Figure S6.
Brief 10-min inhibition of VPS34 does not affect the formation of SNX17- and SNX27-positive puncta or their recruitment to dendritic spines. (A) DIV16 hippocampal neurons were co-transfected with mNeonGreen-SNX17 and mCherry (filler). 24 h later, cells were incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mNeonGreen-SNX17–positive puncta in 30 µm of dendritic spines was quantified. DMSO: 0.291 ± 0.014, N = 30 neurons; VPS34-IN1: 0.281 ± 0.014, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (B) Same as B, but the mean intensity of mNeonGreen-SNX17 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mNeonGreen-SNX17 mean intensity in the dendritic shaft. DMSO: 0.691 ± 0.035, N = 30 neurons; VPS34-IN1: 0.683 ± 0.037, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (C) DIV16 hippocampal neurons were co-transfected with mScarlet-SNX27 and eGFP (filler). 24 h later, cells were incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mScarlet-SNX27-positive puncta in 30 µm of dendritic spines was quantified. DMSO: 0.291 ± 0.014, N = 30 neurons; VPS34-IN1: 0.279 ± 0.014, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (D) Same as C, but the mean intensity of mScarlet-SNX27 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mScarlet-SNX27 mean intensity in the dendritic shaft. DMSO: 0.750 ± 0.036, N = 30 neurons; VPS34-IN1: 0.781 ± 0.038, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (E) DIV16 hippocampal neurons were transfected with mNeonGreen-SNX17 and mCherry (filler). 24 h later, cells were treated with or without cLTP stimulation. cLTP-treated cells were washed, and incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mNeonGreen-SNX17–positive puncta in 30 µm of dendritic spines was quantified. Baseline: 0.253 ± 0.013, N = 30 neurons; cLTP+DMSO: 0.343 ± 0.020, N = 30 neurons; cLTP+VPS34-IN1: 0.319 ± 0.024, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01. Error bars are SEM. (F) DIV16 hippocampal neurons were transfected with mScarlet-SNX27 and eGFP (filler). 24 h later, cells were treated with or without cLTP stimulation. cLTP-treated cells were washed and incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mScarlet-SNX27–positive puncta in 30 µm of dendritic spines was quantified. Baseline: 0.246 ± 0.013, N = 30 neurons; cLTP+DMSO: 0.373 ± 0.020, N = 30 neurons; cLTP+VPS34-IN1: 0.361 ± 0.021, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

Brief 10-min inhibition of VPS34 does not affect the formation of SNX17- and SNX27-positive puncta or their recruitment to dendritic spines. (A) DIV16 hippocampal neurons were co-transfected with mNeonGreen-SNX17 and mCherry (filler). 24 h later, cells were incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mNeonGreen-SNX17–positive puncta in 30 µm of dendritic spines was quantified. DMSO: 0.291 ± 0.014, N = 30 neurons; VPS34-IN1: 0.281 ± 0.014, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (B) Same as B, but the mean intensity of mNeonGreen-SNX17 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mNeonGreen-SNX17 mean intensity in the dendritic shaft. DMSO: 0.691 ± 0.035, N = 30 neurons; VPS34-IN1: 0.683 ± 0.037, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (C) DIV16 hippocampal neurons were co-transfected with mScarlet-SNX27 and eGFP (filler). 24 h later, cells were incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mScarlet-SNX27-positive puncta in 30 µm of dendritic spines was quantified. DMSO: 0.291 ± 0.014, N = 30 neurons; VPS34-IN1: 0.279 ± 0.014, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (D) Same as C, but the mean intensity of mScarlet-SNX27 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mScarlet-SNX27 mean intensity in the dendritic shaft. DMSO: 0.750 ± 0.036, N = 30 neurons; VPS34-IN1: 0.781 ± 0.038, N = 30 neurons. Unpaired two-tailed Student’s t test determined that there are no significant differences between the DMSO and VPS34-IN1 conditions. Error bars are SEM. (E) DIV16 hippocampal neurons were transfected with mNeonGreen-SNX17 and mCherry (filler). 24 h later, cells were treated with or without cLTP stimulation. cLTP-treated cells were washed, and incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mNeonGreen-SNX17–positive puncta in 30 µm of dendritic spines was quantified. Baseline: 0.253 ± 0.013, N = 30 neurons; cLTP+DMSO: 0.343 ± 0.020, N = 30 neurons; cLTP+VPS34-IN1: 0.319 ± 0.024, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01. Error bars are SEM. (F) DIV16 hippocampal neurons were transfected with mScarlet-SNX27 and eGFP (filler). 24 h later, cells were treated with or without cLTP stimulation. cLTP-treated cells were washed and incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the number of mScarlet-SNX27–positive puncta in 30 µm of dendritic spines was quantified. Baseline: 0.246 ± 0.013, N = 30 neurons; cLTP+DMSO: 0.373 ± 0.020, N = 30 neurons; cLTP+VPS34-IN1: 0.361 ± 0.021, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

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