Journal: Oncogene

Article Title: FBXO28 suppresses liver cancer invasion and metastasis by promoting PKA-dependent SNAI2 degradation

doi: 10.1038/s41388-023-02809-0

Figure Lengend Snippet: A Hep3b cells stably expressing shCtrl, shFBXO28 alone or in combination with shSNAI2 were subjected to transwell assays. Scale bar: 10 μm. Data were presented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01 and *** P < 0.001 (one-way ANOVA). B Hep3b cells (5 × 10 6 ) stably expressing shCtrl, shFBXO28 alone or in combination with shSNAI2 were injected into NOD/SCID mice via tail vein, and the number of lung metastatic nodules was counted after mice were sacrificed at 8 weeks after injection ( n = 4–5). Scale bar: 4 mm, * P < 0.05 (one-way ANOVA). C Hematoxylin and eosin staining of the indicated lung metastatic nodules. The scale bars in left and right panel represent 500 μm and 100 μm, respectively.

Article Snippet: pcDNA3.1-SNAI2-Myc (#31698) was obtained from Addgene. pCMV6-FBXO28-Myc-Flag (#RC206490), pLenti-C-mGFP-P2A (#PS100093), pLenti-FBXO28-mGFP-P2A (#RC206490L4), pRS-shSNAI2 (#TR309225B) and pRS-shRNA control (#TR30012) plasmids were acquired from Origene Technologies. pcDNA4.0-SNAI2-HA and pLKO.1-shFBXO28 plasmids, were constructed in our lab. FBXO28 and SNAI2 deletion or point mutants were obtained from Sangon Biotech Co. Ltd, China.

Techniques: Stable Transfection, Expressing, Injection, Staining

Journal: Oncogene

Article Title: FBXO28 suppresses liver cancer invasion and metastasis by promoting PKA-dependent SNAI2 degradation

doi: 10.1038/s41388-023-02809-0

Figure Lengend Snippet: A Huh7 cells transfected with FBXO28-Flag or vector for 48 h (left panel) or Hep3b infected with lentivirus expressing shFBXO28 or shCtrl for 72 h (right panel) were subjected to IB analyses of the EMT pathway-related markers. B Total cellular RNA was extracted for RT-qPCR analysis in the same conditions as in ( A ). Data were presented as mean ± SD from three independent experiments, * P < 0.05 and ** P < 0.01 (independent T -test). C Huh7 cells expressing FBXO28-Flag or vector for 72 h by transient transfection were treated with MG132 for 6 h prior to IB analyses. D , E Huh7 cells transfected with FBXO28-Flag or a control vector ( D ) or Hep3b cells expressing shFBXO28 or shCtrl for 48 h ( E ) were treated with CHX (10 μg/mL) for the indicated time interval and subjected to IB analyses. The SNAI2 protein levels in ( D and E ) were quantified, respectively, and shown on the right panel. F Huh7 cells stably expressing FBXO28-mGFP or a control vector were transfected with SNAI2-HA for 48 h; or were transfected with SNAI2-HA or a control vector for 24 h prior to transfection with FBXO28-Flag for 24 h, followed by migration and invasion assay, respectively. Representative images are shown. Scale bar: 10 μm. Data were expressed as mean ± SD from three independent experiments. * P < 0.05 and ** P < 0.01 (one-way ANOVA).

Article Snippet: After washing with lysis buffer, the SCF FBXO28 immunocomplexes were added into the incubation reaction system comprised of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugation enzyme, SNAI2 protein (#TP760008, Origene), ATP and Ubiquitin according to instructions in the Ubiquitination Kit (#BML-UW9920, Enzolife).

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Quantitative RT-PCR, Control, Stable Transfection, Migration, Invasion Assay

Journal: Oncogene

Article Title: FBXO28 suppresses liver cancer invasion and metastasis by promoting PKA-dependent SNAI2 degradation

doi: 10.1038/s41388-023-02809-0

Figure Lengend Snippet: A , B Huh7 cells were transfected with indicated plasmids for 72 h, and then subjected to IP and IB analyses. C Huh7 cells were subjected to IP with FBXO28 antibody or normal rabbit IgG and IB analyses. D Co-localization of FBXO28 and SNAI2 by immunofluorescence staining in Hep3b cells. Representative images are shown. Scale bar: 8 μm. E HEK293T cells co-expressing FBXO28 and full-length or deletion mutants of SNAI2 (with schematic diagram shown on the upper panel in F ) were cultured for 48 h prior to IP and IB analyses. F Upper panel: A schematic representation of SNAI2 deletion mutants. Lower panel: HEK293T cells transfected with indicated plasmids for 48 h were subjected to IB analyses. Arrows indicate the target band. G In vitro ubiquitination assay using cell immunocomplex pulled down with anti-Myc beads from HEK293T cell extracts after transfection with FBXO28-Flag plasmid for 48 h. The polyubiquitination was detected by IB with anti-SNAI2 antibody. H HEK293T cells were co-transfected with indicated plasmids for 72 h. The polyubiquitylated proteins were purified by Ni-NTA beads and detected with anti-HA antibody. I HEK293T cells were transfected with indicated plasmids for 72 h followed by Ni-NTA beads purification and IB with anti-HA antibody. J HEK293T cells were transfected with indicated plasmids for 48 h prior to IP and IB analyses. SE short exposure. LE long exposure.

