Journal: Journal of Virology

Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals

doi: 10.1128/JVI.00769-18

Figure Lengend Snippet: Evolutionary analyses of divergent mammalian HBV X proteins. (A) Phylogenetic analysis of the X proteins from hepadnaviruses that naturally infect mammals. The viral X proteins tested in our in vitro functional assays (Fig. 5 to ​to7)7) are indicated by an asterisk. Phylogenetic analysis of orthohepadnaviral X proteins was performed using a 161-amino-acid alignment obtained with MUSCLE (see supplemental data set 2 at https://figshare.com/articles/DatasetS2_Orthohepadnaviral_HBx_amino_acid_alignment_interleaved_phylip_format_/6194825) and the tree was built with PhyML and a JTT+I+G model with 1,000 bootstrap replicates. Bootstrap values (>800/1,000) are indicated at the nodes. The tree was rooted for representation purposes according to the work of Drexler et al. (52) (but the outgroup of orthohepadnavirus is still under debate [2]). The scale bar indicates the number of amino acid substitutions per site. We analyzed the X proteins from HBVs from the ground squirrel (GSHBV), arctic squirrel (ASHBV), and woodchuck (WHV), three bat viruses naturally infecting Hipposideros cf. ruber (roundleaf bat), Rhinolophus alcyone (horseshoe bat), and Uroderma bilobatum (tent-making bat), respectively (RBHBV, HBHBV, and TBHBV), wooly monkey HBV (WMHBV), human HBV, and HBVs from other indicated hominoids. (B) Amino acid alignment of the viral X proteins used for Fig. 5 to ​to7.7. The black-to-white gradient depicts high-to-low sequence identity (Geneious). The open reading frames (ORFs) overlapping with HBx are shown, as well as the DDB1-binding region in the human viral HBx protein (72).

Article Snippet: The membranes were probed with 1:5,000 mouse monoclonal anti-GFP antibody (Roche; 11814460001) to detect the GFP-tagged X proteins, 1:1,000 mouse monoclonal antibodies against Smc6 (Abgent; AT3956a), 1:500 rabbit polyclonal antibodies against Smc6 (a gift from A. R. Lehmann) (NIH 3T3 [ ]) ( 64 ), 1:1,000 rabbit polyclonal anti-Nse4 (Abgent; AP9909A), 1:10,000 mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma-Aldrich; G8795), and 1:500 goat polyclonal anti-DDB1 (Everest Biotech) antibodies.

Techniques: In Vitro, Functional Assay, Sequencing, Binding Assay

Journal: Journal of Virology

Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals

doi: 10.1128/JVI.00769-18

Figure Lengend Snippet: Highly divergent mammalian HBV X proteins show a conserved property of recruiting human DDB1 and antagonizing human Smc5/6 restriction. (A and B) Degradation of the human Smc5/6 complex by mammalian hepadnavirus X proteins. Human hepatoma HepG2 cells (A) and 293T cells (B) were transduced with a lentivector expressing only GFP (control) or the GFP-fused X protein from diverse hepadnaviruses (Fig. 4) or a mock control. Western blot analysis of the endogenous Smc6 and Nsmce4A was performed (see Materials and Methods). GAPDH served as a loading control. (C) Effect of mammalian X proteins on transiently transfected reporter gene activity. HepG2 cells were transfected with a luciferase reporter construct and the next day transduced with lentiviral vectors expressing the indicated proteins as described above. At days 5 to 7, the luciferase activity was measured; the fold increase of relative light units (RLU) versus the GFP control condition (set at 1) is shown. The means from three independent experiments are shown, along with SDs. *, P value = 0.1. P values correspond to the Wilcoxon Mann-Whitney test against the null hypothesis of no difference in the luciferase activity between the GFP control and GFP-X conditions. Of note, the same six X proteins unfused to GFP (i.e., in their native forms) also retained this activity (data not shown). (D) Interaction with human DDB1 protein was conserved for all hepadnaviral X proteins tested. The presence of DDB1 and GFP-fused protein (IP) was assessed by Western blotting. The viral X proteins could all interact with human DDB1, except for the DDB1 binding-deficient HBx mutant (R96E) that was used as a control. Note that GFP migrates to a position near the immunoglobulin light chain.

