eosin (H&E - upper images) and Ki67 expression (Ki67 - bottom images) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in H. 4X and 20X magnification are shown. Right graph reports the percentage of Ki67 positive cells. Each dot represents one tumor. Data are expressed as mean (±SD) and unpaired t-test was used to verify the statistical significance.

In the figure A.U. = Arbitrary Units, *p<0.05; **p<0.01; ***p<0.001.