Figure 1. INPP4B overexpression has minimal effect on ER+ breast cancer sensitivity to 4-OHT or alpelisib in monolayer culture. (a–d) MCF-7 (a,b) or T47D (c,d) cells expressing GFP-vector or GFP-INPP4B were treated with 1–20 µM 4-OHT or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (a,c), or plotted as a 4-parameter logistical regression (b,d) ± SD (n = 3 experiments). (e–h) MCF-7 (e,f) or T47D (g,h) cells expressing GFP-vector or GFP-INPP4B were treated with 0.2–10 µM alpelisib or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (e,g), or plotted as a 4-parameter logistical regression (f,h) ± SD (n = 3 experiments). (i–l) MCF-7 (i,j) or T47D (k,l) cells expressing GFP-vector or GFP-INPP4B were treated with 10 µM 4-OHT and 0.1–5 µM alpelisib or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (i,k), or plotted as a 4-parameter logistical regression normalized to 4-OHT-treated cells (j,l) ± SD (n = 3 experiments). p values were determined by two-tailed unpaired t test of the area under the curve in (b,d,f,h,j,l).

Figure 1. INPP4B overexpression has minimal effect on ER+ breast cancer sensitivity to 4-OHT or alpelisib in monolayer culture. (a–d) MCF-7 (a,b) or T47D (c,d) cells expressing GFP-vector or GFP-INPP4B were treated with 1–20 µM 4-OHT or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (a,c), or plotted as a 4-parameter logistical regression (b,d) ± SD (n = 3 experiments). (e–h) MCF-7 (e,f) or T47D (g,h) cells expressing GFP-vector or GFP-INPP4B were treated with 0.2–10 µM alpelisib or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (e,g), or plotted as a 4-parameter logistical regression (f,h) ± SD (n = 3 experiments). (i–l) MCF-7 (i,j) or T47D (k,l) cells expressing GFP-vector or GFP-INPP4B were treated with 10 µM 4-OHT and 0.1–5 µM alpelisib or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (i,k), or plotted as a 4-parameter logistical regression normalized to 4-OHT-treated cells (j,l) ± SD (n = 3 experiments). p values were determined by two-tailed unpaired t test of the area under the curve in (b,d,f,h,j,l).

Figure 2. INPP4B-overexpressing MCF-7 cells are more sensitive to pyrvinium in monolayer culture. MCF-7 cells expressing GFP-vector or GFP-INPP4B were treated with 0.5–20 µM LGK-974 (a,b), 0.25–7.5 µM PRI-724 (c,d), 1–50 µM celecoxib (e,f), 10–500 nM pyrvinium (g,h) or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (a,c,e,g), or plotted as a 4-parameter logistical regression (b,d,f,h) ± SD (n = 3 experiments). p values were determined by two-tailed unpaired t test of the area under the curve in (b,d,f,g).

Figure 2. INPP4B-overexpressing MCF-7 cells are more sensitive to pyrvinium in monolayer culture. MCF-7 cells expressing GFP-vector or GFP-INPP4B were treated with 0.5–20 µM LGK-974 (a,b), 0.25–7.5 µM PRI-724 (c,d), 1–50 µM celecoxib (e,f), 10–500 nM pyrvinium (g,h) or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (a,c,e,g), or plotted as a 4-parameter logistical regression (b,d,f,h) ± SD (n = 3 experiments). p values were determined by two-tailed unpaired t test of the area under the curve in (b,d,f,g).

Figure 3. INPP4B-overexpressing MCF-7 cells are more sensitive to 4-OHT-pyrvinium combination treatment in monolayer culture. MCF-7 cells expressing GFP-vector or GFP-INPP4B were treated with 10 µM 4-OHT ± 0.5–20 µM LGK-974 (a,b), 0.25–7.5 µM PRI-724 (c,d), 1–50 µM celecoxib (e,f), 10–500 nM pyrvinium (g,h) or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (a,c,e,g), or plotted as a 4-parameter logistical regression normalized to 4-OHT-treated cells (b,d,f,h) ± SD (n = 3 experiments). p values were determined by two-tailed unpaired t test of the area under the curve in (b,d,f,g).

