Figure S4.
Decreased PI(3)P levels block the cLTP-dependent increase in SNX17- and SNX27-positive puncta. (A) Representative confocal images of DIV17 hippocampal neurons that were transfected at DIV16 with mNeonGreen-SNX17 and mScarlet-SNX27. 24 h later, neurons were treated with either DMSO or 1 μM VPS34-IN1 for 30 min. Neurons were either fixed in the absence of cLTP (baseline) or treated with cLTP for 5 min and fixed 10 or 30 min after the cLTP stimulus. DMSO or VPS34-IN1 were maintained during the course of the experiment. Scale bar, 5 µm. (B) The number of mNeonGreen-SNX17 puncta in the first 30 μm of secondary dendrites was quantified at the indicated time points. DMSO baseline: 0.238 ± 0.013, N = 30 neurons; DMSO cLTP 10 min: 0.362 ± 0.013, N = 30 neurons; DMSO cLTP 30 min: 0.379 ± 0.014, N = 30 neurons; VPS34-IN1 baseline: 0.090 ± 0.007, N = 30 neurons; VPS34-IN1 cLTP 10 min: 0.114 ± 0.010, N = 30 neurons; VPS34-IN1 cLTP 30 min: 0.119 ± 0.010, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (C) The number of mScarlet-SNX27 puncta in the first 30 μm of secondary dendrites was quantified at the indicated time points. DMSO baseline: 0.243 ± 0.012, N = 30 neurons; DMSO cLTP 10 min: 0.367 ± 0.013, N = 30 neurons; DMSO cLTP 30 min: 0.389 ± 0.014, N = 30 neurons; VPS34-IN1 baseline: 0.101 ± 0.007, N = 30 neurons; VPS34-IN1 cLTP 10 min: 0.124 ± 0.011, N = 30 neurons; VPS34-IN1 cLTP 30 min: 0.124 ± 0.010, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) DIV16 hippocampal neurons were transfected with mNeonGreen-SNX17 and mScarlet-SNX27. 24 h later, neurons were either fixed in the absence of cLTP (baseline) or treated with cLTP for 5 min and fixed 0, 10, 30 or 60 min after the cLTP stimulus. The percentage of SNX17 that overlaps with SNX27 at the different time points was determined using Mander’s colocalization coefficient (×100). Baseline: 80.94 ± 2.058%, N = 26 neurons; 0 min after cLTP: 80.42 ± 1.678%, N = 28 neurons; 10 min after cLTP: 83.64 ± 1.582%, N = 30 neurons; 30 min after cLTP: 85.79 ± 1.474%, N = 30 neurons; 60 min after cLTP: 79.57 ± 1.512%, N = 24 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM. (E) Same as D, but the percentage of SNX27 that overlaps with SNX17 at the different time points was determined using Mander’s colocalization coefficient (×100). Baseline: 90.52 ± 2.066%, N = 26 neurons; 0 min after cLTP: 89.63 ± 1.605%, N = 28 neurons; 10 min after cLTP: 91.05 ± 1.689%, N = 30 neurons; 30 min after cLTP: 90.43 ± 1.525%, N = 30 neurons; 60 min after cLTP: 88.47 ± 2.162%, N = 24 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM. DIV, days in vitro.

Decreased PI(3)P levels block the cLTP-dependent increase in SNX17- and SNX27-positive puncta. (A) Representative confocal images of DIV17 hippocampal neurons that were transfected at DIV16 with mNeonGreen-SNX17 and mScarlet-SNX27. 24 h later, neurons were treated with either DMSO or 1 μM VPS34-IN1 for 30 min. Neurons were either fixed in the absence of cLTP (baseline) or treated with cLTP for 5 min and fixed 10 or 30 min after the cLTP stimulus. DMSO or VPS34-IN1 were maintained during the course of the experiment. Scale bar, 5 µm. (B) The number of mNeonGreen-SNX17 puncta in the first 30 μm of secondary dendrites was quantified at the indicated time points. DMSO baseline: 0.238 ± 0.013, N = 30 neurons; DMSO cLTP 10 min: 0.362 ± 0.013, N = 30 neurons; DMSO cLTP 30 min: 0.379 ± 0.014, N = 30 neurons; VPS34-IN1 baseline: 0.090 ± 0.007, N = 30 neurons; VPS34-IN1 cLTP 10 min: 0.114 ± 0.010, N = 30 neurons; VPS34-IN1 cLTP 30 min: 0.119 ± 0.010, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (C) The number of mScarlet-SNX27 puncta in the first 30 μm of secondary dendrites was quantified at the indicated time points. DMSO baseline: 0.243 ± 0.012, N = 30 neurons; DMSO cLTP 10 min: 0.367 ± 0.013, N = 30 neurons; DMSO cLTP 30 min: 0.389 ± 0.014, N = 30 neurons; VPS34-IN1 baseline: 0.101 ± 0.007, N = 30 neurons; VPS34-IN1 cLTP 10 min: 0.124 ± 0.011, N = 30 neurons; VPS34-IN1 cLTP 30 min: 0.124 ± 0.010, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) DIV16 hippocampal neurons were transfected with mNeonGreen-SNX17 and mScarlet-SNX27. 24 h later, neurons were either fixed in the absence of cLTP (baseline) or treated with cLTP for 5 min and fixed 0, 10, 30 or 60 min after the cLTP stimulus. The percentage of SNX17 that overlaps with SNX27 at the different time points was determined using Mander’s colocalization coefficient (×100). Baseline: 80.94 ± 2.058%, N = 26 neurons; 0 min after cLTP: 80.42 ± 1.678%, N = 28 neurons; 10 min after cLTP: 83.64 ± 1.582%, N = 30 neurons; 30 min after cLTP: 85.79 ± 1.474%, N = 30 neurons; 60 min after cLTP: 79.57 ± 1.512%, N = 24 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM. (E) Same as D, but the percentage of SNX27 that overlaps with SNX17 at the different time points was determined using Mander’s colocalization coefficient (×100). Baseline: 90.52 ± 2.066%, N = 26 neurons; 0 min after cLTP: 89.63 ± 1.605%, N = 28 neurons; 10 min after cLTP: 91.05 ± 1.689%, N = 30 neurons; 30 min after cLTP: 90.43 ± 1.525%, N = 30 neurons; 60 min after cLTP: 88.47 ± 2.162%, N = 24 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM. DIV, days in vitro.

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