Article Snippet: The membranes were probed with 1:5,000 mouse monoclonal anti-GFP antibody (Roche; 11814460001) to detect the GFP-tagged X proteins, 1:1,000 mouse monoclonal antibodies against Smc6 (Abgent; AT3956a), 1:500 rabbit polyclonal antibodies against Smc6 (a gift from A. R. Lehmann) (NIH 3T3 [ ]) ( 64 ), 1:1,000 rabbit polyclonal anti-Nse4 (Abgent; AP9909A), 1:10,000 mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma-Aldrich; G8795), and 1:500 goat polyclonal anti-DDB1 (Everest Biotech) antibodies.

Techniques: Transduction, Expressing, Western Blot, Transfection, Activity Assay, Luciferase, Construct, MANN-WHITNEY, Binding Assay, Mutagenesis

Journal: Frontiers in Immunology

Article Title: AMBRA1 drives gastric cancer progression through regulation of tumor plasticity

doi: 10.3389/fimmu.2024.1494364

Figure Lengend Snippet: KD of AMBRA1 triggered the FoxO3A/p53/CRK/CDKN1B signaling pathway. (A) AMBRA1 pathway network derived from the Pathway Interaction Database which was curated by STRING. (B) GSEA of transcriptomic data from gastric cancer samples of The Cancer Genome Atlas (TCGA-STAD), senescence gene sets in AMBRA1_Low vs. AMBRA1_High subgroups (n = 16 for each subgroup). (C) Correlation between AMBRA1 and FOXO3, DDB1, CRK, CDKN1B, CDK4, CCND1, BCL2L11 in TCGA-STAD on TIMER2, with Spearman’s rho value representing the correlation degree. D–I) The expression of DDB1, FOXO3, DDB1, p53, CRK, CDKN1B and Tubulin in AGS and KD-AMBRA1 cells under CHX (1 μM), MG132 (0.5 μM) and MLN4924(5 μM)-treated for 24 hours were detected by western blotting. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Rabbit antibodies, including anti-AMBRA1 (Catalog #38182), anti-p27 (Catalog #41299), anti-p38 (Catalog #33149), anti-FoxO3A (Catalog #40937), anti-CUL4A (Catalog #38477), anti-CyclinD1 (Catalog #39315), anti-DDB1 (Catalog #38485), anti-Histone H2AX (Catalog #54667), anti-Phospho-Histone H2A.X (S139) (Catalog #13343), anti-Histone H3K9me3 (Catalog #HW029), anti-FoxO3A (Catalog #40937), anti-p53 (Catalog #HW109), anti-Caspase-3 (Catalog #9662), anti-Cleaved Caspase-3 (Catalog #9661), and anti-Phospho-Thr286 CyclinD1 (Catalog #11968), were procured from Signalway Antibody LLC (SAB).

Techniques: Derivative Assay, Expressing, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

doi: 10.1038/s41392-022-01253-y

Figure Lengend Snippet: CRL4 CUL4A/DDB 1 is critical for cisplatin resistance and mitochondrial morphology maintenance of OCCs. a Protein expression of DDB1 and CUL4A in normal ovaries and OC tissue samples based on the CPTAC dataset. Raw data were obtained from the OncoLnc database. * p < 0.01, ** p < 0.01. The significance of expression level difference was determined using a t -test. b Relative mRNA expression of DDB1 and CUL4A detected by qPCR in COC1/DDP and COC1 cell lines. Data represent mean ± SEM normalized to 18S. * p < 0.01, ** p < 0.01, *** p < 0.001. The significance of expression level difference was determined using a t -test. c Relative DDB1 and CUL4A protein expression levels in COC1/DDP and COC1 cell lines detected by Western Blot. GAPDH was used as a loading control. d , e Box-and-whiskers plot showing a relationship between (left panel) DDB1 and (right panel) CUL4A expression and d serous tumor stage and e platinum (Plat) resistance. n = 113 for DDB1 staining ( n = 8 for platinum-resistant, n = 105 for platinum-sensitive) n = 120 for CUL4A staining ( n = 9 for platinum-resistant, n = 111 for platinum-sensitive). f Immunohistochemical staining analysis of (upper panel) DDB1 and (lower panel) CUL4A expression in tumor cells in Plat-sensitive (Sens) and Plat-resistant (Res) serous tumors. n = 113 for DDB1 staining ( n = 8 for platinum-resistant, n = 105 for platinum-sensitive) n = 120 for CUL4A staining ( n = 9 for platinum-resistant, n = 111 for platinum-sensitive). g Overall survival (OS) and h Diseases free survival (DFS) in patients with serous ovarian cancer expressing high (>60% positive cells) or low (<60% positive cells) levels of (left) DDB1 or (right) CUL4A. n = 113 for DDB1 staining ( n = 8 for platinum-resistant, n = 105 for platinum-sensitive) n = 120 for CUL4A staining ( n = 9 for platinum-resistant, n = 111 for platinum-sensitive). i Representative images showing mitochondrial morphology in (upper panel) A2780CP and (lower panel) COC1/DDP cells with CRL4-knockdown by transmission electron microscope assay

