Supplementary FigureS1

Supplementary FigureS1.Expression and localization of TMEM88 in epithelial malignancies and non-small cell lung cancer (NSCLC) cell lines

(A)TMEM88 localized on the membrane (a, 200× and inset, 400×) or in the nucleus (b, 200× and inset, 400×) in few lung cancer tissues.

(B) Western blot analysis of TMEM88 expression in 40 paired fresh lung cancer specimens.TMEM88 showstwo bands(17 kDa and 25 kDa), and the 17-kDa isoform (red solid triangle) was highly expressed in 90%(36/40) oflung cancer tissues compared to noncancerous tissues.

(C)The amino acid sequencesofthe two TMEM88 isoforms (17-kDa TMEM88CRA-a;25-kDa TMEM88 CRA-b).The 17-kDa isoform contains the VWV motif in its C-terminus, which could interact with DVL,and the 25-kDa isoform lacks this motif.

(D)The normalized level of the 25-kDa isoform in lung cancer revealed no significant changes relative to paired normal tissues.

(E)TMEM88protein expressionin breast cancer (b, 200×), colon carcinoma (d, 200×), gastric carcinoma (f, 200×), and hepatocellular carcinoma (h, 200×) issignificantly higher than in the paired normal tissues (a, c, e, and g, 200×; p=0.004, p=0.007, p=0.001, and p=0.003, respectively). Cytoplasmic staining is observedin all of the neoplastic sections, suggesting primarily cytosolic localization of the protein.

(F) TMEM88 is localized tothe cytoplasm in eight NSCLC cell lines and a normal bronchial epithelial cell line (HBE), whereasit is primarily localized on the cell membrane only in the LK2 cell line, 600× magnification.

Supplementary FigureS2

Supplementary FigureS2.Effect of TMEM88 on canonical Wnt signaling and expression of its downstream target genes

(A)Western blot results showed two isoforms ofendogenous TMEM88 (25 kDa and 17 kDa)in A549 cells using an anti-TMEM88 antibody. After transfecting myc-tagged TMEM88 (TMEM88 CRA-a), we didnot find the two endogenous bands detected by the Myc-tag antibody but observedtwo bands above 17 kDa.We proposed that the band close to 25 kDamay result from targeting by two tags(Myc and DKK), while the other bands may be caused by post-translational modification of the protein. After transfection of TMEM88-∆C, both bands were lower,likely due to sequence truncation.

In cells expressingcytosolic TMEM88, the canonical Wnt signaling activity (B and E) and the protein (C and F) and mRNA (D and G) expression of the target genes showedno significant changes upon transfection ofTMEM88 or TMEM88-ΔC in A549 cells or siRNA knockdown ofTMEM88 in SPC cellswith or without Wnt3a treatment.

Supplementary FigureS3

Supplementary FigureS3.Co-localization of andinteraction between TMEM88 and DVLSin different cellular compartments

TMEM88 is co-localized with DVLS on the membrane of LK2 cells (A) or in the cytoplasm of H1299 cells (B) when TMEM88 is overexpressed in these cells, following the intrinsic subcellular distribution of the protein in each cell line. In contrast, the co-localization of the two proteins is abolished forTMEM88-ΔC,600× magnification.

(C)Co-immunoprecipitationwith DVLS antibodies in H1299 cells shows that myc-TMEM88 may interact with all three isoforms of DVLS, with a weaker interaction betweenTMEM88 andDVL-2.

(D) Co-immunoprecipitation assays performed in SPC cells show that 17-kDaTMEM88 interacts with DVLS.The 25-kDaisoformof TMEM88 shows nointeraction with DVLS.

(E)Immunofluorescence assays performed in SPC cells show that TMEM88 co-localized with DVLS in the cytoplasm,600×magnification.

Supplementary FigureS4

Supplementary FigureS4.Effects of TMEM88 on proliferation, migration, and invasion of NSCLC cells with cytosolic TMEM88

TMEM88 overexpression orsiRNA knockdown in A549 orH1299 andSPC cells, respectively,shows no significant changes in proliferation (A–C) orcolonyformation(D–F). However, migration (G–I) and invasion (J–L) are significantly enhanced withoverexpression in A549 cells and decreased with siRNAknockdown in H1299 and SPC cells.*p<0.05, **p<0.01, ***p<0.001.

