Additional file 1 of Organelle resolved proteomics uncovers PLA2R1 as a novel cell surface marker required for chordoma growth
Creators: name: Khan, Shahbaz
name: Zuccato, Jeffrey A.
name: Ignatchenko, Vladimir
name: Singh, Olivia
name: Govindarajan, Meinusha
name: Waas, Matthew
name: Mejia-Guerrero, Salvador
name: Gao, Andrew
name: Zadeh, Gelareh
name: Kislinger, Thomas
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1. Fig. S1: Protein detection and subtractive proteomics. a) Bar plot showing the number of proteins detected in each organelle fraction with the dots on the bar representing number of replicates. b) Principal component analysis (PCA) showing proteomic separation among the organelle fractions. c-f) Bar plot showing the number of proteins detected in the four clusters (through hierarchal clustering) corresponding to organelle selective proteins from different databases. Fig. S2: Differential expression analysis between organelle fractions. a) Venn diagram showing overlap between the plasma membrane fraction with all the other organelle fractions in the four chordoma cell lines. Proteins significantly enriched in plasma membrane fractions with a Geniescore of &gt;20 and SPC score of 3 and the following cut-off values (FDR 1) are indicated in red. b) Upset plot showing the overlap of differentially expressed proteins with Genie score and SPC cut-off in the four chordoma cell lines. Fig. S3: Expression of PLA2R1 in HPA and publicly available proteomics and RNA-seq datasets. a) IHC staining of PLA2R1 in normal tissue as reported by HPA. 1 = Low expression; 2 = medium expression; 3 = high expression. b) Bar plot shows log2 iBAQ intensities of PLA2R1 in different normal tissues in the Wang et al. [44] proteomics dataset. c) Boxplots show log2 normalised protein abundance of PLA2R1 in the GTEx proteomics dataset, with bar plot showing number of samples the protein was detected in. D) Boxplots show log2 TPM+1 of PLA2R1 in GTEx transcriptomics dataset, with bar plot showing number of samples the protein was detected in different normal tissues. Fig. S4: Validation of PLA2R1. a) Immunoblots showing expression of PLA2R1 in an additional chordoma cell line, UM-Chor1 and in a negative control ovarian cancer cell line, Ovcar8. b) Representative IHC image showing expression of PLA2R1 localized to cell membrane. c) Expression of PLA2R1 in U-CH17M, U-CH17S and UM-Chor1 cell line after 24 and 48 hours of siRNA knockdown. d) Digital images of colony formation assay after siRNA KD of PLA2R1. Fig. S5: a) Boxplot with jitters showing almarBlue fluorometric intensity of the spheroids (p-values calculated with t-test, between sg1 and sg2 compared against LacZ). b) Immunoblots showing expression of total PARP and caspases in PLA2R1 KD cells and controls. c) Immunoblots showing that cleaved PARP, Caspase 3 and Caspase 7 expression are not observed after PLA2R1 KD. d) Immunoblots for total and phosphorylated AKT1 and its downstream substrate. The immunoblots show no change in phosphorylation of AKT and its downstream substrates. Fig. S6: Effect of PLA2R1 knockdown on cell proliferation. a) Plot showing cell proliferation by cell counting for a period of 16 days. The PLA2R1 KD cells show a slow down in growth compared to controls. b) Stacked barplot from EdU cell proliferation assay (n = 3) showing percentage (mean values from three experiments) of cells in different phases of cell cycle G0-G1 phase, S-phase and G2-M phase of PLA2R1 KD cells (sg1 and sg2) and controls (NT and LacZ). There is a higher percentage of cells in G0-G1 phase in PLA2R1 KD cells compared to control (LacZ) (p- values calculated with t-test comparison between sg1 and sg2 compared against LacZ). Lower percentage of cells in S-phase of cell cycle (p-values calculated with t-test comparison  between sg1 and sg2 compared against LacZ). c–f) Representative images of EdU flow cytometry experiment of PLA2R1 KD cells (sg1 and sg2) and control cells (NT and LacZ).