Article Snippet: The following antibodies were used in this study: anti-CUL4A (Proteintech, 14851-1-AP, 1:2000), anti-DDB1 (HuaBio, ET1706-22, 1:2000), anti-DRP1 (C-Terminal, Proteintech, 12957-1-AP, 1:2000), anti-p62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:3000), anti-ATG5 (Proteintech, 10181-2-AP, 1:2000), anti-ATG14L (Proteintech, 19491-1-AP, 1:2000), anti-BCL-2 (Proteintech, 12789-1-AP, 1:1000), anti-MFF (Proteintech, 17090-1-AP, 1:1000), anti-phospho-MFF Ser172/Ser146 (Affinity, AF2365, 1:1000) anti-Beclin-1 (Proteintech, 11306-1-AP, 1:1000), anti-phospho-DRP1-Ser616 (CST, #3455, 1:1000), anti-phospho-DRP1-Ser637 (ABclonal, AP0812, 1:1000), anti-phospho-AMPKα-Thr172 (CST, #2535 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:2000), anti-AMPK alpha 1 (HuaBio, ET1608-40, 1:1000), anti-Parkin (HuaBio, ET1702-60, 1:1000), anti-PINK1 (HuaBio, ER1706-27, 1:1000), anti-BNIP3 (Abclonal, A5683, 1:1000), anti-BNIP3L (Abclonal, A6283, 1:1000), anti-FUNDC1 (Abclonal, A16318, 1:1000), anti-PGC-1α (Abclonal, A12348, 1:1000), anti-Tomm20 (Proteintech, 11802-1-AP, 1:2000), anti-Tim23 (Proteintech, 11123-1-AP, 1:2000).

Techniques: Expressing, Western Blot, Staining, Immunohistochemical staining, Transmission Assay, Microscopy

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

doi: 10.1038/s41392-022-01253-y

Figure Lengend Snippet: Knocking down CRL4 CUL4A/DDB1 significantly impaired the mitochondrial function of cisplatin-resistant OCCs. a Mitochondrial membrane potential in A2780CP cells determined with JC-1 probe after CRL4 CUL4A/DDB1 knockdown. CCCP (Carbonyl cyanide 3-chlorophenylhydrazone) was used as a positive control. Normal mitochondrial membrane potential (MMP) is shown in red with JC-1 dimers (JC-1 aggregates) and depolarized membrane potential is shown in green in JC-1 monomers, where a fluorescent color change from red to green indicates a decrease of MMP. Scale Bar: 20 μm. b Mitochondrial membrane potential detected by flow cytometric analysis in intact cells treated as in ( a ) and the quantification. Normal mitochondrial membrane potential (MMP) is shown in JC-1 dimers (JC-1 aggregates) and depolarized membrane potential is shown in JC-1 monomers. Data represent mean ± SEM with three replicates, ** p < 0.01, *** p < 0.001, **** p < 0.0001. c The ROS accumulation levels detected by DCAF probe using flow cytometry in A2780CP cells lacking CRL4 CUL4A/DDB1 . d The ATP level detected by multifunctional imaging enzyme labeling in A2780CP cells knocking down CRL4 CUL4A/DDB1 . e , g A representative graph of the oxygen consumption rate (OCR) throughout the mitochondrial-stress test using Seahorse XF24 analyzer in e A2780CP and g COC1/DDP CRL4-knockdown cells. Mitochondrial inhibitors were added to the media to assess respiratory parameters with indicated time. f , h Oxygen consumption rates (OCR) of basal respiration, proton leak, ATP production, and mitochondrial maximal respiration quantified from the mitochondrial-stress test are shown f A2780CP and h COC1/DDP CRL4-knockdown cells. Data were presented as the mean ± SEM Group differences are analyzed by the two-tailed Student’s t -test. i Transcriptional levels of TCA- and oxidative phosphorylation-associated enzymes based on RNA-seq data analysis

