Jiang Et Al, TMEM43/Lumais a Keysignaling Component Mediating EGFR-Induced NF- B Activation

Jiang et al,TMEM43/LUMAis a keysignaling component mediating EGFR-induced NF-B activation and tumor progression

Supplemental Figures

Figure S1. Tissue distribution of TMEM43 in mouse. Mouse tissues or organs were dissected from8-week-old B6 mice. The tissues or organs were homogenized and lysed and the lysates were subjected to immunoblots with indicated antibodies.

Figure S2. TMEM43 is a critical signaling component in mediating NF-B activation. (A)Constructs of CARMA3 WT and deletion mutants were shown and their ability to bind to TMEM43 was also shown in the right. (B)Constructs of TMEM43WT and deletion mutants were shown and their ability to bind to CARMA3 was also shown in the right. (C and D) HEK293T cells were transfected with the constructs as indicated and co-immunoprecipitation assay was performed. Results from immunoblotting were shown. (E) HEK293T cells transduced with shTMEM43 or shGFP were stimulated with vehicle, PMA plus ionomycin, or TNFα for the indicated time. Cell lysates were prepared and subjected to immunoblots with indicated antibodies. (F and G) TMEM43(F) and CARMA3 (G) mRNA levels were measured by qPCR in HEK293T cells transduced with shGFP, shTMEM43 (F) or shCARMA3 (G).

Figure S3. TMEM43 is required for EGFR/HER2-induced NF-B activation. A431 (A), MDA468 (B) or SkBr3 (C) cells transduced with shGFP and shTMEM43 were stimulated with EGF (A and B) or HRG (C) for 60 minutes or TNFα (A-C) for 30 minutes. Nuclear extracts were isolated and subjected to EMSA using 32P-labelled NF-B probe. Oct-1 serves as a loading control.

Figure S4. TMEM43 deficiency significantly affects the characteristics of cancer cells in SkBr3 cells in vitro. (A and B) SkBr3 cells transduced with shGFP, shTMEM43 and shCARMA3 were seeded in 96 wells and cultured with (right panel) or without (left panel) 5ng/ml of HRG for the indicated time. The cell proliferation was measured using MTT assay. The mean OD values were plotted. (B-C) SkBr3 cells transduced with shGFP, shTMEM43 and shCARMA3 were subjected to transwell migration (B) and invasion assay (C) using 50 ng/ml of HRG (left panel) or 10% FBS (right panel) as chemoattractant for 16 hours (C) or 48 hours (D). The cells were fixed, stained and imaged using inverted microscope. The representative images were shown. (D-E) SkBr3 cells transduced with shGFP, shTMEM43 and shCARMA3 were subjected to colony formation assay in soft agar with (right panel) or without (left panel) 5ng/ml of HRG for 2 weeks. The numbers of colonies with size bigger than 40 µm were plotted (D) and representative images were shown (E).

Figure S5. TMEM43 deficiency significantly affects tumor progression in vivo. (A-C) A431 cells transduced with shGFP or shTMEM43 were injected subcutaneously into nude mice and the tumor growth was monitored twice a week. The tumor volume were calculated and plotted (A). At the end point, the tumors were dissected. RNA was isolated from aliquots of tumor and TMEM43 mRNA were measured by quantitative PCR and plotted (B). Aliquots of tumor were homogenized and lysed and the lysates were subjected to immnoblotting using the indicated antibodies (C). (D)TMEM43-V5 and endogenous expression in vector or TMEM43-V5 overexpressing A431 cells was shown in western blotting.

Figure S6. Clinical relevance of TMEM43 in brain tumor malignancy.The Kaplan-Meier plot represents the overall disease-free survival rate in patients with LGG (left) or patients with GBM (right) according to TMEM43 expression status after surgery removal of primary tumor. High and lowTMEM43 mRNA levels were the top 25% (112 LGG and 27 GBM) and the bottom 75% (368 LGG and 88 GBM), respectively.The long-rank test P values were generated using the long-rank test.

Figure S7. TMEM43 deficiency significantly affects the characteristics of brain tumor cells in LN229 cells in vitro. (A and B)TMEM43expression was detected by western blots in U87 cells (A) or LN229 cells (B) as indictated. (C and D) LN229 cells transduced with shCtl, shTMEM43_1 or shTMEM43_2 were seeded in 96 wells and cultured with (D) or without (C) 10ng/ml of EGF for the indicated time (days). The cell proliferation was measured using MTT assay. The mean OD values were plotted. (E and F) LN229 cells transduced with shCtl, shTMEM43_1 or shTMEM43_2 were subjected to colony formation assay in soft agar with (F) or without (E) 10ng/ml of EGF for 4 weeks. The numbers of colonies with size bigger than 40 µm were plotted. (G and H) LN229 cells transduced with shCtl, shTMEM43_1 or shTMEM43_2 were subjected to transwell migration (G) and invasion assay (H) using EGF as chemoattractant for 16 hours (G) or 48 hours (H). The cells were fixed, stained and imaged using inverted microscope. (I) CARMA3 mRNA levels were measured by qPCR in U87 cells transduced with shGFP or shCARMA3. (J) U87 cells transduced with shCtl and shCARMA3 were subjected to transwell migration (left panel) and invasion assay (right panel) using EGF as chemoattractant for 16 hours (Left panel) or 48 hours (right panel). The cells were fixed, stained and imaged using inverted microscope.Representative images were shown. (K and L) U87 cells transduced with shCtl or shCARMA3 were seeded in 96 wells and cultured with (L) or without (K) 10ng/ml of EGF for the indicated time (days). The cell proliferation was measured using MTT assay. The mean OD values were plotted. (M and N) U87 cells transduced with shCtl and shCARMA3 were subjected to colony formation assay in soft agar with (N) or without (M) 10ng/ml of EGF for 4 weeks. The numbers of colonies with size bigger than 40 µm were plotted.