Supplementary Figures 1 - 8, Tables 1 - 3 from Conserved Oncogenic Behavior of the FAM83 Family Regulates MAPK Signaling in Human Cancer
Authors: Cipriano Rocky , Miskimen Kristy L.S. , Bryson Benjamin L. , Foy Chase R. , Bartel Courtney A. , Jackson Mark W. 
Published: April 2023

<p>PDF file - 1239KB, S1. Expression of exogenous FAM83B relative to endogenous FAM83B. Western blot of control GFP-expressing HME1 cells and FAM83B-expressing HME1 cells using a FLAG antibody or a FAM83B antibody. S2. Expression of FAM83A, FAM83B and FAM83D in lung cancer subtypes. The samples included normal lung (Norm - 38 samples), squamous cell carcinoma (SCC - 30 samples), Adenocarcinoma (Ad - 33 samples), Adenosquamous carcinoma (AdS - 4 samples), non-small cell carcinoma (NSC - 9 samples), large cell carcinoma (LC - 12 samples), small cell carcinoma (SC - 3 samples). The significant p-values defined as follows, p<0.005 (*) and p<0.05 (#). S3. FAM83 member expression in normal tissues. Real-time PCR analysis was performed for FAM83 members A-E. The relative expression of each FAM83 member is presented from all normal, associated tissues from an Origene TissueScan Cancer Survey Panel. The expression of each FAM83 member in each tissue was normalized to its expression in the adrenal gland, which was set equal to 1. S4. FAM83 member expression in normal tissues. The expression of FAM83A-E were examined in 353 various normal tissues were analyzed on Affymetrix U133 Plus 2.0 microarrays in a study by Roth et al. Neurogenetics. 2006 May,7(2):67-80. S5. FAM83A or FAM83D knock-down impairs MCF7 cell growth. MCF7 cells were infected with lentiviruses encoding a shRNA targeting GFP, FAM83A, or FAM83D. Western and Real Time PCR analysis confirmed the suppression of FAM83A protein (A) and FAM83D mRNA (B). MCF7 cells expressing shRNAs were plated, grown for 8 days, and the cell number was determined (C). S6. Specificity of FAM83 shRNAs. MDA468 cells were infected with lentiviruses expressing shRNAs targeting GFP (shGFP), FAM83A (sh83A2), FAM83B (sh83B2), or FAM83D (sh83D2). Real-time PCR was performed to analyze FAM83A, FAM83B, or FAM83D mRNA levels in the infected cells to confirm the specificity of each shRNA construct. S7. Analysis of RAS activation in FAM83-expressing cells. RAS activation assay was performed according to the procedures recommended by Upstate. Lysates from cells expressing Vector, FAM83A, FAM83B, or FAM83D were precipitated using a glutathione S-transferase (GST) fusion protein containing the RAS-binding domain (RBD) of CRAF (GST-CRAF-RBD) bound to agarose beads, and the bead-associated proteins were analyzed by SDS page electrophoresis followed by immunoblotting using anti-RAS and anti-GST antibodies. S8. Multiple FAM83 members are coordinately overexpressed in cancer. Heatmaps showing relative expression of FAM83A, FAM83B, FAM83C, or FAM83D in the panel of cancer specimens presented in Figure 3. RAN-BP3 and EGFR were examined in Breast and Lung specimens, respectively. The sample sizes for each tissue type are as follows: Ovarian: N=13, C=76. Breast: N=16, C=191. Lung: N=38, C=98. Bladder: N=3, C=18. Table S1. FAM83 member identification by mass spectrometry. Table S2. Relative expression of FAM83 members in HME1 cells. Table S3. Summary of FAM83 member expression in tumor microarray datasets.</p>