Article Snippet: After washing with lysis buffer, the SCF FBXO28 immunocomplexes were added into the incubation reaction system comprised of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugation enzyme, SNAI2 protein (#TP760008, Origene), ATP and Ubiquitin according to instructions in the Ubiquitination Kit (#BML-UW9920, Enzolife).

Techniques: Transfection, Immunofluorescence, Staining, Expressing, Cell Culture, In Vitro, Ubiquitin Assay, Plasmid Preparation, Purification

Journal: Oncogene

Article Title: FBXO28 suppresses liver cancer invasion and metastasis by promoting PKA-dependent SNAI2 degradation

doi: 10.1038/s41388-023-02809-0

Figure Lengend Snippet: A Huh7 cells transiently expressing FBXO28-Flag were treated with DMSO or 0.2 μM H89, 4 μM CHIR-99021, 5 μM CID755673, 0.5 μM PF-3758309, 2 nM Dinaciclib for 24 h, followed by IB analyses. B Huh7 cells transiently expressing FBXO28-Flag or a control vector were treated with DMSO or 0.5 μM H89 for 24 h prior to IB analyses. C HEK293T cells were transfected with indicated plasmids for 24 h, and then treated with DMSO or 0.2 μM H89 for 24 h, followed by IP and IB analyses. D HEK293T cells were transfected with indicated plasmids for 48 h prior to treatment with DMSO or 0.2 μM H89 for 24 h. The polyubiquitylated proteins were purified by Ni-NTA beads and detected with anti-HA antibody. E Huh7 cells expressed FBXO28-Flag or a control vector for 24 h were treated in the absence or presence of 0.5 μM H89 or 1 μM KT5720 for another 24 h, followed by transwell assays. Scale bar: 10 μm. Data were presented as mean ± SD ( n = 3). ** P < 0.01 and *** P < 0.001 (one-way ANOVA). F Illustration of conservative amino acids sequences in the C-terminal of SNAI2 across different species using UniProt dataset. Predicted phosphorylation sites by PKA are highlighted. G , H HEK293T cells were transfected with indicated plasmids for 48 h and subjected to IP and IB analyses in ( G ) or Ni-NTA beads purification in ( H ). The asterisks represent non-specific band. I Huh7 cells stably expressing FBXO28-mGFP or a control vector were transfected with SNAI2-HA or SNAI2 S3A-HA for 48 h and treated with CHX for the indicated time interval, followed by IB analyses. J Huh7 cells were expressed with SNAI2 S3A-HA or a control vector for 24 h prior to transfection with FBXO28-Flag for another 24 h, followed by transwell assays. Scale bar: 10 μm. Data were shown as mean ± SD ( n = 3). * P < 0.05 (one-way ANOVA).

Article Snippet: After washing with lysis buffer, the SCF FBXO28 immunocomplexes were added into the incubation reaction system comprised of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugation enzyme, SNAI2 protein (#TP760008, Origene), ATP and Ubiquitin according to instructions in the Ubiquitination Kit (#BML-UW9920, Enzolife).

Techniques: Expressing, Control, Plasmid Preparation, Transfection, Purification, Stable Transfection

Journal: Oncogene

Article Title: FBXO28 suppresses liver cancer invasion and metastasis by promoting PKA-dependent SNAI2 degradation

doi: 10.1038/s41388-023-02809-0

Figure Lengend Snippet: A Protein levels of FBXO28, PKAα, p-CREB and SNAI2 in paired HCC specimens by IB analyses. Arrows indicate target band. B Quantification data of ( A ) were expressed as mean ± SD ( n = 8). * P < 0.05, ** P < 0.01 and *** P < 0.001 (paired T -test). C Expression of PRKACA mRNA levels in HCC and normal cohorts in TCGA dataset. ** P < 0.01 (independent T -test). D Correlation of PRKACA expression and overall survival (OS) via Kaplan–Meier survival analysis of liver cancer patients. E Working model for the regulatory function of FBXO28 during HCC EMT process by degrading SNAI2 via UPS.

