Figure S1.
cLTP promotes the formation of PI(3)P- and VPS34-positive puncta, which is dependent on the NMDAR–calcium–CaMKII pathway. (A) Rat hippocampal neurons were transfected at DIV16 with mCherry as a filler. 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 min after the cLTP stimulus. DMSO or VPS34-INH were maintained during the course of the experiment. After fixation, cells were immunostained with the PI(3)P recombinant biosensor conjugated to Alexa488 (SNAP488). Scale bar, 5 µm. (B) The number of PI(3)P-positive puncta in the first 30 μm of secondary dendrites was quantified for DMSO-treated cells. Baseline: 0.385 ± 0.023, N = 31 neurons; cLTP: 0.645 ± 0.031, N = 31 neurons. Three independent experiments. Statistical significance was determined using Student’s unpaired t test, ****P < 0.001. Error bars are SEM. (C) DIV16 hippocampal neurons were co-transfected at DIV16 with HA-VPS34 and mCherry as a filler. 24 h later, neurons were either fixed in the absence of cLTP (baseline) or treated with cLTP for 5 min and fixed 10 min after the cLTP stimulus, followed by immunostaining with an anti-HA antibody. Scale bar, 5 µm. (D) The number of HA-VPS34–positive puncta in the first 30 μm of secondary dendrites was quantified. Baseline: 0.250 ± 0.021, N = 30 neurons; cLTP: 0.353 ± 0.022, N = 30 neurons. Three independent experiments. Statistical significance was determined using Student’s unpaired t test, **P < 0.01. Error bars are SEM. (E) DIV17 rat cortical neurons were either left untreated (baseline) or treated with cLTP for 5 min. Extracts were collected 10 min after cLTP and analyzed by western blot for the levels of VPS34, and GAPDH was used as a loading control. (F) VPS34 levels were quantified and normalized to the baseline protein levels. Baseline: 100; cLTP: 109.50 ± 13.24. Four independent experiments. Statistical significance was determined using Student’s unpaired t test. Error bars are SEM. (G) DIV16 hippocampal neurons were co-transfected at DIV16 with HA-VPS34 and mCherry as a filler. 24 h later, neuron cultures were treated with DMSO, 10 μM BAPTA-AM, 100 μM D-APV, 10 μM nifedipine, 10 uM AIP, 2 μM KT5720, or 10 μM U0126 for 30 min, followed by a 5-min cLTP stimulus in the presence of compounds where indicated. Neurons were further incubated in the presence of the indicated compounds for 10 min before fixation, followed by permeabilization and incubation with an anti-HA antibody. The number of HA-VPS34–positive puncta in the first 30 μm of secondary dendrites was quantified. DMSO: 0.243 ± 0.019, N = 30 neurons; DMSO + cLTP: 0.337 ± 0.023, N = 30 neurons; BAPTA-AM + cLTP: 0.237 ± 0.017, N = 30 neurons; D-APV + cLTP: 0.242 ± 0.013, N = 30 neurons; nifedipine + cLTP: 0.340 ± 0.018, N = 30 neurons; AIP + cLTP: 0.247 ± 0.012, N = 30 neurons; KT5720 + cLTP: 0.339 ± 0.018, N = 30 neurons; U0126 + cLTP: 0.338 ± 0.021, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. (H) DIV16 rat hippocampal neurons were either left untreated (baseline) or treated with cLTP for 5 min. Extracts were collected 10 min after cLTP and used for immunoprecipitation with an anti-VPS34 antibody, followed by immunoblotting with an antibody against pan-phospho-Ser/Thr. (I) The ratios of pan-phospho-Ser/Thr to VPS34 were quantified and normalized to the baseline group. Baseline: 100; cLTP: 97.95 ± 5.22. Three independent experiments. Statistical significance was determined using Student’s unpaired t test. Error bars are SEM. DIV, days in vitro. Source data are available for this figure: SourceData FS1.

