Journal: bioRxiv

Article Title: A lipid transfer-dependent feedback loop activates ATG9A compartments in autophagy initiation

doi: 10.1101/2025.08.16.670665

Figure Lengend Snippet: ( A ) Immunofluorescence staining of HeLa WT and ΔATG2 cells for autophagy-related proteins: ATG9A, LC3B, ATG16L1, p62, ULK1, and WIPI2. ΔATG2 cells show aberrant accumulation of autophagy markers in distinct foci. ( B ) Rescue of foci phenotype in ΔATG2 cells 24 h after transient transfection with a wild-type ATG2A construct. ATG9A localization resembles that of WT HeLa cells, indicating restoration of normal trafficking. The merged images show ATG9A staining (magenta), p40PX staining (yellow), and DNA (turquoise). (C) Quantification of co-localization between ATG9A and FYVE domains in HeLa WT, ΔATG2 cells, and ΔATG2 cells transfected with a wild-type ATG2A construct (shown in B ), assessed using Pearson’s correlation coefficient (PCC). Statistical significance was determined using an unpaired t -test (N = 4). Bars represent mean ± s.d. ( D ) Immunofluorescence staining of HeLa WT and ΔATG2 cells expressing EGFP-2xFYVE, treated with 2 µM VPS34-IN1 (a PI3KC3-C1 inhibitor) for 120 min. Inhibitor treatment abrogates FYVE puncta formation, confirming the PI3P-dependence of the observed signal. ( E ) Immunofluorescence staining of HeLa ATG9A knockout (ΔATG9A) cells as a negative control for background co-localization between ATG9A and FYVE signals. ( F ) Quantification of ATG9A–FYVE co-localization in HeLa ΔATG9A cells (N = 3) analyzed by PCC. Statistical significance was assessed using an unpaired t -test. Bars represent mean ± s.d.

Article Snippet: For the purification of GST- and mCherry-tagged glycine-exposed GABARAP (Addgene_244946), GST- and mCherry-tagged full-length GABARAP (Addgene_244947), GST-tagged full-length GABARAP (Addgene_244948), GST- and His-tagged glycine-exposed LC3B (Addgene_244950), and the GST-tagged LIR motif of p62 (amino acids 334– 342; Addgene_244939), cell pellets were resuspended in lysis buffer containing 50 mM HEPES pH 7.4, 300 mM NaCl, 1% Triton X-100, 5% glycerol, 2 mM MgCl2, 1 mM DTT, 2 mM β-mercaptoethanol, 1× cOmpleteTM EDTA-free protease inhibitor cocktail, Phosphatase Inhibitor Cocktail, and DNase I.

Techniques: Immunofluorescence, Staining, Transfection, Construct, Expressing, Knock-Out, Negative Control

Journal: bioRxiv

Article Title: A lipid transfer-dependent feedback loop activates ATG9A compartments in autophagy initiation

doi: 10.1101/2025.08.16.670665

Figure Lengend Snippet: ( A ) Immunofluorescence staining of HeLa WT and ATG2 DKO cells 24 h after transient transfection with EGFP-WIPI4. An antibody against EGFP was used to visualize WIPI4. Compared to other autophagy proteins , WIPI4 shows minimal localization to ATG9A-positive foci in ATG2 DKO cells, indicating that little or no PI3P is present at these sites. ( B ) FLAG Trap beads coated with 3×FLAG-ATG2A were incubated with 6×HIS-tagged WIPI4, LC3B, or GFP (negative control). After one wash with buffer containing 300 mM NaCl, signals were detected for all proteins (low-salt condition). Following additional washes, the GFP signal was lost, whereas WIPI4 and LC3B signals persisted (high-salt condition). Compared to the input, signals of bound proteins were relatively weak. ( C ) Superose 6 size-exclusion chromatography (SEC) profiles of ATG2A, WIPI4, or a mixture of ATG2A (4 µM) and WIPI4 (4.8 µM). ( D ) Western blot analysis of SEC fractions from ATG2A, WIPI4, or ATG2A–WIPI4 mixtures. WIPI4 shows partial co-elution with the ATG2A peak, but most remains unbound. ( E ) Schematic of the experimental setup shown in F . ( F ) GFP-Trap agarose beads coated with native GFP-ATG9A compartments were incubated with mCherry-GABARAPΔL117 (GABARAP-GLY) in the presence of the ATG8 lipidation machinery: E1 (ATG7), E2 (ATG3), and E3 (a complex of ATG12–ATG5-ATG16L1), along with ATP, to assess lipidation. mCherry fluorescence on the bead surface indicates successful conjugation of GABARAP to ATG9A compartments. As negative controls, beads were incubated with either mCherry-GABARAP full-length (FL), which cannot be lipidated, or mCherry alone.