Supplementary FigureS5

Supplementary FigureS5.Effects of TMEM88 on proliferation and invasion of non-small cell lung cancer cells

In cell lines expressingcytosolic TMEM88,the average tumor volume (A and B,G and H) and weight (C and I) for nude mice subcutaneously injected with A549-TMEM88(+) or SPC-TMEM88(-) revealed no significant differences comparedwith the control.Mice injected with A549-TMEM88(+)viathe tail vein developed more metastases than the control group (D–F,40×, arrows indicate lung metastatic nodules), and mice injected with SPC-TMEM88(-)developedfewerlung metastases than the control group(J–L, 40×, arrows indicate small metastases).*p<0.05

Supplementary FigureS6

Supplementary FigureS6.Effects of cytosolic TMEM88 on expression of epithelial-mesenchymal transition (EMT) proteins

(A–B) TMEM88 overexpression in A549 cells down-regulates Snail protein and up-regulates occludin and ZO-1 protein (A) and mRNA expressions (B, p=0.009and p=0.045).SiRNA knockdown of TMEM88 in SPC cells decreases Snailexpression and increases occludin and ZO-1protein (A) and mRNA expressions (B, p=0.043 and p=0.036).The mRNA expression of Snail was not altered by manipulation of TMEM88 expression in these two cell lines despite thechanges in protein level. Furthermore, there is no significant effect on the expressions of Claudin-1, E-cadherin, N-cadherin, and Vimentin(C–D)or on the cytomorphologyofthe two cell lines (E).*p<0.05, **p<0.01.

Supplementary FigureS7

Supplementary FigureS7.Effect of TMEM88 andDVLSco-transfection on expression of Snail and invasion in NSCLC cells

(A)Co-transfection of TMEM88 and DVLS in A549 and H1299 cells synergistically up-regulates Snail,synergistically down-regulatesoccludin and ZO-1, and synergistically enhances invasion (B and C;A549 cells: p = 0.002invasive cells in TMEM88+DVL-1 co-transfection versus empty plasmid, p = 0.002 invasive cells inTMEM88+DVL-3co-transfection versus empty plasmid, p = 0.006 invasive cells inTMEM88+DVL-1 co-transfection versus TMEM88 alone, p=0.007invasive cells inTMEM88+DVL-3co-transfection versus TMEM88 alone; H1299 cells: p = 0.002 invasive cells in TMEM88+DVL-1co-transfection versus empty plasmid, p = 0.001invasive cells in TMEM88+DVL-3co-transfection versus empty plasmid, p = 0.01 invasive cells in TMEM88+DVL-1 co-transfection versus TMEM88 alone, p = 0.006 invasive cells in TMEM88+DVL-3 co-transfection versus TMEM88 alone).The expressions of occludin and ZO-1 are not decreased by co-transfection when SnailsiRNA is present (D).*p<0.05, **p<0.01.

Supplementary FigureS8

Supplementary FigureS8.Effects of co-transfecting TMEM88 and DVLS on proteins involved in Snail stabilization

(A) The expressions of p-P38, p-GSK3β (Thr390), and p-ATF2 are enhanced when TMEM88 and DVLS are co-transfected in both H1299 (left column) and A549 (rightcolumn) cells, while those of p-ERK, p-JNK, p-AKT, p-GSKβ (Ser9), and p-NF-κB (Ser536) showno significant changes(B).

(C)Snail is down-regulated,occludin and ZO-1 are up-regulated after co-transfection of TMEM88,DVLS, and GSK3β.

(D) The expressions of p-P38 and p-GSK3β (Thr390) are significantly down-regulated after transfecting TMEM88-ΔC alone or co-transfecting TMEM88-ΔC and DVLS in H1299 cells compared with TMEM88 alone or co-transfection of TMEM88 and DVLS.

(E and F)The cytosolic and nuclear protein (E) and mRNA (F) levels of NF-κBshowno significant changes uponco-transfection of TMEM88 and DVLS. In contrast, nuclear Snail protein significantly increased despite althoughits transcript abundance showed no significant change after co-transfecting TMEM88 and DVLS. *p<0.05,**p<0.01.

(G)Immunohistochemistry assays show that expressions of TMEM88, p-P38, and Snail are significantly correlatedin individual cases among 50 NSCLC specimens, 200×.

Supplementary FigureS9

Supplementary FigureS9.Overexpression of TMEM88 did not influence expression of Snail,occludin,orZO-1 in cells with membrane-bound TMEM88

In LK2 cells, transfection of TMEM88 and TMEM88-ΔC showed no obvious effect on the expression of Snail,occludin, and ZO-1 or on thephosphorylation of P38 and GSK3β.