Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 regulates SRC transcriptionally. (A) reverse-phase protein array (RPPA) data analysis of SRC in KIRC-TCGA (clear cell renal cell carcinoma, ccRCC) stratified by BAP1 and/or PBRM1 mutation status. (B) RPPA data analysis in UVM-TCGA (uveal melanoma, UM) stratified by BAP1 mutation status. (C,D) Representative western blotting analysis of BAP1-deficient ccRCC UMRC-6 cell lines (C) and UM UPMM2 cell lines (D) reconstituted with an empty vector (EV), wild-type BAP1 , or p.C91S catalytically inactive BAP1 mutant. (E) Kaplan-Meier curves of overall survival in KIRC-TCGA depending on SRC expression levels, stratified by quartile. Intermediate SRC expression is the combination of the second and third quartiles. (F,G) qRT-PCR analysis of SRC mRNA expression in these reconstituted BAP1-deficient ccRCC (F) and UM (G) cell lines. (H) chromatin immunoprecipitation (ChIP) to assess BAP1 occupancy on the SRC promoter. Error bars represent the average ± SE ( n = 3–4). HR, hazard ratio; CI, confidence interval; n.s ., non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Protein Array, Mutagenesis, Western Blot, Plasmid Preparation, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 loss suppresses autophagy. The autophagic activity of UMRC-6 cells reconstituted with an empty vector (EV) or BAP1 (wild-type or p.C91S mutant) was analyzed using the following methods: (A) western blotting, (B,C) quantification of GFP-LC3 puncta ( n = 50), (D) western blotting of samples treated with Bafilomycin A1 (BafA1; 100 nM, 3 h), (E,F) quantification of autolysosomes vs . autophagosomes, (G) the number of WIPI2 dots per cell, (H) quantification of HiBiT-LC3 luminescence, and (I) the kinase activity of BECN1-bound VPS34. (J-L) Autophagy in UMRC-6 cells reconstituted with an empty vector (EV), wild-type BAP1 , or multiple BAP1 mutants observed in tumors were analyzed by microscopy of GFP-LC3 puncta formation (J,K) and western blotting (L) in cells treated with Bafilomycin A1 (100 nM, 3 h) or the DMSO control (vehicle). Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests. Non-overlapping letters (e.g., “a” vs . “b”) represent significant differences ( p < 0.05) using ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis, Western Blot, Microscopy, Control

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 loss leads to SRC-mediated suppression of autophagy. The effects of SRC inhibition on autophagy were assessed by treatment with dasatinib, and samples were analyzed by (A) western blotting, (B) HiBiT-LC3 luminescence, (C) GFP-LC3 puncta and (D) their quantification ( n = 50), (E) number of autophagosomes and autolysosomes ( n = 50), (F,G) number of GFP-FYVE puncta ( n = 50) treated with 1 µM dasatinib for 24 h or DMSO control, as well as by SRC silencing (H,I) or overexpression of a V5-tagged SRC construct (J-L). Bafilomycin A1 was added at 100 nM for 3 h and dasatinib at 1 µM for 24 h. Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests. Non-overlapping letters represent significant differences ( p < 0.05) by ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Inhibition, Western Blot, Control, Over Expression, Construct

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: SRC binds to and phosphorylates BECN1 to regulate autophagy and proliferation. (A) BECN1 immunoprecipitation (IP) in UMRC-6 cells that overexpress SRC -V5 or an empty vector control (EV), containing plasmids for either constitutive SRC knockdown (sh SRC ) or a scrambled control (shSc). The arrow indicates the SRC -V5 band, and the bands below correspond to the heavy chain of IgG. (B) SRC -V5 was immunoprecipitated from UMRC-6 cells that overexpress SRC -V5 and were transfected with FLAG-BECN1 (or the corresponding empty vector control). (C) BECN1 (or IgG control) IP of UMRC-6 cells, followed by western blot analysis of phosphorylated tyrosine residues. (D) BECN1 IP of UMRC-6 cells overexpressing SRC -V5 (or empty vector control, EV) along with BAP1 (or EV) treated with 1 µM dasatinib for 24 h or DMSO control (vehicle). Red arrows indicate the bands for phosphorylated BECN1 (top) and VPS34 (bottom). (E,F) in vitro kinase assays of FLAG-BECN1 (or vector control) using in vitro transcribed/translated HA-SRC (E) or a kinase-dead version (F). The arrows in (E) indicate IgG heavy chains and the red asterisks (*) indicate the phosphorylated (top) and total (bottom) BECN1. (G-K) UMRC-6 cells stably expressing wild-type (WT) FLAG-BECN1, tyrosine phosphorylation mutants (non-phosphorylatable [BECN1 Y229/233/352F, BECN1-3F] or phospho-mimetic [BECN1 Y229/233/352E, BECN1-3E]) or an empty vector control (EV) were analyzed for autophagic flux by the number of GFP-LC3 puncta (G,H), the number of autolysosomes and autophagosomes (I), western blotting (J) or cell proliferation by Hoechst staining of the nuclei (K). Baf A1, 100 nM Bafilomycin A1 for 3 h; IP, immunoprecipitation; WCL, whole cell lysate. Error bars represent the average ± SE ( n = 50). n.s ., non-significant; *, p < 0.05; ***, p < 0.001 by t -tests. Non-overlapping letters represent significant differences ( p < 0.05) by ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Immunoprecipitation, Plasmid Preparation, Control, Knockdown, Transfection, Western Blot, In Vitro, Stable Transfection, Expressing, Phospho-proteomics, Staining