Article Snippet: The following antibodies were used in this study: anti-CUL4A (Proteintech, 14851-1-AP, 1:2000), anti-DDB1 (HuaBio, ET1706-22, 1:2000), anti-DRP1 (C-Terminal, Proteintech, 12957-1-AP, 1:2000), anti-p62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:3000), anti-ATG5 (Proteintech, 10181-2-AP, 1:2000), anti-ATG14L (Proteintech, 19491-1-AP, 1:2000), anti-BCL-2 (Proteintech, 12789-1-AP, 1:1000), anti-MFF (Proteintech, 17090-1-AP, 1:1000), anti-phospho-MFF Ser172/Ser146 (Affinity, AF2365, 1:1000) anti-Beclin-1 (Proteintech, 11306-1-AP, 1:1000), anti-phospho-DRP1-Ser616 (CST, #3455, 1:1000), anti-phospho-DRP1-Ser637 (ABclonal, AP0812, 1:1000), anti-phospho-AMPKα-Thr172 (CST, #2535 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:2000), anti-AMPK alpha 1 (HuaBio, ET1608-40, 1:1000), anti-Parkin (HuaBio, ET1702-60, 1:1000), anti-PINK1 (HuaBio, ER1706-27, 1:1000), anti-BNIP3 (Abclonal, A5683, 1:1000), anti-BNIP3L (Abclonal, A6283, 1:1000), anti-FUNDC1 (Abclonal, A16318, 1:1000), anti-PGC-1α (Abclonal, A12348, 1:1000), anti-Tomm20 (Proteintech, 11802-1-AP, 1:2000), anti-Tim23 (Proteintech, 11123-1-AP, 1:2000).

Techniques: Positive Control, Flow Cytometry, Imaging, Labeling, Two Tailed Test, RNA Sequencing Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

doi: 10.1038/s41392-022-01253-y

Figure Lengend Snippet: Knocking down CRL4 CUL4A/DDB1 leads to mitochondrial fragmentation in cisplatin-resistant OCCs. a (Left panel) Representative images of mitochondrial morphology in (upper panel) A2780CP and (lower panel) COC1/DDP cells with CRL4 CUL4A/DDB1 knockdown stained with MitoTracker Green. Images were captured by a laser confocal microscope. Results represent the mean from three independent experiments, measured in quadruplicate. (Right panel) Quantification analyses of the mitochondrial footprint in CRL4-knockdown (upper panel) A2780CP and (lower panel) COC1/DDP cells. The significance of differences was calculated using Student’s t -test (*** p < 0.001). b DRP1 expression level in CRL4-knockdown cells detected by western blot, GAPDH serves as an internal loading control. Protein levels were quantified with Image J software and normalized to the control group (NT). c Immunoblot detection of ACC1, p-ACC1, CUL4A, DDB1, DRP1, p-DRP1 Ser616 , p-DRP1 Ser637 , AMPK-α1, and p-AMPK-α1 in (left panel) A2780CP and (right panel) COC1/DDP cells with CRL4 CUL4A/DDB1 knockdown. β-actin serves as an internal loading control. d Immunoblot detection of MFF, p-MFF, CUL4A, DDB1, AMPK-α1, and p-AMPK-α1 in (left panel) A2780CP and (right panel) COC1/DDP cells with CRL4 CUL4A/DDB1 knockdown. e Immunoblotting analysis of DRP1 and MFF in the cytosolic (Cyto) and mitochondrial (Mito) fractions of A2780CP cells with CRL4 CUL4A/DDB1 knockdown. f Immunoblot detection of DRP1, AMPK-α1, and VDAC1 in (upper panel) input or (lower panel) immunoprecipitated samples in the NT control or CRL4 CUL4A/DDB1 knockdown (left panel) A2780CP and (right panel) COC1/DDP cells. β-actin serves as a loading control