Figure 3. INPP4B-overexpressing MCF-7 cells are more sensitive to 4-OHT-pyrvinium combination treatment in monolayer culture. MCF-7 cells expressing GFP-vector or GFP-INPP4B were treated with 10 µM 4-OHT ± 0.5–20 µM LGK-974 (a,b), 0.25–7.5 µM PRI-724 (c,d), 1–50 µM celecoxib (e,f), 10–500 nM pyrvinium (g,h) or DMSO as a vehicle control for 48 h, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated cells (a,c,e,g), or plotted as a 4-parameter logistical regression normalized to 4-OHT-treated cells (b,d,f,h) ± SD (n = 3 experiments). p values were determined by two-tailed unpaired t test of the area under the curve in (b,d,f,g).

Figure 4. 4-OHT-pyrvinium treatment selectively enhances INPP4B-overexpressing ER+ breast cancer cell death. (a–d) MCF-7 (a,b) or T47D (c,d) cells expressing GFP-vector or GFP-INPP4B were treated with 50 nM pyrvinium and/or 10 µM 4-OHT or DMSO as a vehicle control for 48 h, then incubated with 1 µg/mL propidium iodide (PI) and 1 µg/mL Hoechst 33,342 for 30 min (a,c). Data represent the percentage of PI-positive cells ± SD (n = 3 experiments, >300 cells/experiment) (b,d). Scale bar is 100 µm in a, c. + indicates where treatment was added, and – indicates where no treatment was added. p values were determined by one-way ANOVA with Tukey post hoc test in (b,d).

Figure 4. 4-OHT-pyrvinium treatment selectively enhances INPP4B-overexpressing ER+ breast cancer cell death. (a–d) MCF-7 (a,b) or T47D (c,d) cells expressing GFP-vector or GFP-INPP4B were treated with 50 nM pyrvinium and/or 10 µM 4-OHT or DMSO as a vehicle control for 48 h, then incubated with 1 µg/mL propidium iodide (PI) and 1 µg/mL Hoechst 33,342 for 30 min (a,c). Data represent the percentage of PI-positive cells ± SD (n = 3 experiments, >300 cells/experiment) (b,d). Scale bar is 100 µm in a, c. + indicates where treatment was added, and – indicates where no treatment was added. p values were determined by one-way ANOVA with Tukey post hoc test in (b,d).

Figure 5. INPP4B-overexpressing ER+ breast cancer spheroids are more sensitive to pyrvinium or 4-OHT-pyrvinium. (a–c) MCF-7 cells expressing GFP-vector or GFP-INPP4B were cultured in Matrigel for 4 days to form 3D spheroids, then treated with 50 nM pyrvinium and/or 10 µM 4-OHT or DMSO as a vehicle control for 72 h. Representative brightfield images of 3D spheroids are shown (a). Data represent the area of the largest 32 spheroids (b), or the relative area of the largest 32 spheroids normalized to untreated cells (c) ± SD (n = 3 experiments). (d–f) T47D cells were cultured in Matrigel for 4 days to form 3D spheroids, then treated with 10 nM pyrvinium ± 5 µM 4-OHT or DMSO as a vehicle control for 72 h. Representative brightfield images of 3D spheroids are shown (d). Data represent the area of the largest 32 spheroids (e), or the relative area of the largest 32 spheroids normalized to untreated cells (f) ± SD (n = 3 experiments). Scale bar is 100 µm in (a,d). + indicates where treatment was added, and – indicates where no treatment was added. p values were determined by one-way ANOVA with Tukey post hoc test in (b,c,e,f).

Figure 5. INPP4B-overexpressing ER+ breast cancer spheroids are more sensitive to pyrvinium or 4-OHT-pyrvinium. (a–c) MCF-7 cells expressing GFP-vector or GFP-INPP4B were cultured in Matrigel for 4 days to form 3D spheroids, then treated with 50 nM pyrvinium and/or 10 µM 4-OHT or DMSO as a vehicle control for 72 h. Representative brightfield images of 3D spheroids are shown (a). Data represent the area of the largest 32 spheroids (b), or the relative area of the largest 32 spheroids normalized to untreated cells (c) ± SD (n = 3 experiments). (d–f) T47D cells were cultured in Matrigel for 4 days to form 3D spheroids, then treated with 10 nM pyrvinium ± 5 µM 4-OHT or DMSO as a vehicle control for 72 h. Representative brightfield images of 3D spheroids are shown (d). Data represent the area of the largest 32 spheroids (e), or the relative area of the largest 32 spheroids normalized to untreated cells (f) ± SD (n = 3 experiments). Scale bar is 100 µm in (a,d). + indicates where treatment was added, and – indicates where no treatment was added. p values were determined by one-way ANOVA with Tukey post hoc test in (b,c,e,f).