Article Snippet: For the purification of GST- and mCherry-tagged glycine-exposed GABARAP (Addgene_244946), GST- and mCherry-tagged full-length GABARAP (Addgene_244947), GST-tagged full-length GABARAP (Addgene_244948), GST- and His-tagged glycine-exposed LC3B (Addgene_244950), and the GST-tagged LIR motif of p62 (amino acids 334– 342; Addgene_244939), cell pellets were resuspended in lysis buffer containing 50 mM HEPES pH 7.4, 300 mM NaCl, 1% Triton X-100, 5% glycerol, 2 mM MgCl2, 1 mM DTT, 2 mM β-mercaptoethanol, 1× cOmpleteTM EDTA-free protease inhibitor cocktail, Phosphatase Inhibitor Cocktail, and DNase I.

Techniques: Immunofluorescence, Staining, Transfection, Incubation, Negative Control, Size-exclusion Chromatography, Western Blot, Co-Elution Assay, Fluorescence, Conjugation Assay

Journal: bioRxiv

Article Title: A lipid transfer-dependent feedback loop activates ATG9A compartments in autophagy initiation

doi: 10.1101/2025.08.16.670665

Figure Lengend Snippet: ( A ) Western blot analysis of samples collected during the isolation of native ATG9A compartments. Shown are the supernatant (SUP), the input fraction for FLAG-Trap incubation (INPUT), the FLAG-Trap elution (ELUTION), and the final vesicle fraction bound to GFP-Trap beads (BOUND) from HAP1 WT, HAP1 ATG9A–GFP, and HAP1 ATG9A–GFP ΔATG2 cells. ATG9A is enriched in the BOUND fraction of both ATG9A–GFP and ΔATG2 cells compared to WT. LC3B signal is increased in the ATG9A-containing vesicles from ΔATG2 cells, suggesting the accumulation of LC3-positive, unsealed membranes. ( B ) Western blot analysis of GABARAP conjugation to ATG9A vesicles. GFP-Trap agarose beads coated with ATG9A vesicles were incubated with GABARAP, ATG7 (E1), ATG3 (E2), ATG12–5–16L1 complex (E3), and ATP. The lipidation reaction was controlled by individually omitting each essential component of the lipidation machinery (E1, E2 or E3). Lipidated GABARAP (GABARAP-II) exhibits a characteristic mobility shift relative to the unlipidated form (GABARAP-I). PE-containing SUVs served as a positive control for lipidation. GABARAP-GLY: GABARAPΔL117 mutant exposing the penultimate glycine for conjugation; FL: full-length GABARAP, which cannot undergo lipidation; M: mock-purified control from HAP1 wild-type cells ( C ) Western blot analysis of isolated autophagosomes from HAP1 ATG9A-GFP and HAP1 ATG9A-GFP ΔATG2 cells before and after Proteinase K (PK) digestion. In ATG9A–GFP cells, approximately 30% of the p62 signal is lost, indicating the presence of closed, protease-protected vesicles. In contrast, ΔATG2 cells show ∼91% loss of p62, consistent with disrupted or open autophagosomes. SUPERNATANT refers to the soluble fraction collected during the final isolation step, whereas PELLET refers to the unwashed AP fraction. INPUT corresponds to the washed APs used for the PK digest. ( D ) Quantification of p62 signal following PK digestion, as shown in C (N = 5). Statistical significance was determined using an unpaired t -test. Bars represent mean ± s.d. ( E ) Schematic of the experimental workflow for PI3P detection on isolated autophagosomes shown in F. Autophagosomes were isolated from HAP1 ATG9A-GFP and HAP1 ATG9A-GFP ΔATG2 cells following 2-h treatment with EBSS and 100 nM Bafilomycin A1 (BafA) and incubated with GST-LIR-coated GSH beads. ( F ) Immunofluorescence detection of PI3P on autophagosomes from HAP1 ATG9A-GFP and HAP1 ATG9A–GFP ΔATG2 bound to GST-LIR beads using anti-PI3P antibody (Echelon, Cat# Z-P003; mouse) and anti-mouse AF546 secondary antibody. ( G ) Quantification of AF546 fluorescence intensity representing PI3P levels on autophagosomes shown in F (N = 3). Total fluorescence signals were background-corrected and normalized within each replicate to the respective maximum value. Statistical significance was assessed using an unpaired t -test. While this does not reach statistical significance (P < 0.05), it indicates a trend. Bars represent mean ± s.d.