Article Snippet: The following antibodies were used in this study: anti-CUL4A (Proteintech, 14851-1-AP, 1:2000), anti-DDB1 (HuaBio, ET1706-22, 1:2000), anti-DRP1 (C-Terminal, Proteintech, 12957-1-AP, 1:2000), anti-p62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:3000), anti-ATG5 (Proteintech, 10181-2-AP, 1:2000), anti-ATG14L (Proteintech, 19491-1-AP, 1:2000), anti-BCL-2 (Proteintech, 12789-1-AP, 1:1000), anti-MFF (Proteintech, 17090-1-AP, 1:1000), anti-phospho-MFF Ser172/Ser146 (Affinity, AF2365, 1:1000) anti-Beclin-1 (Proteintech, 11306-1-AP, 1:1000), anti-phospho-DRP1-Ser616 (CST, #3455, 1:1000), anti-phospho-DRP1-Ser637 (ABclonal, AP0812, 1:1000), anti-phospho-AMPKα-Thr172 (CST, #2535 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:2000), anti-AMPK alpha 1 (HuaBio, ET1608-40, 1:1000), anti-Parkin (HuaBio, ET1702-60, 1:1000), anti-PINK1 (HuaBio, ER1706-27, 1:1000), anti-BNIP3 (Abclonal, A5683, 1:1000), anti-BNIP3L (Abclonal, A6283, 1:1000), anti-FUNDC1 (Abclonal, A16318, 1:1000), anti-PGC-1α (Abclonal, A12348, 1:1000), anti-Tomm20 (Proteintech, 11802-1-AP, 1:2000), anti-Tim23 (Proteintech, 11123-1-AP, 1:2000).

Techniques: Staining, Microscopy, Expressing, Western Blot, Software, Immunoprecipitation

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

doi: 10.1038/s41392-022-01253-y

Figure Lengend Snippet: CRL4 knockdown induces autophagy in cisplatin resistance OCCs. a Immunoblotting analysis of LC3B, ATG5, and p62 expression in (left panel) A2780CP or (right panel) COC1/DDP cells with DDB1 or CUL4A knockdown. b Representative images of the transmission electron microscope in A2780CP cells treated as in ( a ). c Quantification of the LC3 puncta number in ( b ) (*** p < 0.001, **** p < 0.0001, t -test). d Immunofluorescence analysis of LC3 in (upper panel) A2780CP and (lower panel) COC1/DDP cells with or without CRL4 knockdown. Scale Bar: 10 μm. e Quantification of autophagic vesicles in ( d ). Data represent mean ± SEM with three replicates, ** p < 0.01, *** p < 0.001, **** p < 0.0001. f , g The interaction between BECN1 and BCL2, ATG14L was detected by Co-IP assay in f A2780CP and g COC1/DDP cells with CRL4 knockdown. h Immunofluorescence analysis of CRL4-knockdown A2780CP cells transiently transfected with tandem mRFP-GFP-tagged LC3B, followed by treatment with 10 μM chloroquine for another 24 h. Scale Bar: 10 μm. i Quantification of the ratio of autolysosome (AL, red puncta) versus autophagosome (AP, yellow puncta). * p < 0.05, ** p < 0.01; *** p < 0.001, t -test. j Colocalization of mitochondria and autolysosomes analyzed by staining A2780CP cells with Lyso-Tracker Red and MitoTracker Green. Red indicates staining with Lyso-Tracker Red, green represents staining with MitoTracker Green, and yellow stands for the two colors merged together. Yellow puncta were counted as mitochondria having autolysosomes. White arrows indicate colocalization points. Scale bar: 10 μm. k Immunoblotting of mitochondrial outer membrane protein Tomm20 and inner membrane protein Tim23 in A2780CP or COC1/DDP cells transfected with lentivirus carrying shRNA targeting CUL4A (right panel) or DDB1 (left panel). GAPDH was used as a loading control for Tomm20, Tim23, DDB1, and CUL4A. Representative images were selected from three independent experiments. * p < 0.05, ** p < 0.01; *** p < 0.001, t -test. l Representative images of CRL4 knockdown OCCs incubated with BODIPY-conjugated bovine serum (DQ-BSA, red) for 1 h, or with serum- and glucose-free medium (starvation). Scale bar: 20 μm

