MOESM1 of Contextual tumor suppressor function of T cell death-associated gene 8 (TDAG8) in hematological malignancies

Additional file 1: Figure S1. Restoration of TDAG8 gene expression results in increased phosphorylation of CREB at serine 133 in U937 and Ramos cells. (A) Restoration of TDAG8 gene expression in U937 cells results in stimulation of CREB phosphorylation at serine 133 indicating the TDAG8 activity level is increased. (B) TDAG8 gene expression is restored in Ramos cells to a level that is physiologically relevant. (C) Restoration of TDAG8 gene expression in Ramos cells results in stimulation of CREB phosphorylation at serine 133 indicating the TDAG8 activity level is increased. **P < 0.01. Figure S2. TDAG8 gene expression restoration reduces c-myc oncogene expression in U937 cells. (A, B) Restoration of TDAG8 gene expression reduces c-myc oncogene expression at the mRNA level in U937 cells. (C) Over 14 days U937/TDAG8 GFP expression is reduced at physiological pH 7.4 while U937/Vector GFP is stable. (D) Reduction of U937/TDAG8 GFP expression is further augmented by activation of TDAG8 with acidic pH 6.9 treatment while the U937/Vector GFP is stable. ns: P > 0.05, *P < 0.05, ***P < 0.001. Figure S3. Restoration of TDAG8 gene expression in RPMI 8226 myeloma cells inhibits cell proliferation. (A) The empty vector does not substantially affect RPMI 8226 cell proliferation at physiological pH 7.4 in comparison to the RPMI 8226 parental cells. (B) Restoration of TDAG8 gene expression significantly reduces RPMI 8226 cell proliferation at physiological pH 7.4 in comparison to the RPMI 8226 parental cells. (C) The empty vector does not substantially affect RPMI 8226 cell proliferation at acidic pH 6.9 in comparison to the RPMI 8226 parental cells. (D) Restoration of TDAG8 gene expression significantly reduces RPMI 8226 cell growth at acidic pH 6.9 in comparison to the RPMI 8226 parental cells. ***P < 0.001. Figure S4. Restoration of TDAG8 gene expression increases apoptosis signaling. (A, B) Restoration of TDAG8 gene expression stimulates cleaved caspase 3 in U937 cells at physiological pH 7.4 and acidic pH 6.4. (A and C) Restoration of TDAG8 gene expression increases cleaved caspase 9 in U937 cells at physiological pH 7.4. (A and D) Restoration of TDAG8 gene expression increases cleaved PARP in U937 cells at acidic pH 6.4. ns: P > 0.05, *P < 0.05. Figure S5. Restoration of TDAG8 gene expression in Ramos lymphoma cells reduces primary tumor growth in SCID mice. (A) Representative image of a mouse subcutaneously injected with Ramos/Vector (left flank) and Ramos/TDAG8 (right flank) cells. (B) Restoration of TDAG8 in Ramos cells significantly delays primary tumor growth in SCID mice starting day 9 after injection. (C) Restoring TDAG8 gene expression in Ramos cells moderately reduces overall tumor mass after necropsy on day 21. (D) Representative image of Ramos/Vector and Ramos/TDAG8 tumors excised from SCID mice. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Figure S6. TDAG8 stimulates apoptotic signaling through Gα13/Rho signaling. (A, B) Inhibition of Gα13 signaling in U937/TDAG8 cells reduces cleaved caspase 3 and activation of Rho in U937/TDAG8 cells stimulates cleaved caspase 3. (A and C) Inhibition of Gα13 signaling in U937/TDAG8 cells reduces cleaved caspase 9 and activation of Rho in U937/TDAG8 cells stimulates cleaved caspase 9. (A and D) Inhibition of Gα13 signaling in U937/TDAG8 cells reduces cleaved PARP and activation of Rho in U937/TDAG8 cells stimulates cleaved PARP. ns: P > 0.05, *P < 0.05. Figure S7. Restoration of TDAG8 gene expression in U937 cells reduces attachment to a HUVEC monolayer and reduces migration toward a chemoattractant. (A) Restoration of TDAG8 gene expression reduces overall U937 cell attachment to a HUVEC monolayer while extracellular acidosis increases cell attachment. (B) Restoration of TDAG8 gene expression significantly reduces U937 cell migration toward a chemoattractant (SDF-1α) while extracellular acidosis reduces overall U937 cell migration. (C) In vivo imaging of a SCID mouse injected with U937/Vector-Luc cells that presented with hind limb paralysis and metastasis throughout the body. *P < 0.05, **P < 0.01, ***P < 0.001.