Supplementary Figures from Transmembrane and Coiled-Coil Domain 1 Impairs the AKT Signaling Pathway in Urinary Bladder Urothelial Carcinoma: A Characterization of a Tumor Suppressor
Authors: Li Chien-Feng , Wu Wen-Ren , Chan Ti-Chun , Wang Yu-Hui , Chen Lih-Ren , Wu Wen-Jeng , Yeh Bi-Wen , Liang Shih-Shin , Shiue Yow-Ling 
Published: April 2023

<p>Figure S1. High-resolution oligonucleotide-based array comparative genomic hybridization shows frequent DNA copy number gain at loci spanning TMCO1 gene at 1q24.1 (9/40, 22.5%; including 1 exhibited high-level gain). The lack of deletion involving TMCO1 gene locus suggested genome deletion is less likely the inactivation mechanism of TMCO1. Figure S2. Alteration the TMCO1 levels changes the ratios of pCDKN1A(T145) (inactive form)/CDKN1A and CDN1B(T157) (inactive)/CDKN1B but TP53 mRNA levels were changed inconsistently. Overexpression of the TMCO1 gene in BFTC905 cells (A) notably downregulated phospho/inactive CDKN1A and CDKN1B to ~25%, yet was not able to induce TP53 mRNA levels (B). Co-transfection of pTMCO1-HaloTag and a constitutive active AKT1 plasmid, pHRIG-AKT1, recovered phospho/inactive CDKN1A and CDKN1B to ~66% and 49%, respectively (A). Knockdown of the TMCO1 gene with 2 distinct shRNAi clones (C) downregulated phospho/inactive CDKN1A and CDKN1B to ~70 to 92%, however, upregulated TP53 mRNA levels. (D) Overexpression pTMCO1-HaloTag carrying wild-type (WT) or another 3 mutants T33A, S60A and S84A, were not able to alter the TP53 mRNA levels. Figure S3. Stable knockdown of the TMCO1 gene with two distinct shRNAi clones in J82 cells downregulated TMCO1 mRNA and protein levels (A); decreased and increased cells in G1 and S phases, respectively (B); enhanced cell viability (C), cell proliferation (D), colony formation/anchorage-independent cell growth (E, F). Protein levels of RB1, TP53, pTP53(S15), CDKN1A, CDKN1B, CDK4 were downregulated; CCND1, CCNE1, CDK2, pAKT1(S473), MDM2, pMDM2(S166) protein levels and pCDKN1A(T145)/CDKN1A and pCDKN1B(T157)/CDKN1B ratios were notably upregulated (G). All experiments were performed in triplicate and results are expressed as the mean ï,± SEM. For immunoblotting analysis, on representative image is shown. Pan-actin served as a loading control. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001. Figure S4. Immunoblotting analyses on a series of cyclin-dependent kinase inhibitors after stable TMCO1 overexpression and knockdown showed that CDKN1C, CDKN2B, CDKN2A, CDKN2C and CDKN2D protein levels were not able to be induced (A) and suppressed (B) in TSGH8301 and RT4 cells, respectively. All experiments were conducted in triplicated and one representative immunoblotting image is shown. GAPDH served as a loading control. Figure S5. Wound healing, transwell migration and transwell invasion assays demonstrated that knockdown of the TMCO1 gene in RT4 cells enhanced cell migration (A, B) and invasion (B). Figure S6. BFTC905 cells (2 ï,' 105) were seeded overnight, starved (0.5% FBS) for 2 h and treated with a pan-PHLPP inhibitor, NSC117079 for 35 min with different concentrations. Immunoblot analysis identified 15 ï�­M of NSC117079 profoundly upregulated pAKT1(S473) protein levels. Figure S7. Immunocytochemistry (ICC) shows that TMCO1 and PHLPP1 proteins were only partially colocalized in BFTC905 and RT4 cells. Blue: DAPI (nuclear); red: TMCO1 (cell membrane & cytoplasma), green: PHLPP1 (membrane). One representative ICC image is shown for each cell line. Figure S8. Tumor growth curve and relative volumes of SCID/NOD mice bearing BFTC905 cell xenografts. Tumor growth curve of mice stably carrying pHaloTag (control), wild-type TMCO1 [pTMCO1(WT)-HaloTag], and another three mutants: pTMCO1(T33A)-HaloTag, pTMCO1(S60A)-HaloTag and pTMCO1(S84A) plasmids. Figure S9. In vitro and in vivo studies show that TMCO1 is a tumor suppressor in HTB-33 epithelial cells. Stable overexpression of the TMCO1 gene in HTB-33 cells (cervix, derived from metastatic site, omentum) (A) induced the expression of TMCO1-DDK (Flag) protein, (B) induce G1 cell cycle arrest, reduced cells in the S phase; suppressed cell viability (C), cell proliferation (D), colony formation (E), anchorage-independent cell growth (F); altered the expression levels of several cell cycle regulators, downregulated phospho/inactive CDKN1A and CDKN1B ratios (G), downregulated pAKT1(S473), MDM2 and pMDM2(S166) protein level (H), upregulated nuclear CDKN1A, both nuclear and cytosolic CDKN1B (I), yet did not change the TP53 mRNA level (J), enhanced cell migration and invasion (K, L), accompanied with CD44 and VIM downregulation (M). Exogenous TMCO1 expression was not able to upregulate PHLPP1 and PHLPP2 protein levels (N). However, one pan-PHLPP inhibitor, NSC117079, restored TMCO1-suppressed pAKT1(S473) level (O). Immunocytochemistry further indicated that TMCO1 and PHLPP2 are colocalized in cytoplasm and cell membrane (P). Co-transfection of the pTMCO1-HaloTag and pcDNA3-HA-PHLPP2 plasmids, notably downregulated pAKT1(S473) (R) and AKT kinase activity (S) compared to transfection with the pTMCO1-HaloTag or the pcDNA3-HA-PHLPP2 plasmid alone. TMCO1-overexpressed HTB-33 cells along with SCID/NOD mice models show that TMCO1 suppresses tumor growth in vivo (T, U). Mice were sacrificed on day 28, control xenografts (cells carrying pHaloTag) displayed a carcinoma with high cellularity, while the TMCO1-overexpressing group (pTMCO1-HaloTag) showed large areas of necrosis and stromal hyalinization and a much low percentage of cancer components.</p>