Article Snippet: The following antibodies were used in this study: anti-CUL4A (Proteintech, 14851-1-AP, 1:2000), anti-DDB1 (HuaBio, ET1706-22, 1:2000), anti-DRP1 (C-Terminal, Proteintech, 12957-1-AP, 1:2000), anti-p62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:3000), anti-ATG5 (Proteintech, 10181-2-AP, 1:2000), anti-ATG14L (Proteintech, 19491-1-AP, 1:2000), anti-BCL-2 (Proteintech, 12789-1-AP, 1:1000), anti-MFF (Proteintech, 17090-1-AP, 1:1000), anti-phospho-MFF Ser172/Ser146 (Affinity, AF2365, 1:1000) anti-Beclin-1 (Proteintech, 11306-1-AP, 1:1000), anti-phospho-DRP1-Ser616 (CST, #3455, 1:1000), anti-phospho-DRP1-Ser637 (ABclonal, AP0812, 1:1000), anti-phospho-AMPKα-Thr172 (CST, #2535 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:2000), anti-AMPK alpha 1 (HuaBio, ET1608-40, 1:1000), anti-Parkin (HuaBio, ET1702-60, 1:1000), anti-PINK1 (HuaBio, ER1706-27, 1:1000), anti-BNIP3 (Abclonal, A5683, 1:1000), anti-BNIP3L (Abclonal, A6283, 1:1000), anti-FUNDC1 (Abclonal, A16318, 1:1000), anti-PGC-1α (Abclonal, A12348, 1:1000), anti-Tomm20 (Proteintech, 11802-1-AP, 1:2000), anti-Tim23 (Proteintech, 11123-1-AP, 1:2000).

Techniques: Western Blot, Expressing, Transmission Assay, Microscopy, Immunofluorescence, Co-Immunoprecipitation Assay, Transfection, Staining, shRNA, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

doi: 10.1038/s41392-022-01253-y

Figure Lengend Snippet: Knocking down CRL4 CUL4A/DDB1 stimulates mitophagy by inducing mitochondrial Parkin translocation in cisplatin-resistant OCCs. a The colocalization of mitochondria and autophagosome was determined by immunofluorescence staining. Briefly, A2780CP cells were observed 24 h after CRL4 CUL4A/DDB1 was knocked down, followed by staining with GFP-LC3 plasmids for 24 h and staining with MitoTracker Red for 30 min. Scale Bar:10 μm. b Graphs show the Pearson’s R values using whole cells by Image J. ( n = 4–6 fields). c Immunoblotting analysis of LC3 in the cytosolic (Cyto) and mitochondrial (Mito) fractions of A2780CP (upper panel) and COC1/DDP (lower panel) cells with or without CRL4 CUL4A/DDB1 knockdown. d Western blot analysis of Parkin, PINK1, DDB1, and CUL4A protein levels in A2780CP and COC1/DDP cells with or without CRL4 CUL4A/DDB1 knockdown. e , f Immunoblotting analysis of Parkin and PINK1 in the cytosolic (Cyto) and mitochondrial (Mito) fractions of (left panel) A2780CP and (right panel) COC1/DDP cells with or without CRL4 CUL4A/DDB1 knockdown. g Immunofluorescence analysis of the colocalization of endogenous Parkin and COX4 in A2780CP cells with or without CRL4 CUL4A/DDB1 knockdown. Scale Bar:10 μm. h Immunofluorescence analysis of the colocalization of endogenous COX4 and PINK1 in A2780CP cells with or without CRL4 CUL4A/DDB1 knockdown. Scale Bar:10 μm

