Fig 2: AMPK signaling is downregulated in RC-kd. (a) Schematic representation of the pathway presumably linking AMPK with MCOLN1 activity. (b) Western blot analysis of whole-cell extracts (first 2 lanes) as well as extracts obtained from AMPK immunoprecipitation, using a pan-antibody against PRKAA1 and PRKAA2. Lanes 3–4 show two independent experiments of AMPK immunoprecipitate in control cells, and lanes 5–6 show 2 independent experiments of AMPK immunoprecipitate in RC-kd cells. Lanes 7–8 show the unbound eluate, and lane 9 is the negative control. The proteins detected include AMPK and PIKFYVE, which is readily detected in the AMPK immunoprecipitate. GAPDH is detected in the input samples and in the unbound eluate, but not in the AMPK immunoprecipitate, demonstrating specificity of the interaction between AMPK-PIKFYVE. (c) Decreased phosphorylation of AMPK targets in RC-kd. Western blot analysis of whole-cell extracts for UQCRC1, AMPK, p-AMPK (T172), TSC2. p-TSC2 (S1387) and PIKFYVE in RC-kd and scrambled control HeLa cells, using GAPDH as loading control. The bands for UQCRC1, p-AMPK and p-TSC2 are decreased in RC-kds, while there is no change in PIKFYVE protein amount, as shown in the plots on the right side. Column plot shows average±s.d. of 3 independent experiments, with duplicates in each experiment. T-test p-value *p < 0.05 (d) Decreased PtdIns(3,5)P2 levels in the lysosomes of RC-kd cells, returned to control levels by reactivation of AMPK. Representative equatorial plane confocal images of HeLa cells (scrambled with vehicle control DMSO; scrambled with PIKFYVE inhibitor YM201636 1 µM 4 h; RC-kds with vehicle control DMSO; RC-kd treated with AMPK activator A769662 100 µM 4 h), cotransfected with ML1N*2-GFP and LAMP1-RFP. The localization of the ML1N*2-GFP probe to the lysosome is related to the lysosomal PtdIns(3,5)P2 content. Scale bar: 2 µm. The plot presents the quantification of PtdIns(3,5)P2 fluorescence on microscopy images of HeLa, scrambled and RC-kd, cotransfected with ML1N*2-GFP and LAMP1-RFP. Control cells were also treated with the PIKFYVE inhibitor YM201636 1 µM 4 h, and RC-kd were treated with AMPK activator A769662 100 µM 4 h (or DMSO as vehicle control). The plot shows the average±s.e.m ML1N*2-GFP intensity on the spots marked with LAMP1-RFP, across 45 cells for each condition, obtained from 3 independent experiments. ANOVA p-value **p < 0.005; ***p < 0.001; n.s., non-significant.

Fig 3: LASSO regression and multivariate COX regression analysis graph. (A), Tenfold cross-validation of LASSO regression model adjustment parameter selection. (B), LASSO coefficient curves for the seven analyzed genes included in the analysis. (C), forest plot of multifactorial Cox regression analysis. P-value < 0.05 from multifactorial Cox regression analysis of TRIM68, PIKFYVE and DYNLL2. “*” represents P < 0.05, “**” represents P < 0.01.

Fig 4: PIKfyve regulates the recycling of SNX17-Retriever-CCC-WASH cargoes and colocalizes with SNX17 and subunits of the Retriever, CCC, and WASH complexes.(A) HeLa cells were treated with DMSO or 1 µM apilimod for 1 hr and then the levels of SNX17 cargoes, ß1-integrin, and a5-integrin were determined using a surface biotinylation assay. Surface biotinylation assay was similarly performed on HUH7 cells and low-density lipoprotein receptor-related protein 1 (LRP1) levels were measured. (B) Quantification of western blots from three independent surface biotinylation experiments. (C) HEK293 cells expressing 3xHA-endogenously tagged PIKfyve were fixed, permeabilized, and incubated with antibodies against the HA tag and antibodies against either SNX17, the Retriever complex subunit (VPS35L), CCC complex subunits (CCDC93 and COMMD1), or WASH complex subunits (FAM21 and Strumpellin). Bar: 10 µm. Arrows indicate examples of puncta showing colocalization. (D) The percentage of PIKfyve colocalizing with the indicated proteins was determined using Mander’s colocalization coefficient analysis from three independent experiments. Data presented as mean ± SE. *p < 0.05, ***p < 0.005, and ****p < 0.001. Figure 6—source data 1.Contains numerical and uncropped western blot source data for Figure 6.

Fig 5: Inhibition of PIKfyve results in a defect in ß1-integrin recycling.(A–J) HeLa cells were incubated with ß1-integrin antibody for 1 hr at 37°C to allow the antibody-labeled integrin to internalize. Cells were then acid washed to remove surface ß1-integrin-bound antibodies, and were either fixed (A and F) or incubated with DMSO or 1 µM apilimod containing media at 37°C for the indicated times. Cells were either fixed (B–E) or fixed after a second acid wash to remove antibodies that returned to the surface (G–J). Fixed cells were permeabilized and immunostained with Alexa-Fluor-488-conjugated anti-mouse secondary antibodies. Flow diagram (top) outlines the experiment. (K) Surface levels of ß1-integrin were inferred from the intensity of ß1-integrin within 0.8 µm from the cell border. The levels of ß1-integrin that recycled back to the surface (for images B–E) were quantified as percentage of the total labeled integrin. (L) Intensity of non-recycled ß1-integrin was quantified from cells treated as indicated in (G–J). All values were normalized to the average of the 0 min time point (F). Data presented as mean ± SE. Statistical significance from three independent experiments were analyzed using two-way ANOVA and Sidak’s multiple comparisons tests. (K–L). ***p < 0.005 and ****p < 0.001. Bar: 10 µm. Figure 4—source data 1.Contains numerical source data for Figure 4.