cLTP promotes the formation of PI(3)P- and VPS34-positive puncta, which is dependent on the NMDAR–calcium–CaMKII pathway. (A) Rat hippocampal neurons were transfected at DIV16 with mCherry as a filler. 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 min after the cLTP stimulus. DMSO or VPS34-INH were maintained during the course of the experiment. After fixation, cells were immunostained with the PI(3)P recombinant biosensor conjugated to Alexa488 (SNAP488). Scale bar, 5 µm. (B) The number of PI(3)P-positive puncta in the first 30 μm of secondary dendrites was quantified for DMSO-treated cells. Baseline: 0.385 ± 0.023, N = 31 neurons; cLTP: 0.645 ± 0.031, N = 31 neurons. Three independent experiments. Statistical significance was determined using Student’s unpaired t test, ****P < 0.001. Error bars are SEM. (C) DIV16 hippocampal neurons were co-transfected at DIV16 with HA-VPS34 and mCherry as a filler. 24 h later, neurons were either fixed in the absence of cLTP (baseline) or treated with cLTP for 5 min and fixed 10 min after the cLTP stimulus, followed by immunostaining with an anti-HA antibody. Scale bar, 5 µm. (D) The number of HA-VPS34–positive puncta in the first 30 μm of secondary dendrites was quantified. Baseline: 0.250 ± 0.021, N = 30 neurons; cLTP: 0.353 ± 0.022, N = 30 neurons. Three independent experiments. Statistical significance was determined using Student’s unpaired t test, **P < 0.01. Error bars are SEM. (E) DIV17 rat cortical neurons were either left untreated (baseline) or treated with cLTP for 5 min. Extracts were collected 10 min after cLTP and analyzed by western blot for the levels of VPS34, and GAPDH was used as a loading control. (F) VPS34 levels were quantified and normalized to the baseline protein levels. Baseline: 100; cLTP: 109.50 ± 13.24. Four independent experiments. Statistical significance was determined using Student’s unpaired t test. Error bars are SEM. (G) DIV16 hippocampal neurons were co-transfected at DIV16 with HA-VPS34 and mCherry as a filler. 24 h later, neuron cultures were treated with DMSO, 10 μM BAPTA-AM, 100 μM D-APV, 10 μM nifedipine, 10 uM AIP, 2 μM KT5720, or 10 μM U0126 for 30 min, followed by a 5-min cLTP stimulus in the presence of compounds where indicated. Neurons were further incubated in the presence of the indicated compounds for 10 min before fixation, followed by permeabilization and incubation with an anti-HA antibody. The number of HA-VPS34–positive puncta in the first 30 μm of secondary dendrites was quantified. DMSO: 0.243 ± 0.019, N = 30 neurons; DMSO + cLTP: 0.337 ± 0.023, N = 30 neurons; BAPTA-AM + cLTP: 0.237 ± 0.017, N = 30 neurons; D-APV + cLTP: 0.242 ± 0.013, N = 30 neurons; nifedipine + cLTP: 0.340 ± 0.018, N = 30 neurons; AIP + cLTP: 0.247 ± 0.012, N = 30 neurons; KT5720 + cLTP: 0.339 ± 0.018, N = 30 neurons; U0126 + cLTP: 0.338 ± 0.021, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. (H) DIV16 rat hippocampal neurons were either left untreated (baseline) or treated with cLTP for 5 min. Extracts were collected 10 min after cLTP and used for immunoprecipitation with an anti-VPS34 antibody, followed by immunoblotting with an antibody against pan-phospho-Ser/Thr. (I) The ratios of pan-phospho-Ser/Thr to VPS34 were quantified and normalized to the baseline group. Baseline: 100; cLTP: 97.95 ± 5.22. Three independent experiments. Statistical significance was determined using Student’s unpaired t test. Error bars are SEM. DIV, days in vitro. Source data are available for this figure: SourceData FS1.

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