Article Snippet: The following antibodies were used in this study: anti-CUL4A (Proteintech, 14851-1-AP, 1:2000), anti-DDB1 (HuaBio, ET1706-22, 1:2000), anti-DRP1 (C-Terminal, Proteintech, 12957-1-AP, 1:2000), anti-p62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:3000), anti-ATG5 (Proteintech, 10181-2-AP, 1:2000), anti-ATG14L (Proteintech, 19491-1-AP, 1:2000), anti-BCL-2 (Proteintech, 12789-1-AP, 1:1000), anti-MFF (Proteintech, 17090-1-AP, 1:1000), anti-phospho-MFF Ser172/Ser146 (Affinity, AF2365, 1:1000) anti-Beclin-1 (Proteintech, 11306-1-AP, 1:1000), anti-phospho-DRP1-Ser616 (CST, #3455, 1:1000), anti-phospho-DRP1-Ser637 (ABclonal, AP0812, 1:1000), anti-phospho-AMPKα-Thr172 (CST, #2535 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:2000), anti-AMPK alpha 1 (HuaBio, ET1608-40, 1:1000), anti-Parkin (HuaBio, ET1702-60, 1:1000), anti-PINK1 (HuaBio, ER1706-27, 1:1000), anti-BNIP3 (Abclonal, A5683, 1:1000), anti-BNIP3L (Abclonal, A6283, 1:1000), anti-FUNDC1 (Abclonal, A16318, 1:1000), anti-PGC-1α (Abclonal, A12348, 1:1000), anti-Tomm20 (Proteintech, 11802-1-AP, 1:2000), anti-Tim23 (Proteintech, 11123-1-AP, 1:2000).

Techniques: Translocation Assay, Immunofluorescence, Staining, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

doi: 10.1038/s41392-022-01253-y

Figure Lengend Snippet: Knocking down CRL4 CUL4A/DDB1 inhibits OCC growth by inducing mitophagy. a Cell proliferation determined by CCK8 assay in A2780CP cells after CRL4 CUL4A/DDB1 knockdown with or without shATG5. b Verification of ATG5 knockdown in A2780CP cells using shRNA. c , d Cell proliferation determined by CCK8 assay in A2780CP ( c ) and COC1/DDP ( d ) cells after CRL4 CUL4A/DDB1 knockdown with or without 1 mM of 3-MA treatment for 36 h. e Cell proliferation determined by colony formation assay in A2780CP cells (10,000 cells) after CRL4 CUL4A/DDB1 knockdown with or without 1 mM of 3-MA treatment for 36 h. f Cell proliferation determined by colony formation assay in A2780CP cells (3000 cells) after CRL4 CUL4A/DDB1 knockdown with or without 4 µM of CQ treatment for 24 h. g Cell proliferation determined by CCK8 assay in (left panel) A2780CP and (right panel) COC1/DDP cells after treatment with 0.5 µM MLN4924 for 24 h with or without 1 mM of 3-MA treatment for 36 h. h Cell proliferation determined by CCK8 assay in (left panel) A2780CP and (right panel) COC1/DDP cells after treatment with 0.5 µM MLN4924 for 24 h with or without 4 µM of CQ treatment for 24 h. i Cell proliferation determined by CCK8 assay in A2780CP cells after treatment with 0.5 µM MLN4924 for 24 h with or without 10 µM of Wortmannin treatment for 24 h. j Cell proliferation determined by CCK8 assay in A2780CP cells after ATG5 knockdown with or without 0.5 µM of MLN4924 treatment for 24 h. k Representative image of isolated A2780CP tumor xenografts from mice in cohorts. Female immunodeficient nude mice received a subcutaneous injection of 5 × 10 6 A2780CP cells containing either shDDB1/shCUL4A or negative control constructs (NT). End-point tumors isolated after four weeks of growth are shown. l Quantification of (left panel) total tumor weight and (right panel) tumor volume for A2780CP xenografts ( n = 5 per group). Statistical significance was determined using a t -test (* p < 0.05). m Immunohistochemical staining analysis of hematoxylin-eosin stain, DDB1, CUL4A, DRP1, and Parkin staining in NT or shCUL4A/DDB1 tumors. n Representative ventral bioluminescent images of nude mice injected with A2780CP-luc-puro cells transduced with indicated shRNA constructs on days 0, 7, 14, 21, and 28. o Longitudinal ventral bioluminescence measurement for all mice injected with A2780CP-luc-puro transduced with indicated shRNA. Data represent mean total counts (photons/sec) for each group ± SEM. ( n = 6 mice per group). p Representative end point necropsy showing differential tumor size and distribution in mice injected with A2780CP cells. Scale bar, 1 cm. Visible tumors are circled in yellow. (For all panels in this figure, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, t -test)

