Journal: Thoracic Cancer

Article Title: Effects of Autophagy Inhibition by SAR405 , a Selective VPS34 Inhibitor, on Pleural Mesothelioma Cells

doi: 10.1111/1759-7714.70255

Figure Lengend Snippet: No synergistic effect was seen for the combination of SAR405 and cisplatin. (A) Mesothelioma cells were cultured for 72 h with 1.0–5.0 μM cisplatin, 2.5–10.0 μM SAR405, or dimethyl sulfoxide (DMSO) as a control. Cell viability was assessed by the reduction of water‐soluble tetrazolium 8 (WST‐8). The results show the proportion of viable cells after 72 h treatment with either cisplatin combined with SAR405 or DMSO. The proportion of viable cells was lower after combined treatment with cisplatin and SAR405 than after treatment with DMSO. Data are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control. (B) Drug interactions of cisplatin with either SAR405, PIK‐III, or Vps34‐IN‐1 in mesothelioma cell lines. H28, H2452, and 211H cells were cultured for 72 h with 0.1–100 μM cisplatin and 0.1–100 μM SAR405, PIK‐III, or Vps34‐IN‐1. Drug interactions were evaluated using SynergyFinder (version 3.0) using the zero interaction potency (ZIP) model. A ZIP synergy score > 10 is considered synergistic; a score between −10 and 10 is considered additive; and a score less than −10 is considered antagonistic.

Article Snippet: SAR405 (28 012; MedChemExpress, Monmouth Junction, NJ, USA), PIK‐III (22 452; MedChemExpress), Vps34‐IN‐1 (24 001; MedChemExpress), chloroquine diphosphate (28 916; Sigma‐Aldrich), cisplatin (033–20 091; FUJIFILM Wako Pure Chemical), and/or dimethyl sulfoxide (DMSO; 046–21 981; FUJIFILM Wako Pure Chemical), as a control, were added to the cell culture media.

Techniques: Cell Culture, Control

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , A schematic of the lysosomal phosphoinositide conversion pathway linking MTM1 phosphatase activity to RagGTPase–mTORC1 signalling. PI3P and PI(3,5)P 2 are dynamically regulated by PI3KC2β/Vps34, MTM1 and PIKfyve at the lysosome. b , c , MTM1 phosphatase activity is required to repress mTORC1 signalling and support myogenic differentiation. Representative immunoblots of mTORC1 activity (p-S6K/S6K) ( b ) and representative images with quantification of fusion index and myotube area ( c ) in MTM1 -KO Cas9 myotubes expressing wild-type MTM1 (FL) or phosphatase-dead MTM1 (C375S). Data are shown as mean ± s.d.; n = 20 myotubes (fusion index) and n = 22 myotubes (area) from three biologically independent experiments; one-way ANOVA with Dunnett’s multiple-comparisons test; scale bar, 100 µm. d , MTM1 phosphatase activity is necessary for normal lysosomal abundance of the LAMTOR–RagGTPase complex. Representative immunoblots showing that MTM1-C375S does not restore lysosomal LAMTOR and RagA levels in MTM1 -KO Cas9 myotubes (three biologically independent experiments). e , The quantification of total cellular and lysosomal phosphoinositide species (PIPx) in CTRL and MTM1 -KO Cas9 myotubes. Data are shown as mean ± s.d. from three biologically independent experiments for whole-cell extracts and six biologically independent Lyso-IP preparations for lysosomal fractions. Two-sided Welch’s t- test. f , The lipidomic profiling of PI, PI3P, PI5P and PI(3,5)P 2 species in total cell and lysosomal fractions from CTRL and MTM1 -KO Cas9 myotubes. Data are shown as log 2 FC relative to CTRL; circle size reflects −log 10 P values. Two-sided Welch’s t- test; P < 0.01. g , PI3KC2β contributes to mTORC1 hyperactivation in MTM1 -KO Cas9 myotubes. Representative immunoblots showing reduced p-S6K/S6K following PI3KC2β knockdown using two independent shRNAs. Data are shown as mean ± s.d. from three biologically independent experiments. One-way ANOVA with Dunnett’s multiple-comparisons test. h , The pharmacological inhibition of PIKfyve partially suppresses mTORC1 signalling in MTM1 -KO Cas9 myotubes. Representative immunoblots of p-S6K/S6K following Apilm treatment. Data are shown as mean ± s.d. from three biologically independent experiments; one-way ANOVA with Dunnett’s multiple-comparisons test. i , Combined PI3KC2β knockdown and PIKfyve inhibition normalize lysosomal PI3P and PI(3,5)P 2 levels in MTM1 -KO Cas9 myotubes. Data are shown as mean ± s.d. of six biologically independent Lyso-IP preparations; one-way ANOVA with Dunnett’s multiple-comparisons test. j , The reduced lysosomal recruitment of the LAMTOR–Rag–mTORC1 complex following PI3KC2β depletion, with partial restoration upon PIKfyve inhibition. Representative Lyso-IP immunoblots showing LAMTORs, RagA, Raptor and mTOR in MTM1 -KO Cas9 myotubes. Two biologically independent experiments. For b , d , g , h and j , protein molecular weight is indicated in kDa. Illustration in a created in BioRender; Karim, H. https://biorender.com/n6ucxpz (2026).

Article Snippet: The following drugs were diluted in DMSO to obtain the appropriate concentration: the pan-PI3Ks inhibitors wortmannin (5 mM; W1628, Sigma) and LY294002 (5 mM; L9908, Sigma), the mTORC1-ATP competitors torin 1 (200 nM; 475991, Sigma) and AZD8055 (200 nM; S1555, Selleckchem), the p70 ribosomal S6 kinase (S6K1) inhibitor PF-4708671 (10 mM; PZ0143, Sigma), the eIF2α inhibitor salubrinal (10 mM; SML0951, Sigma), the Vps34 inhibitor IN1 (0.25–1 mM; S7980, Selleck) and the PikFyve inhibitor Apilm (0.05–1 mM; S0785, Selleck).

Techniques: Activity Assay, Cell Characterization, Western Blot, Expressing, Knockdown, Inhibition, Molecular Weight

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , Representative immunoblots showing the effect of VPS34 inhibition (IN1) on mTORC1 downstream signaling, assessed by p-S6K levels. b , Representative images showing the impact of VPS34 inhibition (IN1) on the differentiation capacity of MTM1-KO cas9 myotubes, quantified as fusion index. Scale bar, 100 µm. c , d , Representative images of MHC-labelled myotubes illustrating the effects of PI3KC2β knockdown (shRNA Sh1 and Sh2) and apilimod (Apilm) treatment on differentiation capacity of MTM1-KO cas9 myotubes, quantified as fusion index ( c ) and myotube area ( d ). Scale bar, 100 µm. e , Relative abundance of individual phosphoinositide (PIPₓ) species in lysosome immunoprecipitation (Lyso-IP) samples from MTM1-KO cas9 cells following PI3KC2β knockdown (shRNA-Sh2), apilimod treatment, or the combined condition. Lipid levels are expressed as FC relative to CTRL cells and normalized to Lyso-Tag (HA) content. All graphs show mean ± s.d. Individual data points represent individual myotubes or independent Lyso-IP preparations. For b , n = 15 myotubes per condition; for c , d , n = 24 myotubes per condition; all derived from three biologically independent experiments. For a and e, data represent three biologically independent experiments (Lyso-IP preparations). One-way ANOVA followed by Dunnett’s multiple-comparisons test was used throughout ( a – e ). Molecular weights in a are indicated in kDa. Exact P values are reported in the figures, except when P < 0.0001.

Article Snippet: The following drugs were diluted in DMSO to obtain the appropriate concentration: the pan-PI3Ks inhibitors wortmannin (5 mM; W1628, Sigma) and LY294002 (5 mM; L9908, Sigma), the mTORC1-ATP competitors torin 1 (200 nM; 475991, Sigma) and AZD8055 (200 nM; S1555, Selleckchem), the p70 ribosomal S6 kinase (S6K1) inhibitor PF-4708671 (10 mM; PZ0143, Sigma), the eIF2α inhibitor salubrinal (10 mM; SML0951, Sigma), the Vps34 inhibitor IN1 (0.25–1 mM; S7980, Selleck) and the PikFyve inhibitor Apilm (0.05–1 mM; S0785, Selleck).

Techniques: Western Blot, Inhibition, Knockdown, shRNA, Immunoprecipitation, Derivative Assay

Journal: bioRxiv

Article Title: Dynamin-2 promotes Atg9A retrieval from phagophores during autophagy

doi: 10.64898/2026.03.11.711183

Figure Lengend Snippet: Dnm2 promotes Atg9A retrieval from phagophores. ( A ) Examples of Dnm2-RFP wedged between spots with GFP-Atg9A, and BFP-LC3 with fluorescence intensities for the transect plotted below. Frames in live cell imaging were separated by 350-400 msec. Scale bar is 1 μm. (B) Mean frequencies of Dnm2-RFP spots transiently wedged between GFP-Atg9A and BFP-LC3 spots in untreated cells and cells treated with Dynole 34-2 or Ryngo 1-23. The frequencies were low because only a small part of a cell was observed for a limited time (20 sec), but their transient nature and the opposing effects of a dynamin inhibitor and an activator suggest a functional connection with fission events that separate Atg9A and LC3. Average and SD are shown for three biological replicates, each determined with 4-7 technical repeats. Statistical analysis used a one-way ANOVA with Tukey’s post hoc comparisons across biological replicates. (C) Delayed dissociation of Atg9A and p62 in Dnm2 KO cells. A buildup of Atg9A and p62 was induced for 16h with the Vps34 inhibitor Vps34-IN1. This was followed by inhibitor washout for the indicated durations. Association and dissociation of Atg9A and p62 were monitored by immunofluorescence microscopy of endogenous proteins in WT and Dnm2 KO HeLa cells. These cells were fixed with paraformaldehyde before imaging. Average and SD are shown for three biological replicates, each determined with five technical repeats. Statistical analysis used an unpaired Student’s t-test on the biological replicates for each treatment. (D,E) Colocalization of RFP-Atg9A and GFP-LAMP1 after treatment with CCCP is increased by adding the p97 inhibitor NMS-873. Cells were incubated for 4 hr with DMSO or 10 μM NMS-973 and DMSO or 10 μM CCCP as indicated. Scale bar is 10 μm. Average and SD are shown for three biological replicates, each determined with five technical repeats. Statistical analysis used one-way ANOVA with Tukey’s post hoc comparisons of the biological replicates for each treatment. (F,G) Western blot showing decreased amounts of Atg9A protein in CCCP-treated Dnm2 KO cells. The lower panel shows quantification of Atg9A protein levels in blots of wild-type and Dnm2 KO cells. Levels decrease in Dnm2 KO cells after treatment with CCCP, but this decrease is partially prevented by Bafilomycin A, suggesting that Atg9A is degraded by autophagy in Dnm2 KO cells. Band intensities were determined with Licor software. Average and SD are shown for four biological replicates. Statistical analysis used one-way ANOVA with Tukey’s post hoc comparisons of the biological replicates for each cell line. (H) Functions of Dnm2 and EndoB1 on phagophores. Autophagy is triggered by activation of ULK1 kinase, which is associated with the scaffold protein FIP200 and with Atg9A-containing vesicles. The phagophore then grows from the Atg9A vesicles by Atg2-mediated lipid transfer from other organelles, such as the ER, and these lipids are equilibrated between the leaflets of the phagophore membrane by the scramblase function of Atg9A. Vps34-mediated lipid phosphorylation and subsequent LC3 lipidation promote phagophore encapsulation of cargo. Once the phagophore is complete, EndoB1 and Dnm2 mediate fission to form a vesicle that retrieves Atg9A. For the sake of simplicity, only a subset of autophagy proteins is shown.

Article Snippet: VPS34-IN1 (Selleckchem) was used at 10μM.

Techniques: Fluorescence, Live Cell Imaging, Functional Assay, Immunofluorescence, Microscopy, Imaging, Incubation, Western Blot, Software, Activation Assay, Membrane, Phospho-proteomics, Encapsulation