Figure S2 Repression of Mir-101 Is Involved in the Tumorigenic Activity of IL-1B in NSCLC Cells

Figure S1 No significant difference in the serum levels of IL-4, IL-6, TNF-α and TGF-β between NSCLC patients and healthy controls.

Comparison of pro-inflammatory cytokines levels in serum from healthy donors and NSCLC patients. The concentration of IL-4 (A), IL-6 (B), TNF-α (C) and TGF-β (D) in serum from 37 healthy donors and 37 NSCLC patients was measured by ELISA. The significance of the differences between the healthy donors and patients was assessed using the Student’s t test.

1 Figure S2 Repression of mir-101 is involved in the tumorigenic activity of IL-1 in NSCLC cells.

(A) IL-1 exerts a tumor-promoting effect in NSCLC cells H460 (top) or H1299 cells (bottom). Left, MTT assays; right, transwell migration assays. The assays were performed 24 h post-treatment of IL-1.

(B) The effect of IL-1β treatment on expression of miR-101 in H1299 cells. The level of miR-101 was determined by quantitative real-time PCR (qRT-PCR) assays 48 h after IL-1β treatment, with U6 serving as an internal normalized reference.

(C) Restoring mir-101 expression in IL-1-treated H460 cells. IL-1 treated-H460 cells were respectively transfected with 0.5 nmol/L, 1 nmol/L or 2.5 nmol/L of miR-101 mimics, and qRT-PCR analyses of miR- 101 levels were performed 24 h post-transfection.

(D) Ectopic miR-101 overrode the tumorigenic effect of IL-1 in H1299 cells. IL-1 treated H1299 cells were respectively transfected with Ctrl RNA or miR-101 mimics, and the MTT (left) and transwell migration assays (right) were performed 24 h post-transfection.

The average values ± s.d. of three separate experiments were plotted. *P<0.05, **P<0.01. Results shown are representative of three independent experiments.

2 Figure S3 IL-1β down-regulates mir-101 via the COX-2/HIF-1α pathway in NSCLC cells.

(A) qRT-PCR assays of the effect of IL-1β on primary and mature mir-101 expression in H460 cells. Cells were treated with IL-1β, and the assays were performed 24 h post treatment. -actin mRNA and U6 respectively served as internal normalized references for pri-miR-101 and miR-101.

3 (B) Immunostaining of HIF-1α protein (green) in H460 cells. Cells were treated with CoCl2 (middle) or IL- 1β (bottom). Representative staining images are shown on the left, and quantification of nuclear HIF-1 staining fluorescent densities is shown at the right (mean ± SD). Nuclei were stained with DAPI (blue); scale bar: 20 m.

(C) qRT-PCR assays of the effect of CoCl2 on primary and mature mir-101 expression in H460 cells. Cells were treated with CoCl2, and the assays were performed 5 h post-treatment. -actin mRNA and U6 respectively served as internal normalized references for pri-miR-101 and miR-101.

(D) Western blot assays of the effect of IL-1 on HIF-1 protein expression (lane 2), with -actin serving as a loading reference.

(E) Knockdown of HIF-1 completely abolished the repression of mir-101 by IL-1 in H460 cells. IL-1- treated cells were transfected with scrambled siRNA (Scr-siR) or HIF-1 siRNA (HIF-1-siR), and the assays were performed 24 h post-transfection. Top, qRT-PCR assays of the effect of HIF-1-siR on IL-1- mediated repression of mir-101; bottom, western blot assays of HIF-1 protein, with -actin serving as a loading reference.

(F) Overexpression of COX-2 strongly repressed mir-101 expression. Top, qRT-PCR analysis of miR-101 level, with U6 serving as an internal normalized reference; bottom, western blotting assays of COX-2 protein, with -actin serving as a loading reference.

(G) Knockdown of COX-2 significantly increased mir-101 expression. Top, qRT-PCR analysis of miR-101 level, with U6 serving as an internal normalized reference; bottom, Western blotting assays of COX-2 protein, with -actin serving as a loading reference.