Article Snippet: The following antibodies were used in this study: anti-CUL4A (Proteintech, 14851-1-AP, 1:2000), anti-DDB1 (HuaBio, ET1706-22, 1:2000), anti-DRP1 (C-Terminal, Proteintech, 12957-1-AP, 1:2000), anti-p62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:3000), anti-ATG5 (Proteintech, 10181-2-AP, 1:2000), anti-ATG14L (Proteintech, 19491-1-AP, 1:2000), anti-BCL-2 (Proteintech, 12789-1-AP, 1:1000), anti-MFF (Proteintech, 17090-1-AP, 1:1000), anti-phospho-MFF Ser172/Ser146 (Affinity, AF2365, 1:1000) anti-Beclin-1 (Proteintech, 11306-1-AP, 1:1000), anti-phospho-DRP1-Ser616 (CST, #3455, 1:1000), anti-phospho-DRP1-Ser637 (ABclonal, AP0812, 1:1000), anti-phospho-AMPKα-Thr172 (CST, #2535 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:2000), anti-AMPK alpha 1 (HuaBio, ET1608-40, 1:1000), anti-Parkin (HuaBio, ET1702-60, 1:1000), anti-PINK1 (HuaBio, ER1706-27, 1:1000), anti-BNIP3 (Abclonal, A5683, 1:1000), anti-BNIP3L (Abclonal, A6283, 1:1000), anti-FUNDC1 (Abclonal, A16318, 1:1000), anti-PGC-1α (Abclonal, A12348, 1:1000), anti-Tomm20 (Proteintech, 11802-1-AP, 1:2000), anti-Tim23 (Proteintech, 11123-1-AP, 1:2000).

Techniques: CCK-8 Assay, shRNA, Colony Assay, Isolation, Injection, Negative Control, Construct, Immunohistochemical staining, Staining, Transduction

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

doi: 10.1038/s41392-022-01253-y

Figure Lengend Snippet: The schematic diagram showing the underlying mechanisms involved in mitophagy repression by CRL4 CUL4A/DDB1 in chemoresistant ovarian cancer. Downregulation of CRL4 CUL4A/DDB1 promotes mitophagy by modulating the Parkin-PINK1 axis, MFF phosphorylation, DRP1 Ser637 dephosphorylation, as well as the interaction between DRP1 and VDAC1, eventually inducing mitochondrial fission and selectively removing fragmented mitochondrial in chemoresistant ovarian cancer cells. [created with BioRender.com ( https://biorender.com/ )]

Article Snippet: The following antibodies were used in this study: anti-CUL4A (Proteintech, 14851-1-AP, 1:2000), anti-DDB1 (HuaBio, ET1706-22, 1:2000), anti-DRP1 (C-Terminal, Proteintech, 12957-1-AP, 1:2000), anti-p62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:3000), anti-ATG5 (Proteintech, 10181-2-AP, 1:2000), anti-ATG14L (Proteintech, 19491-1-AP, 1:2000), anti-BCL-2 (Proteintech, 12789-1-AP, 1:1000), anti-MFF (Proteintech, 17090-1-AP, 1:1000), anti-phospho-MFF Ser172/Ser146 (Affinity, AF2365, 1:1000) anti-Beclin-1 (Proteintech, 11306-1-AP, 1:1000), anti-phospho-DRP1-Ser616 (CST, #3455, 1:1000), anti-phospho-DRP1-Ser637 (ABclonal, AP0812, 1:1000), anti-phospho-AMPKα-Thr172 (CST, #2535 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:2000), anti-AMPK alpha 1 (HuaBio, ET1608-40, 1:1000), anti-Parkin (HuaBio, ET1702-60, 1:1000), anti-PINK1 (HuaBio, ER1706-27, 1:1000), anti-BNIP3 (Abclonal, A5683, 1:1000), anti-BNIP3L (Abclonal, A6283, 1:1000), anti-FUNDC1 (Abclonal, A16318, 1:1000), anti-PGC-1α (Abclonal, A12348, 1:1000), anti-Tomm20 (Proteintech, 11802-1-AP, 1:2000), anti-Tim23 (Proteintech, 11123-1-AP, 1:2000).

Techniques: De-Phosphorylation Assay