36

Figure 9 ERK3 is highly upregulated in human lung carcinomas. (A). ERK3 gene expression is

highly upregulated in squamous cell lung carcinomas versus normal lung tissues. ERK3 gene

expression in squamous cell lung carcinomas (n=21) and normal lung samples (n=17) was

analyzed on Affimetrix U95A microarrays (Bhattacharjee et al, 2001). P value was calculated

using unpaired two-tailed t test. (B). Immunohistological analysis of ERK3 protein expression in

NSCLC tissue microarray (TMA) that contains 302 lung adenocarcinomas and 154 squamous

cell lung carcinomas. Statistical significance was determined by unpaired two-tailed t test.

Supplemental Figure 1 Generation of purified ERK3 protein kinase. Myc-tagged ERK3 protein

kinase was expressed in 293T cells, purified using anti-Myc beads, and eluted off the beads

using Myc peptide. The purity of protein samples was examined by Coomassie staining (A) and

Western blotting (B). Protein lysate of H1299 cells were used as a positive control for the

expression of ERK3 and ERK1/2 proteins.

Supplemental Figure 2 Knockdown of SRC-3 and ERK3 inhibits lung cancer cell invasion.

H441 (A) and H520 (B) cells were stably transduced with lentiviral non-targeting control shRNA

(shCtrl), shSRC-3, shERK3, or both shSRC-3 and shERK3. Cell invasion was determined by

transwell matrigel cell invasion assay. Depletion of SRC-3 or ERK3 inhibits the invasion of both

H441 (A) and H520 (B) cells. Values are means ± s.e of four separate experiments. “*” indicates

significant difference (Student’s t test).

Supplemental Figure 3 ERK3 promotes H520 cell invasion in a SRC-3 dependent manner and

S857 phosphorylation of SRC-3 is critical in promoting cell invasion. (A). Transwell matrigel

cell invasion assay of H520 cells transiently transfected with either Myc-ERK3 or the empty

vector, or together with either SRC-3 siRNA (siSRC-3) or non-targeting control siRNA (siCtrl).

(B). Transwell matrigel cell invasion assay of H520 cells transiently transfected with either

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lentiviral SRC-3Flag, SRC-3S857AFlag, or Myc-ERK3, or co-transfected with two of the

constructs as indicated. Intact S857 is required for SRC-3 to promote cell invasion.

Supplemental Figure 4 Knockdown of SRC-3 or ERK3 does not change actin polymerization.

Protein lysates were prepared from H1299 cells stably transduced with lentiviral non-targeting

control shRNA (shCtrl), shSRC-3, or shERK3. Actin polymerization was measured at different

time points and represented as relative fluorescence intensity normalized by protein content

(cps/µg).

Supplemental Figure 5 ERK3 and SRC-3 cooperatively regulate MMP gene expression. (A, B,

and C). Depletion of SRC-3 or ERK3 significantly decreased MMP2 (A and C) and MMP10 (B

and D) gene expression. MMP gene expression in A549 (A and B) and H441 (C and D) cell

pools stably expressing shSRC-3, shERK3, or shCtrl was determined by RT-qPCR analysis. (E).

SRC-3 and ERK3 cooperatively promote MMP2 promoter-driven luciferase activity, in which

S857 of SRC-3 plays an important role. H1299 cells were singly- or co-transfected with the

constructs as indicated. Luciferase activity is represented as relative luciferase units (RLU) on

the Y-axis. (F). Knockdown of SRC-3 significantly decreased ERK3-induced MMP2 promoter-

driven luciferase activity. H1299 cells were transfected with either Myc-ERK3 or the empty

vector, or together with either SRC-3 siRNA (siSRC-3) or non-targeting control siRNA (siCtrl).

Values represent the means ± s.e of three separate experiments. “*” indicates a significant

difference (Student’s t test).

Supplemental Figure 6 ERK3 and SRC-3 regulate the activities of secreted MMP2 and MMP10.

(A and B). Depletion of SRC-3 or ERK3 significantly decreased the activities of secreted MMP2

(A) and MMP10 (B). The activities of secreted MMP2 and MMP10 from H1299 cell pools

stably expressing shSRC-3, shERK3, or shCtrl was determined using the SensoLyte fluorimetric

38

MMP2 and MMP10 activity assay kits (AnaSpec). MMP activity is represented as relative

fluorescence units on the Y-axis. (C). SRC-3 and ERK3 cooperatively promote MMP2 activity,

in which S857 of SRC-3 plays an important role. A549 cells were singly- or co-transfected with

the constructs as indicated. Values represent the means ± s.e of three separate experiments. “*”

indicates a significant difference (Student’s t test).

Supplemental Figure 7 Both SRC-3 and ERK3 are required for the invasiveness of H1299 lung

cancer cells in vivo. 1 x 106 of H1299 cells with stable expression of the control shRNA (shCtrl),

SRC-3 shRNA (shSRC-3), ERK3 shRNA (shERK3), or both shSRC3 and shERK3 were injected

into SCID/Beige mice via tail vein. Tumor formation in the lungs was analyzed by

hematoxylin/eosin (H/E) staining of paraffin-embedded tissue sections. Magnification: 50X.

Supplemental Figure 8 Representative images showing different levels of ERK3

immunostaining (red arrows) in squamous cell lung carcinomas (A-C) and lung adenocarcinomas

(D-F). Magnification 200x.

Coomassie staining

250 kD

150 kD

100 kD75 kD

50 kD37 kD

25 kD

(Myc)6-ERK3

HC of Myc IgG

LC of Myc IgG

ERK3 IB

ERK1/2 IB

Western blotting

A B

Supplemental Figure 1

Supplemental Figure 2

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Supplemental Figure 5

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Supplemental Figure 6

shCtrl

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Supplemental Figure 7

Negative nuclear ERK3

Low nuclear ERK3

High nuclear ERK3

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High nuclear ERK3

F

Squamous cell carcinoma Adenocarcinoma

Supplemental Figure 8