Additional file 1 of The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer
Creators: name: Thies, Katie A.
name: Cole, Matthew W.
name: Schafer, Rachel E.
name: Spehar, Jonathan M.
name: Richardson, Dillon S.
name: Steck, Sarah A.
name: Das, Manjusri
name: Lian, Arthur W.
name: Ray, Alo
name: Shakya, Reena
name: Knoblaugh, Sue E.
name: Timmers, Cynthia D.
name: Ostrowski, Michael C.
name: Chakravarti, Arnab
name: Sizemore, Gina M.
name: Sizemore, Steven T.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Space Science
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Biotechnology
subject: 69999 Biological Sciences not elsewhere classified
subject: 19999 Mathematical Sciences not elsewhere classified
subject: FOS: Mathematics
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Supplemental Figure S1. (A) Representative low magnification images of lungs harvested from mice bearing MDA-MB-231 Ctrl, RALA-KO, or RALB-KO tumors. Scale bar = 2000μm. (B and C) Representative images and quantification of lung nodules resulting from MDA-MB-231 shCtrl (n = 4) and shRALA (n = 4) orthotopic mammary tumors (B) or shCtrl (n = 10) and shRALB (n = 12) tumors (C). Tumor-bearing mice were sacrificed when the respective shCtrl group met ERC. (D) Representative images and H-score quantification of Ki67 immunostaining in lung tumor nodules from mice bearing MDA-MB-231 shCtrl or shRALA tumors. Error bars represent SEM; *, P &lt; 0.05. Supplemental Figure S2. (A) Western blots demonstrating RALA and RALB expression in MDA-MB-468 siRNA control (siCtrl), siRALA, and siRALB cells. (B) Representative images and quantification of MDA-MB-231 shCtrl, shRALA and shRALB cell migration after 6 h (scale bar = 200μm). (C) Representative images and quantification of MDA-MB-231 shCtrl, shRALA and shRALB cell invasion through matrigel coated transwell inserts after 24 h (scale bar = 200μm). (D) Western blots demonstrating RALA and RALB expression for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells ± siCtrl or siRALA/B transient knockdown. (E) Quantification of viability for MDA-MB-231 shCtrl, shRALA, and shRALB cells. (F) Quantification of viability for MVT1 shCtrl and shRALA cells over 72 h. (G) Quantification of BrdU incorporation for MDA-MB-231 shCtrl, shRALA, and shRALB cells over 72 h. (H) Quantification of BrdU incorporation for MVT1 shCtrl and siRALA cells over 72 h. (I) Quantification of Annexin V positivity for MDA-MB-231 shCtrl, shRALA, and shRALB cells. (J) Quantification of Annexin V positivity for MVT1 shCtrl and siRALA cells. (K) Quantification of growth in low adhesion (GILA) conditions for MDA-MB-231 shCtrl, shRALA and shRALB cells for 5 days. (L) Quantification of growth in low adhesion (GILA) conditions for MVT1 shCtrl and shRALA cells for 5 days. (M) Quantification of growth in soft agar for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells over 7 days (relative to controls). (N) Quantification of growth in soft agar for MDA-MB-231 shCtrl, shRALA and shRALB cells over 7 days (relative to shCtrl). Data are presented as mean ± SEM; (*), P &lt; 0.05. Supplemental Figure S3. (A) Normalized RALA and RALB expression in breast cancer cell line data from the Broad Cancer Cell Line Encyclopedia (CCLE). (B) Quantification of normalized RALA and RALB expression in TNBC cell lines classified as TNBC-A (more basal-like) or TNBC-B (more mesenchymal like). Data is from CCLE. (C) Western blots illustrating RALA and RALB expression in representative TNBC-A and TNBC-B lines. (D) Correlation of mRNA and protein expression levels for RALA (top) and RALB (bottom) using data from TCGA. Supplemental Figure S4. Analysis of the prognostic significance of RALA expression in the METABRIC cohort. Patient expression data was stratified at the indicated preselection cutoffs and the resulting Kaplan Meier curves were compared by log-rank. Corresponding Log-rank P-values are shown in each panel. RALA was prognostic of DSS at all preselection points. Supplemental Figure S5. Analysis of the prognostic significance of RALA expression in the TNBC population of the METABRIC cohort over a 5 year follow-up period. TNBC patient samples were dichotomized into high (upper 10th percentile) and low (lower 90th percentile). The resulting Kaplan Meier curves were compared statistically by log-rank. Supplemental Figure S6. Analysis of the prognostic significance of RALB expression in the METABRIC cohort. Patient expression data was stratified at the indicated preselection cutoffs and the resulting Kaplan Meier curves were compared by log-rank. Corresponding log-rank P-values are shown in each panel. RALB was prognostic of DSS when the population was stratified along the median RALB expression (lower left panel), or when the upper two-tertiles were compared to the remaining lowest tertile (bottom, middle panel). A trend toward significance remained when the upper three-quartiles were compared to the lowest quartile (bottom right panel). Supplemental Figure S7. Analysis of the prognostic significance of expression of RAL effectors in the METABRIC cohort. The METABRIC cohort was stratified along median expression for EXOC2 (A), EXOC8 (B), or RALBP1 (C) and the resulting Kaplan Meier curves were compared by log-rank. Supplemental Figure S8. (A and B) Analysis of the prognostic significance of expression of RALGAPA1 and RALGAPA2 in the METABRIC cohort. The METABRIC cohort was stratified along median expression for (A) RALGAPA1 or (B) RALGAPA2 and the resulting Kaplan Meier curves were compared by log-rank. (C and D) RALGAPA1 (C) or RALGAPA2 (D) expression in samples of normal breast, TNBC, or other BC subtypes were compared statistically. (E) The METABRIC cohort was stratified along median expression for RALGAPB and the resulting Kaplan Meier curve was compared by log-rank. (F) RALGAPB expression in the METABRIC dataset was compared statistically across normal breast, TNBC, or other BC subtypes. *; P &lt; 0.05 Supplemental Figure S9. (A - D) Analysis of the prognostic significance of expression of (A) RALGDS, (B) RGL1, (C) RGL2, and (D) RGL3 in the METABRIC cohort. The METABRIC cohort was stratified along median expression for each respective gene and the resulting Kaplan Meier curves were compared by log-rank. (E) RGL3 expression in the METABRIC dataset was compared statically across normal breast, TNBC, or other BC subtypes. *; P &lt; 0.05 Supplemental Figure S10. (A and B) Quantification of MDA-MB-468 cell viability upon treatment with varying doses of BQU57 under (A) normal adherent growth conditions for 72 h or (B) under low adhesion (GILA) conditions for 5 days. (C and D) Mouse body weight was monitored over the course of BQU57 treatment in mice bearing (C) MDA-MB-231 or (D) TNBC PDX tumors.