Article Snippet: After washing with lysis buffer, the SCF FBXO28 immunocomplexes were added into the incubation reaction system comprised of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugation enzyme, SNAI2 protein (#TP760008, Origene), ATP and Ubiquitin according to instructions in the Ubiquitination Kit (#BML-UW9920, Enzolife).

Techniques: Expressing

Journal: Cell Death and Differentiation

Article Title: A novel microRNA regulator of prostate cancer epithelial–mesenchymal transition

doi: 10.1038/cdd.2017.69

Figure Lengend Snippet: miR-3622a inhibits EMT by direct targeting of EMT effectors in PCa. (a) Relative ZEB1, SNAI2 and CTNNB1 mRNA expression upon indicated treatments in PPEC, BPH1 cells and PCa cell lines (PC3, LNCaP) as assessed by real-time PCR. The data were normalized to GAPDH control. (b)Immunoblots for endogenous ZEB1, SNAI2, CTNNB1 protein in anti-miR-CON/ anti-miR-3622a-transduced BPH1 cells (left panels) and miR-CON/ miR-3622a transfected PC3 cells (middle panels). Immunoblots for LNCaP cells are represented in right panels. GAPDH was used a loading control.(c) Schematic representation of ZEB1, CTNNB1, SNAI2 3′-UTRs showing putative miR-3622a target site/sites. For luciferase reporter assays, the potential miR-3622a binding sites were mutated to the sequences shown below. (d) Luciferase reporter assays with the indicated wild type and mutated 3′-UTR constructs or control luciferase construct co-transfected with anti-miR-CON/anti-miR-3622a-transfected BPH1 cells (left panel) and miR-CON/miR-3622a-transfected PC3 and LNCaP cells (middle and right panels, respectively). Firefly luciferase values were normalized to Renilla luciferase activity and plotted as relative luciferase activity

Article Snippet: Trilencer-27 predesigned siRNA (Origene) was used for siRNA-mediated knockdowns of ZEB1 (SR304746), SNAI2 (SR304468), and CTNNB1 (SR301063).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Binding Assay, Construct, Activity Assay

Journal: Cell Death and Differentiation

Article Title: A novel microRNA regulator of prostate cancer epithelial–mesenchymal transition

doi: 10.1038/cdd.2017.69

Figure Lengend Snippet: ZEB1 and SNAI2 are functionally relevant targets of miR-3622a in prostate cancer. PC3 cells were transfected with siRNA specific to ZEB1/SNAI2/CTNNB1 or a nonspecific (NS) control siRNA for 72 h followed by functional assays (a,b). (a) Transwell invasion assay and (b) migration assay in NS/ZEB1/SNAI2/CTNNB1 siRNA-transfected PC3 cells. Scale bar: 100 μm. To determine whether ZEB1 and SNAI2 are the key mediators of EMT induced by miR-3622a inhibition in the non-transformed BPH1 cell line, we performed siRNA-mediated inhibition of ZEB1 and SNAI2 in miR-3622a-inhibited BPHI cells (Anti-miR-3622a BPHI.) (Supplementary Figure S4B) followed by functional assays (c–e). (c) Transwell invasion assay and (d) migration assay in NS/ZEB1/SNAI2 siRNA-transfected BPH1 cells. Scale bar: 200 μm. (e) Relative mRNA expression of CDH1 in NS/ZEB1/SNAI2 siRNA-transfected BPH1 cells as assessed by real time PCR. The data were normalized to GAPDH control. (f) Relative expression levels of ZEB1 and SNAI2 (each normalized to GAPDH) and miR-3622a (normalized to RNU48) as assessed by real-time PCR in prostate cancer clinical tissues

Article Snippet: Trilencer-27 predesigned siRNA (Origene) was used for siRNA-mediated knockdowns of ZEB1 (SR304746), SNAI2 (SR304468), and CTNNB1 (SR301063).

Techniques: Transfection, Functional Assay, Transwell Invasion Assay, Migration, Inhibition, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet: Cell line-dependent reversibility of acquired Talazoparib resistance. ( a ) Immunoblot showing PAR level of PSN1 parental cells, TalaR cells maintained in media with Talazoparib (TalaR-M) and TalaR cells cultured in drug-free media for 4 weeks (TalaR-DF). ( b ) Talazoparib IC 50 chart showing reversibility of acquired resistance in PSN1 cells over 4 weeks after drug withdrawal. ( c – f ) Clonogenic assay showing Talazoparib sensitivity of parental and acquired resistant cells in PSN1, PANC1, SW1990 and HCC1806 cells which were maintained in drug media (Tala-M) and in drug free media for 4 weeks (Tala-DF). ( g , h ) Volcano plots showing differentially expressed genes between TalaR-M and TalaR-DF in PSN1 and HCC1806 cells. ( i , j ) UMAP representations of HCC1806 parental and resistant subpools, colored by cell line ( i ) or cluster identity ( j ). ( k , l ) Violin plots of SNAI2 ( k ) and TWIST1 ( l ) gene expression profile across subpopulation clusters shown in ( j ).

