Fig 1: Forced SPARC enhances the expression or activation of pro-survival and pro-death proteins but promotes death signaling in temozolomide (TMZ), whereas HSP27 inhibition promotes apoptotic signaling (SPARC-independent) and autophagic signaling (SPARC-dependent), but suppresses autophagic signaling in the presence of TMZ and SPARC. A-D. Representative Western blots are illustrated for n = 3 experiments of C1.1 control GFP- or H2 SPARC-GFP-expressing cells treated with control siRNA (Csi) or HSP27 siRNA (HSP27si) for 72 hr. Equal numbers of siRNA-treated cells were plated overnight in growth serum, and then treated with 0 (0.1% DMSO control), 40, 80 or 100 µM TMZ for 2 days before lysing. Arrows indicate = 2-fold increases or decreases due to ± SPARC expression and/or ± HSP27 siRNA treatment. Arrow with brackets indicates 30% reduction. # - Indicates a = 2-fold increase in the ratio of LC-3II/LC-3I. Asterisks indicate = 2-fold increases or decreases due to TMZ treatment.
Fig 2: SPARC does not enhance or suppress radiation therapy (RT). A. Representative fluorescent images of control C1.1 GFP- and H2 SPARC-GFP-expressing clonogenic colonies (×40 magnification). B. Average colony forming efficiency ± SD of untreated C1.1 and H2 cells (left panel). Average surviving fraction ± SD of C1.1 and H2 cells exposed to 0 or 1-5 Gy (middle panel) or 0, 5 or 10 Gy (right panel), plating 3000 cells/60-mm dish. Plating 1000 cells/60-mm dish gave similar results. C. Average colony forming efficiency ± SD of untreated LN443 cells transfected with control (Csi) or SPARC siRNA (SPARCsi) (left panel). Average surviving fraction ± SD of LN443 cells transfected with Csi or SPARCsi exposed to 0, 5 or 10 Gy (right panel) plating 750 cells/60-mm dish. Plating 1500 cells/60-mm dish gave similar results.
Fig 3: Inhibition of pAKT eliminates SPARC-induced survival in temozolomide (TMZ). A. Representative Western blots of C1.1 control GFP- or H2 SPARC-GFP-expressing cells in the absence 0 (0.1% DMSO control) or presence of 100 µM TMZ ± AKT inhibitor IV at indicated concentrations for 48 hr before lysing. Arrows indicate = 2-fold increases or decreases due to ± TMZ treatment and asterisks indicate = 2-fold increases or decreases due to AKT inhibitor IV treatment. B. Means for surviving fraction ± SD of C1.1 or H2 cells treated ± AKT IV inhibitor ± TMZ plating 1500 cells/60-mm dish are presented. For C1.1 * p = 0.0022, **, *** p = 0.0001. For H2 *,**,*** p = 0.0001. C. Means for surviving fraction ± SD of C1.1 or H2 cells treated ± AKT IV inhibitor ± TMZ plating 1500 cells/60-mm dish are presented. * p = 0.014, ** p = 0.0006, *** p = 0.0001, **** p = 0.0003, ***** p = 0.03 except H2 0.25 AKT inhibitor IV (40 vs. 80 = µM TMZ) is not significant, ****** p = 0.008. NS - not significant. Plating 3000 cells gave similar results.
Fig 4: Forced SPARC expression protects against temozolomide (TMZ) and suppresses the ability of HSP27 inhibition to sensitize cells to lower concentrations of TMZ. A. Average colony forming efficiency ± SD of C1.1 and H2 plating 750 cells/60-mm dish. B. Average surviving fraction ± SD of C1.1 GFP- and H2 SPARC-GFP-expressing cells in 0 (0.1% DMSO), 1, 10, or 100 µM TMZ plating 750 cells/60-mm dish. TMZ (100 µM) suppressed survival of both clones relative to 0 drug; p = 0.0048. However, SPARC-expressing cells survived better than control cells in 100 µM TMZ; * p = 0.0022. Plating 375 and 1500 cells gave similar results. C. Average colony forming efficiency ± SD of C1.1 and H2 cells transfected with control (Csi) or HSP27 siRNA (HSP27si), plating 750 cells/60-mm dish. *** p = 0.0001. D. Average surviving fraction ± SD of C1.1 and H2 cells transfected with Csi or HSP27si in the absence (0) or presence of increasing concentrations of TMZ, plating 750 cells/60-mm dish. * H2 + Csi vs. H2 + HSP27si: 100 µM TMZ; p = 0.0020. ** C1.1 + Csi vs. C1.1 + HSP27si: 20 µM TMZ; p = 0.022, 40 µM TMZ; p = 0.0026, 60 µM and 80 µM TMZ; p = 0.0001. *** H2 + Csi vs. C1.1 + Csi: 20 µM TMZ; p = 0.015, 40-100 µM TMZ; p = 0.0001. **** H2 + HSP27si vs. C1.1 + HSP27si: 20-60 µM TMZ; p = 0.0055, 80 µM and 100 µM TMZ; p = 0.0002. B, C. Plating 1500 cells gave similar results.
Fig 5: Suppression of AKT1/2 or AKT3 decreases SPARC-induced pro-death signaling in temozolomide (TMZ); suppression of AKT1/2 increases colony forming efficiency, and has no effect on survival in TMZ. A, B. Representative Western blots of LN443 cells treated with control siRNA (Csi), AKT1/2 siRNA (AKT1/2si) or AKT 3 siRNA (AKT3si) for 72 hr. Equal numbers of siRNA-treated cells were plated overnight in growth serum, and then treated with 0 (0.1% DMSO control), or 40, 80 or 100 µM TMZ (TMZ) for 2 days before lysing. Arrows indicate = 2-fold increases or decreases due to HSP27 siRNA treatment. Arrows with brackets indicate 30% and 40% reduction of AKT1 and AKT2, respectively. # - Indicates a = 2-fold increase in the ratio of LC-3II/LC-3I. Asterisks indicate = 2-fold increases or decreases due to TMZ treatment. C. Means for colony forming efficiency ± SD for LN443 cells ± AKT1/2 or AKT3 siRNA plating 750 cells. *** p = 0.0003. D. Means for surviving fraction ± SD for LN443 cells ± AKT1/2 or AKT3 siRNA ± increasing concentrations of TMZ plating 750 cells/60-mm dish.
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