Article Snippet: For the purification of GST- and mCherry-tagged glycine-exposed GABARAP (Addgene_244946), GST- and mCherry-tagged full-length GABARAP (Addgene_244947), GST-tagged full-length GABARAP (Addgene_244948), GST- and His-tagged glycine-exposed LC3B (Addgene_244950), and the GST-tagged LIR motif of p62 (amino acids 334– 342; Addgene_244939), cell pellets were resuspended in lysis buffer containing 50 mM HEPES pH 7.4, 300 mM NaCl, 1% Triton X-100, 5% glycerol, 2 mM MgCl2, 1 mM DTT, 2 mM β-mercaptoethanol, 1× cOmpleteTM EDTA-free protease inhibitor cocktail, Phosphatase Inhibitor Cocktail, and DNase I.

Techniques: Western Blot, Isolation, Incubation, Conjugation Assay, Mobility Shift, Positive Control, Mutagenesis, Purification, Control, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: A lipid transfer-dependent feedback loop activates ATG9A compartments in autophagy initiation

doi: 10.1101/2025.08.16.670665

Figure Lengend Snippet: ( A ) GSH-Trap beads coated with GST-tagged LC3B were incubated with either mCherry-tagged ATG2A or ATG2A-ΔLIR-mCherry, a mutant lacking the LIR motif (aa 1362–1365; FCIL/AAAA). ( B ) Quantification of mCherry fluorescence signal from ATG2A recruitment in A (N = 3). Quantification was carried out using an AI-based image analysis pipeline. Statistical significance was assessed using an unpaired t -test. Bars represent mean ± s.d. ( C ) Comparison of ATG2A WT and ΔLIR lipid transfer activity in the absence or presence of 6XHIS-LC3B (100 nM). Acceptor liposomes contain 60% DOPC, 15% DOPS, 15% DOPE, 5% PI3P and 5% DGS-NTA. NBD fluorescence (F(LT)) was recorded at 535 nm (excitation at 485 nm) and normalized to the DDM-solubilized maximum fluorescence (F(DDM)). Lines represent mean values (N = 4). ( D ) AlphaFold3-predicted structure of the ATG2A–WIPI4– LC3B complex, with a zoom-in on the predicted interaction interface. Indicated ATG2A residues correspond to regions predicted to interact with WIPI4 , and the LC3-interacting region (LIR) motif of ATG2A involved in binding LC3B . The model has a predicted inter-chain TM-score (ipTM) of 0.79 and a predicted TM-score (pTM) of 0.56. The predicted alignment error (PAE) heatmap is shown in . ( E ) Schematic model of ATG2A-mediated lipid transfer and autophagosome growth. ATG2A is recruited to early autophagic membranes through interactions with ATG9A compartments and membrane-anchored ATG8 proteins. Once localized, ATG2A transfers PI or PI3P from donor membranes into ATG9A-positive compartments. Locally synthesized PI3P recruits WIPI4, which in turn helps to stabilize the membrane association of ATG2A. This positive feedback loop enhances lipid transfer activity, promoting membrane expansion and facilitating the growth of the nascent autophagosome.

Article Snippet: For the purification of GST- and mCherry-tagged glycine-exposed GABARAP (Addgene_244946), GST- and mCherry-tagged full-length GABARAP (Addgene_244947), GST-tagged full-length GABARAP (Addgene_244948), GST- and His-tagged glycine-exposed LC3B (Addgene_244950), and the GST-tagged LIR motif of p62 (amino acids 334– 342; Addgene_244939), cell pellets were resuspended in lysis buffer containing 50 mM HEPES pH 7.4, 300 mM NaCl, 1% Triton X-100, 5% glycerol, 2 mM MgCl2, 1 mM DTT, 2 mM β-mercaptoethanol, 1× cOmpleteTM EDTA-free protease inhibitor cocktail, Phosphatase Inhibitor Cocktail, and DNase I.

Techniques: Incubation, Mutagenesis, Fluorescence, Comparison, Activity Assay, Liposomes, Binding Assay, Membrane, Synthesized

Journal: bioRxiv

Article Title: A lipid transfer-dependent feedback loop activates ATG9A compartments in autophagy initiation

doi: 10.1101/2025.08.16.670665

Figure Lengend Snippet: ( A ) Quantification of mCherry fluorescence signal from a microscopy-based bead assay assessing the recruitment of ATG2A to ATG8 proteins (as shown for LC3B in ). GSH-Trap beads coated with GST-tagged ATG8 family members (GABARAP, GABARAPL1, GABARAPL2, LC3A, LC3B, and LC3C) were incubated with either mCherry-tagged ATG2A or ATG2A-ΔLIR. Statistical significance was assessed using an unpaired t -test. Bars represent mean ± s.d. ( B ) Liposome tethering assay measured by absorbance at 405 nm. Lines represent mean values (N = 4). Conditions include: no protein (buffer only), 250 nM ATG2A wild-type (WT) or mutant (ΔLIR), and 250 nM ATG2A WT or mutant (ΔLIR) in the presence of 100 nM 6XHIS-LC3B or 100 nM 6XHIS-LC3B alone. Data reflect increased turbidity upon liposome clustering, indicative of tethering activity ( C ) The predicted alignment error (PAE) heatmap for the AlphaFold3-predicted structure of the ATG2A–WIPI4–LC3B complex shown in .

Article Snippet: For the purification of GST- and mCherry-tagged glycine-exposed GABARAP (Addgene_244946), GST- and mCherry-tagged full-length GABARAP (Addgene_244947), GST-tagged full-length GABARAP (Addgene_244948), GST- and His-tagged glycine-exposed LC3B (Addgene_244950), and the GST-tagged LIR motif of p62 (amino acids 334– 342; Addgene_244939), cell pellets were resuspended in lysis buffer containing 50 mM HEPES pH 7.4, 300 mM NaCl, 1% Triton X-100, 5% glycerol, 2 mM MgCl2, 1 mM DTT, 2 mM β-mercaptoethanol, 1× cOmpleteTM EDTA-free protease inhibitor cocktail, Phosphatase Inhibitor Cocktail, and DNase I.

Techniques: Fluorescence, Microscopy, Incubation, Mutagenesis, Activity Assay

Journal: Cell

Article Title: Multivalent small molecule pan-RAS inhibitors

doi: 10.1016/j.cell.2017.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Generation of RAS-less Mouse Embryo Fibroblasts expressing BRAF V600E -CAAX A BRAF CAAX plasmid was created by inserting a CAAX motif (cloned from pLL7.0: Venus-iLID-CAAX, Addgene, Plasmid #60411) into pBabe-Puro-BRAF-V600E plasmid (Addgene, Plasmid #15269). pBABE-puro plasmid was obtained from Addgene, Plasmid #1764.

Techniques: Recombinant, Transfection, SYBR Green Assay, Plasmid Preparation, Cell Viability Assay, Mass Spectrometry, Sequencing, Mutagenesis, Clone Assay, Software, Modification, Protease Inhibitor, Labeling, Staining

Journal: Cancers

Article Title: Nuclear Localization of BRAF V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

doi: 10.3390/cancers14020311

Figure Lengend Snippet: Figure 2. Transcriptomic analysis on public datasets highlighted the role of HMOX-1 in aggressiveness and BRAF inhibitor resistance in melanoma. (A,B) Kaplan–Meier curves representing the disease- free survival (DFS) of patients with a high expression of HMOX-1 compared to samples with low expression of HMOX-1 in a TCGA melanoma cohort (406 samples with survival information) (A) and in BRAF mutant samples (165 samples) (B) HMOX-1 expression was stratified as “high” and “low” with an optimized cut-off. (C) Boxplot representing the distribution of HMOX-1 expression in sensitive and resistant mice after A375 cell inoculation and BRAF inhibitor treatment (GSE74729, 4 sensitive and 5 resistant samples). (D) Boxplot representing the distribution of HMOX-1 expression in sensitive and resistant patients in a merge of 3 melanoma patient datasets (43 BRAF inhibitor sensitive (before treatment) and 48 resistant and relapse of disease (after treatment)). FC, fold change; DFS, disease-free survival.