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques: Western Blot, Cell Culture, Clonogenic Assay, Expressing

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet: Talazoparib induces EMT signature and SNAI2 expression which partially drives resistance. ( a , b ) Heatmap and volcano plot of RNAseq showing top differentially expressed EMT genes under 3-day Talazoparib treatment in PSN1 cells. ( c ) Immunoblot showing SNAI2 expression in Talazoparib sensitive and insensitive PDAC cell lines. ( d , e ) qPCR showing dose- and time-dependent induction of SNAI2 under Talazoparib treatment in PSN1 cells. Two independent sets of SNAI2 primers were used. ( f ) qPCR showing Talazoparib at 100 nM induces SNAI2 expression across different cell lines. Two-fold increase was highlighted in red dashed line. ( g ) qPCR showing induction of SNAI2 by different clinical PARPis. ( h ) Immunoblot showing SNAI2 upregulation in cell lines with acquired resistance to Talazoparib. ( i ) qPCR showing SNAI2 mRNA level in parental and TalaR cells. ( j ) Immunoblot showing expression of SNAI2 and other EMT-TF in PSN1 parental, TalaR-M and TalaR-DF cells. ( k , m ) Clonogenic assay showing cells gained resistance to Talazoparib after SNAI2 by CRISPRa in PSN1 and SW1990 cells. ( l , n ) Immunoblot showing induced SNAI2 expression by CRISPRa in PSN1 and SW1990 cells. ( o ) IncuCyte analysis curves showing growth of PSN1 parental and TalaR cells after SNAI2 knockout. Insert shows Indel% and knockout score.

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques: Expressing, Western Blot, Clonogenic Assay, Knock-Out

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet: PARP1 protein directly regulates SNAI2 transcription through transcription suppression. ( a ) qPCR and immunoblot showing KD level and SNAI2 expression in cell lines with PARP1 stable KD. ( b ) Summary of open and closed peaks in ATACseq on PSN1 cells treated with DMSO or Talazoparib for 72 h. Three biological replicates are shown for each group. ( c ) Venn diagram showing the number of overlapped gene between ATACseq open genes and RNAseq up genes. Waterfall plots shows enriched pathways from overlapped genes. ( d ) Representative ATACseq peaks showing chromatin accessibility changes on SNAI2 promoter in PANC1 cells with PARP1 stable KD. Three biological replicates are shown for each group. ( e ) ChIP-qPCR by using IgG or PARP1 antibody pulldown in PSN1 cells treated with DMSO or Talazoparib (50 nM, 72 h). Four pairs of primes surrounding SNAI2 promoter (set-1, -3, -7 and -9) ranging from − 2 to + 0.7 kb TSS were used. Same sets of primers were used below. ( f ) ChIP-qPCR by using RNA polymerase II (RNA PolII) antibody pulldown in PSN1 parental and TalaR cells. ( g , h ) ChIP-qPCR by using PARP1 antibody or H3K4me3 antibody pulldown in PANC1 parental and TalaR cells.

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet: CHD1L depletion re-sensitizes cells with acquired resistance to Talazoparib. ( a ) Clonogenic assay showing growth of PSN1 TalaR-M cells with CHD1L stable knockdown. ( b ) Immunoblot showing expression level of CHD1L and SNAI2 in CHD1L stable knockdown cells as in ( a ). ( c ) IncuCyte curves showing the growth PSN1 TalaR-M with inducible CHD1L knockdown with and without doxycycline induction. ( d ) qPCR showing mRNA level of CHD1L and SNAI2 in PSN1 TalaR-M with inducible CHD1L knockdown with and without doxycycline induction as in ( c ). ( e ) IncuCyte curves showing the growth of PSN1 parental and TalaR-M with CHD1L knockout. ( f ) IncuCyte curves showing the growth of HCC1806 parental and TalaR-M with CHD1L knockout. ( g , h ) Immunoblot and qPCR showing CHD1L and SNAI2 level in HCC1806 parental and TalaR-M with CHD1L knockout as in ( f ).

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques: Clonogenic Assay, Western Blot, Expressing, Knock-Out

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet:

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques:

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet:

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques:

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet:

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques: shRNA

Journal: Scientific Reports

Article Title: PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

doi: 10.1038/s41598-022-16623-3

Figure Lengend Snippet:

Article Snippet: SNAI2 cDNA expressing plasmid was purchased from Origene (RC202365) and transfected into cells by using Fugene HD transfection reagent (Promega).

Techniques: