Journal: Cell

Article Title: Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

doi: 10.1016/j.cell.2020.10.030

Figure Lengend Snippet: RAB7A Loss Results in a Reduced Cell Surface Expression and an Increased Endosomal Accumulation of ACE2 (A) Representative histograms of flow cytometry analysis to determine cell surface expression of ACE2 on A549 cell lines (A549 wild-type [WT], A549 ACE2 , and ACE2 with Cas9 and non-targeting [NT] or RAB7A-targeting guide RNAs). The dashed line indicates the gate between the ACE2-negative and -positive cells. (B) Fraction of ACE2 + cells (using gating shown in A). ACE2 expression level was normalized across all samples to the A549 ACE2 cells transduced with non-targeting (NT) guides (n = 2–3 guide RNA-transduced lines per gene, error bars are SEM). (C) Representative images of immunofluorescence staining of ACE2 on A549 ACE2 transduced with an NT or a RAB7A -targeting guide. In NT cells, ACE2 localizes at the cell membrane and in the cytoplasm, while in RAB7A -targeted cells, ACE2 shows a distinct pattern of localization to vesicles. (D) Percent of cells with ACE2 accumulation in vesicles in NT and RAB7A-transduced A549 ACE2 cells (n = 2 biological replicates, error bars are SEM). (E) Representative images of immunofluorescence co-stained for ACE2, EEA1, and LysoTracker in A549 ACE2 cells with a CRISPR guide RNA targeting RAB7A . ACE2 shows a distinct colocalization with EEA1 (an early endosome marker) and a less frequent colocalization with LysoTracker (a lysosomal maker). (F) Representative histograms of flow cytometry analysis to determine cell surface expression in Calu-3 cells. The dashed line indicates the gate between the ACE2-negative and -positive cells. (G) Fraction of ACE2 + cells (using gating shown in F). ACE2 expression level was normalized across all samples to the Calu-3 cells transduced with an NT guide (n = 3 biological replicates, error bars are SEM). (H) Representative images of immunofluorescence staining of ACE2 on Caco-2 cells transduced with a NT or a RAB7A-targeting guide. In NT cells, ACE2 localizes at the cell membrane and in the cytoplasm, whereas in RAB7A-targeted cells, ACE2 shows a distinct pattern of localization to vesicles. (I) Mean area of ACE2 foci in Caco-2 cells transduced with a NT or a RAB7A-targeting guide (n = 4 biological replicates, 80–105 cells per replicate were scored, error bars are SEM). Significance testing for (B) and (G) was performed with a one-way ANOVA (B: F = 9.8, p < 10 −4 ; G: Calu-3: F = 378, p < 10 −4 , Caco-2: F = 222, p < 10 −4 ) with false-discovery rate-corrected post hoc tests. Significance testing for (D) and (I) was performed with an unpaired t test. For all panels, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. See also Figure S6 .

Article Snippet: For each gene of interest, 3 guide RNAs were designed using GUIDES software and were subsequently cloned into an all-in-one vector with an optimized Cas9 scaffold (pCC_01, Addgene 139086) containing Cas9, a guide RNA cassette and a puro resistant cassette ( ; ).

Techniques: Expressing, Flow Cytometry, Transduction, Immunofluorescence, Staining, Membrane, CRISPR, Marker

Journal: Cell

Article Title: Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

doi: 10.1016/j.cell.2020.10.030

Figure Lengend Snippet:

Article Snippet: For each gene of interest, 3 guide RNAs were designed using GUIDES software and were subsequently cloned into an all-in-one vector with an optimized Cas9 scaffold (pCC_01, Addgene 139086) containing Cas9, a guide RNA cassette and a puro resistant cassette ( ; ).

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, Staining, Reverse Transcription, CRISPR, Infection, RNA Sequencing, 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: 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: Briefly, 786–0 cells were electroporated with sgRNA-Cas9 complexes targeting BAP1 (sgRNA2 BAP1 + 52408564, U*C*A*AAUGGAUCGAAGAGCGC + Synthego modified EZ scaffold; sgRNA4 BAP1 + 52408601, G*G*A*AGAUAAAUCCAUAUACA + Synthego modified EZ scaffold) or Cas9 control using 4D-Nucleofector X Kit (Lonza, V4XC–2032).

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: Briefly, 786–0 cells were electroporated with sgRNA-Cas9 complexes targeting BAP1 (sgRNA2 BAP1 + 52408564, U*C*A*AAUGGAUCGAAGAGCGC + Synthego modified EZ scaffold; sgRNA4 BAP1 + 52408601, G*G*A*AGAUAAAUCCAUAUACA + Synthego modified EZ scaffold) or Cas9 control using 4D-Nucleofector X Kit (Lonza, V4XC–2032).

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: Briefly, 786–0 cells were electroporated with sgRNA-Cas9 complexes targeting BAP1 (sgRNA2 BAP1 + 52408564, U*C*A*AAUGGAUCGAAGAGCGC + Synthego modified EZ scaffold; sgRNA4 BAP1 + 52408601, G*G*A*AGAUAAAUCCAUAUACA + Synthego modified EZ scaffold) or Cas9 control using 4D-Nucleofector X Kit (Lonza, V4XC–2032).

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: Briefly, 786–0 cells were electroporated with sgRNA-Cas9 complexes targeting BAP1 (sgRNA2 BAP1 + 52408564, U*C*A*AAUGGAUCGAAGAGCGC + Synthego modified EZ scaffold; sgRNA4 BAP1 + 52408601, G*G*A*AGAUAAAUCCAUAUACA + Synthego modified EZ scaffold) or Cas9 control using 4D-Nucleofector X Kit (Lonza, V4XC–2032).

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: Briefly, 786–0 cells were electroporated with sgRNA-Cas9 complexes targeting BAP1 (sgRNA2 BAP1 + 52408564, U*C*A*AAUGGAUCGAAGAGCGC + Synthego modified EZ scaffold; sgRNA4 BAP1 + 52408601, G*G*A*AGAUAAAUCCAUAUACA + Synthego modified EZ scaffold) or Cas9 control using 4D-Nucleofector X Kit (Lonza, V4XC–2032).

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: Briefly, 786–0 cells were electroporated with sgRNA-Cas9 complexes targeting BAP1 (sgRNA2 BAP1 + 52408564, U*C*A*AAUGGAUCGAAGAGCGC + Synthego modified EZ scaffold; sgRNA4 BAP1 + 52408601, G*G*A*AGAUAAAUCCAUAUACA + Synthego modified EZ scaffold) or Cas9 control using 4D-Nucleofector X Kit (Lonza, V4XC–2032).

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: Briefly, 786–0 cells were electroporated with sgRNA-Cas9 complexes targeting BAP1 (sgRNA2 BAP1 + 52408564, U*C*A*AAUGGAUCGAAGAGCGC + Synthego modified EZ scaffold; sgRNA4 BAP1 + 52408601, G*G*A*AGAUAAAUCCAUAUACA + Synthego modified EZ scaffold) or Cas9 control using 4D-Nucleofector X Kit (Lonza, V4XC–2032).

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