Article Snippet: MEF (BRAF KO) and MV3 (BRAF wild-type) melanoma cells expressing nuclear localization signal (NLS)-BRAFV600E and BRAFV600E were established by infecting the cells with lentiviral particles expressing human BRAFV600E with NLS sequence pLV[Exp]Neo-CMV>DsRed_Express2: ORF_2373bp/ Myc(hBRAFV600E/3xNLS) or without NLS pLV[Exp]-NeoCMV>DsRed_ Express2:ORF_2301bp/Myc(hBRAFV600E) tagged with DsRed fluorescent protein according to the standard protocols (Cyagen, Santa Clara, CA, USA).

Techniques: Expressing, Mutagenesis

Journal: Cancers

Article Title: Nuclear Localization of BRAF V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

doi: 10.3390/cancers14020311

Figure Lengend Snippet: Figure 3. Nuclear BRAFV600E and HMOX-1 expression in xenograft mouse and human melanoma tissue cores. (A) The plasmid (BRAFV600E) or (3XNLS-BRAFV600E), containing BRAFV600E with nu- clear localization sequences (NLS), was transfected into the melanoma cell line MV3 (BRAFwt/wt) and selected with antibiotic G418. (B) MV3 cells transduced with BRAFV600E, NLS-BRAFV600E, and control cells were injected in mice (n = 5 for each group), and representative tumor images were taken. Xenograft mouse tissues were stained with an anti-HOMX1 antibody. Mag. 400×. Scale bars = 50 µm (C) Human malignant melanoma tissue array: immunoreactivity to BRAFV600E and HMOX1 in the human melanoma cores was detected and counterstained for the nuclear and cy- toplasm location. Mag. 400×. Scale bars = 50 µm. (Figure S3) provides the whole IHC analysis. (D) phosphoERK and phosphoAKT were assessed in MEF stably transfected with BRAFV600E or NLS-BRAFV600E after treatment with 5 µM PLX-4032 at different time points. A representative gel is shown. Two additional independent experiments provided similar results.

Article Snippet: MEF (BRAF KO) and MV3 (BRAF wild-type) melanoma cells expressing nuclear localization signal (NLS)-BRAFV600E and BRAFV600E were established by infecting the cells with lentiviral particles expressing human BRAFV600E with NLS sequence pLV[Exp]Neo-CMV>DsRed_Express2: ORF_2373bp/ Myc(hBRAFV600E/3xNLS) or without NLS pLV[Exp]-NeoCMV>DsRed_ Express2:ORF_2301bp/Myc(hBRAFV600E) tagged with DsRed fluorescent protein according to the standard protocols (Cyagen, Santa Clara, CA, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Transduction, Control, Injection, Staining, Stable Transfection

Journal: Cancers

Article Title: Nuclear Localization of BRAF V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

doi: 10.3390/cancers14020311

Figure Lengend Snippet: Figure 6. Pathway analysis revealed a key role of HMOX-1 in BRAF inhibitor therapy resistance in the xenograft model and in melanoma patients. (A) Dotplot representing the enrichment score obtained for the common pathways identified in the xenograft model and melanoma patients. (B) Heatmap representing the correlation matrix between the 36 genes involved in the 3 selected pathways (ferroptosis, fluid shear stress and atherosclerosis, and hepatocellular carcinoma) common in the xenograft model and melanoma patients. Values are from the patient dataset. (C) Network representation of the 36 genes (green nodes) involved in the 3 selected pathways (light yellow nodes), with their Spearman correlation as edges. Dark red ellipse, genes belonging to “fluid shear stress and atherosclerosis”; orange, genes belonging to “ferroptosis”; blue ellipse, genes belonging to “hepatocellular carcinoma”; thicker edges, correlation related to HMOX1; blue edges, negative correlation; red edges, positive correlation; edge transparency proportional to correlation value; blue node border, negative correlation with HMOX1; red node border, positive correlation with HMOX1. Correlation values are from the patient dataset.

Article Snippet: MEF (BRAF KO) and MV3 (BRAF wild-type) melanoma cells expressing nuclear localization signal (NLS)-BRAFV600E and BRAFV600E were established by infecting the cells with lentiviral particles expressing human BRAFV600E with NLS sequence pLV[Exp]Neo-CMV>DsRed_Express2: ORF_2373bp/ Myc(hBRAFV600E/3xNLS) or without NLS pLV[Exp]-NeoCMV>DsRed_ Express2:ORF_2301bp/Myc(hBRAFV600E) tagged with DsRed fluorescent protein according to the standard protocols (Cyagen, Santa Clara, CA, USA).

Techniques: Shear