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Dasatinib and SW076956 synergistically induce autophagy and reduce tumor growth in ovo and ex vivo in patient-derived tumor organoids. (A-D) Representative tumors of chorioallantoic membrane (CAM) assay (A) and quantification of tumor growth (B) of tumors treated with 10 µM Tat-BECN1 peptide (TB1) or 10 µM Tat-scrambled control (TS) (A,B) or with dasatinib (dasa, 1 µM), SW076956 (SW07, 40 µM) or a combination of both (C,D) for 7 days. (E-G) western blot analysis of several tumors treated with the TB1 or TS peptides (E), dasatinib, SW07 or a combination (F), and a representative patient-derived tumor organoid treated with the indicated compounds (G); BafA1: bafilomycin A1, 100 nM, 3 h; Das+SW07, combination of 1 µM dasatinib and 40 µM SW076956 for 24 h. Error bars represent the average ± SE. *, p < 0.05; ***, p < 0.001, t -test. (H-M) ombination effects of treatment with different concentrations of dasatinib and SW076956 on the viability of patient-derived tumor organoids (PDTOs) of ccRCC with BAP1 loss (H), ccRCC PDTOs with wild-type BAP1 (K), UM PDTOs with BAP1 loss (I,J) and UM PDTOs with wild-type BAP1 (L,M). The synergy/antagonism effects were determined using a Loewe synergy model with Combenefit software from two (I-M) or three (H) independent experiments.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: In Ovo, Ex Vivo, Derivative Assay, Membrane, Chick Chorioallantoic Membrane Assay, Control, Western Blot, Software

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Dasatinib and SW076956 synergistically induce autophagy and decrease cell viability in vitro . (A-D) the effects of dasatinib (at nM concentration) and SW076956 (at µM concentration) treatments on autophagy in UMRC-6 cells were assessed by western blotting alone (left panel) or in combination (right panel) (A), HiBiT-LC3 luminescence (B), and the number of GFP-LC3 puncta ( n = 50) (C,D). Cells were treated with 1 µM dasatinib and/or 40 µM SW076956 for 24 h. Baf A1, 100 nM Bafilomycin A1 for 3 h. Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; *, p < 0.05; ***, p < 0.001 by t -tests. Non-overlapping letters indicate significant differences ( p < 0.05) by ANOVA and Student-newman-keuls test. (E,F) the combination effects of dasatinib and SW076956 treatments on cell viability of UMRC-6 (E) or TFK-1 (F) cells reconstituted with empty vector (EV), wild-type BAP1 , or a p.C91S BAP1 mutant were quantified 72 h after treatment of serial dilutions of single and combined compounds for three independent experiments. The synergy/antagonism effects of dasatinib and SW076956 were determined using a Loewe synergy model with Combenefit software from three independent experiments.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: In Vitro, Concentration Assay, Western Blot, Plasmid Preparation, Mutagenesis, Software

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Schematic representation of the proposed mechanism and platform for stratifying BAP1-loss patients who could benefit from treatment with SRC inhibitors and autophagy inducers. Lethal cancers with BAP1 mutations suppress autophagy through the binding and phosphorylation of BECN1 by the proto-oncogene SRC. Treatments with SRC inhibitors and autophagy inducers exhibited synergism in vitro , in ovo and ex vivo in patient-derived tumor organoids (PDTOs) with BAP1 loss, paving the way for the treatment of BAP1-deficient cancers with a combination of autophagy inducers and kinase inhibitors. The BAP1 immunohistochemistry images were reproduced from [10] with permission from Springer Nature.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Binding Assay, Phospho-proteomics, In Vitro, In Ovo, Ex Vivo, Derivative Assay, Immunohistochemistry

Journal: Cell

Article Title: Multidimensional tracking of GPCR signaling via peroxidase-catalyzed proximity labeling

doi: 10.1016/j.cell.2017.03.028

Figure Lengend Snippet: (A) Internalization kinetics of β2AR (class A GPCR) are different from those of AT1R (class B GPCR). β-arrestin is recruited with the peak at 180 s, but clathrin recruitment is delayed to 600 s. Also, Rab5 enrichment peak is at 600 s, indicating arrestin-receptor complex formation and endosomal entry do not occur simultaneously. Late endosomal marker Rab7 and lysosomal marker LAMP1 enrichment peaks appear afterward.

Article Snippet: Each well was transfected with 20 ng each of a pcDNA5 encoding β2AR-APEX/β2AR wild-type and CRE-SEAP reporter plasmid (BD Biosciences).

Techniques: Marker