MiR-9 modulates and predicts the response to radiotherapy and EGFR-inhibition in HNSCC (2021)
Figure 3

Figure 3. Sp1 expression is positively regulated by miR-9.

A. Dot plot reporting the correlation of miR-9 and Sp1 expression in primary HNSCC samples evaluated by qRT-PCR. The number of analyzed samples (n), the Spearman correlation value (r) and its significance (p) are reported in the graph.

B. qRT-PCR analyses of SP1 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.

C. qRT-PCR analyses of Sp1 expression in control (shCTR) and antimiR-9 FaDu cells transiently transduced with PGK-miR-9 or control vector as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.

D. qRT-PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR-9 or control vector. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance.

E. Clonogenic assay on FaDu cells silenced for Sp1 (shSP1#3 or #5) or scramble sequence (shCTR). Left graph reports the number of colonies. On the right, WB analyses of the indicated protein expression in FaDu cells. Tubulin was used as loading control. Bottom panel, representative images of clones are shown. In the graph data represent the mean (±SD) of three independent experiments performed in duplicate and one-way ANOVA was used to verify the statistical significance.

F. Sphere forming assay of the cells described in E. Left panel reports the mean number of spheres formed in first- and second-generations. Right graph reports the area of second-generation spheres. Data represent the mean (±SD) of three independent experiments performed in duplicate and one-way ANOVA was used to verify the statistical significance. Each dot represents one analyzed sphere. Bottom panels show representative images of the spheres.

G. WB analyses of the indicated protein expression in control (shCTR) and antimiR-9 FaDu cells, overexpressing or not RSV-SP1. Actin and Histone H3 were used as loading control.

H. Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib (GEFI - left panel) or Cetuximab (CTX - right panel) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.

I. Schematic representation of the pGL3-vectors used to test the potential activity miR-9 on Sp1 promoter. Vectors are named based on the maximum distance from SP1 ATG. The lines indicate the putative KLF5 binding sites according the statistical prediction power according to EPD portal (blue lines p=0.001, green lines p=0.0001). Black bars indicate the primer pairs used for the amplification of different region of Sp1 promoter in the ChIP analyses depicted in Figure 4H.

J. Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in control (shCTR) or 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.

In the figure A.U. = Arbitrary Units *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.