(H) Knockdown of Cox-2 significantly attenuated the repression of mir-101 by IL-1 in H460 cells. IL-1- treated cells were transfected with Scr-siR or COX-2 siRNA (COX-2-siR), and the assays were performed 24 h post-transfection. Left, qRT-PCR assays of the effect of COX-2 siR on IL-1-mediated repression of mir-101; right, western blot assays of HIF-1 and COX-2 proteins, with -actin serving as a loading reference.

The average values ± s.d. of three separate experiments were plotted. **P<0.01, ***P<0.001. Results shown are representative of three independent experiments.

4 Figure S4 miR-101 up-regulates let-7 family miRNAs by targeting Lin28B in NSCLC cells.

(A) Transfection of miR-101 mimics into IL-1-treated H460 cells completely overrode the effect of IL-1 on the expression of let-7 family miRNAs. IL-1-treated cells were transfected with miR-101 mimics or Ctrl RNA. qRT-PCR assays of let-7 family miRNA levels 48 h post-transfection, with U6 serving as an internal normalized reference.

(B) Ectopic expression of Flag-Lin28B significantly rescued the effect of miR-101 overexpression on expression of let-7 family miRNAs. H1299 cells were transfected with miR-101 mimics or co-transfected with miR-101 mimics and p3xFlag-Lin28B or control p3xFlag vectors. qRT-PCR assays of let-7 family miRNA levels 48 h post-transfection, with U6 serving as an internal normalized reference.

The average values ± s.d. of three separate experiments were plotted. **P<0.01, ***P<0.001. Results shown are representative of three independent experiments.

5 Figure S5 Comparison of the expression of Lin28B and miR-101 in H460 and H1299 cells. (A) qRT-PCR analyses of endogenous miR-101 expression in H460 and H1299 cells, with U6 serving as an internal reference. (B) Western blot assays of endogenous Lin28B protein levels in H460 and H1299 cells, with -actin serving as a loading control. (C) qRT-PCR analyses miR-101 levels in anti-miR-101-transfected H460 cells (left) or miR-101 mimics- transfected H1299 cells (right). The cells were respectively transfected with 100 nmol/L of anti-miR-101 and 50 nmol/L of miR-101 mimics, and qRT-PCR analyses of miR-101 levels were performed 24 h post- transfection. The average values ± s.d. of three separate experiments were plotted. *P<0.05. Results shown are representative of three independent experiments.

6 Figure S6 miR-101 plays a tumor suppressive role in NSCLC cells. (A) Inhibition of mir-101 promoted the proliferation of H460 cells. Top, MTT assays; bottom, western blot analyses of PCNA and Lin28B proteins, with -actin serving as a loading control.

(B) Inhibition of mir-101 promoted soft-agar colony formation. Top, quantitative results of soft agar foci per field; bottom, representative images.

(C) Inhibition of mir-101 promoted transwell cell migration. Top, quantitative results of migratory cells per

7 field; bottom, representative images.

(D) Inhibition of mir-101 reduced xenograft tumor growth in nude mice. The curves show the time course of tumor growth, and the inset photographs are representative xenografted tumors 25 days after inoculation.

H460 cells were transfected with Ctrl RNA or anti-miR-101, and the assays were performed 24 h post transfection except where indicated otherwise. The average values ± s.d. of three separate experiments were plotted. *P<0.05, **P<0.01, ***P<0.001. Results shown are representative of three independent experiments.

8 Figure S7 Repression of miR-101 is critical to IL-1-mediated induction of Cox-2 and Lin28B in NSCLC cells.

(A) Both activation of NF-B and repression of mir-101 are required for IL-1 to enhance Lin28B expression in NSCLC cells. Left, western blot of Lin28B protein in cells with indicated treatment with - actin serving as a loading control; right, qRT-PCR of Lin28B mRNA levels in cells with indicated treatment.

(B) IL-1β up-regulated Lin28B in NSCLC cells via the IL-1/COX-2 axis. Western blot of Lin28B and COX-2 proteins in cells with indicated treatment, with -actin serving as a loading control.

(C) Knockdown of miR-101 expression restored the stimulatory effect of IL-1 on COX-2 levels in Aspirin- or Celecoxib-treated H460 cells. Western blot of COX-2 proteins in cells with indicated treatment, with - actin serving as a loading control.

9 Table S1 Sequences of chemically synthesized DNA and RNA oligonucleotides