Journal: Cancer research

Article Title: Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake

doi: 10.1158/0008-5472.CAN-19-1957

Figure Lengend Snippet: Identification of ARF4 and VCP as regulators of NIS activity. A, Western blot analysis of whole-cell lysate and PM fraction in MDA-MB-231 (NIS+) cells used in MS/MS. B, Top hits for putative NIS interactors identified by MS/MS (peptides ≥ 6). C and D, Western blot analysis and RAI uptake of MDA-MB-231 (NIS+) cells transfected with esiRNA specific for indicated NIS interactors. NT, nontransfected cells. E and F, Western blot analysis and RAI uptake in MDA-MB-231 (NIS+) cells, TPC-1 (NIS+) cells, and human primary thyrocytes transfected with ARF4 siRNA (E) or ARF4 (F). G and H, Same as E and F, but cells transfected with VCP siRNA (G) or VCP (H). NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Untagged-ARF4 plasmid was purchased from Origene (#SC119092), whereas wild-type (WT) and mutant (QQ) VCP (rat) plasmid were kindly provided by Dr. Yihong Ye (National Institutes of Health, Bethesda, MD; refs. 11 , 12 ).

Techniques: Activity Assay, Western Blot, Tandem Mass Spectroscopy, Transfection, esiRNA

Journal: Cancer research

Article Title: Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake

doi: 10.1158/0008-5472.CAN-19-1957

Figure Lengend Snippet: Involvement of ARF4 in trafficking NIS at the PM. A and B, HILO microscopy images demonstrating trafficking of ARF4-dsRED (red), NIS-GFP (green), and colocalization (yellow) to the PM in HeLa cells. Video capture times (hr:min:sec) are indicated. PM regions in framed areas are magnified in bottom right, which highlight the movement of ARF4 and NIS (white and orange arrowheads; see bottom). Scale bars, 10 μm. C, Representative images of NIS-GFP movement patterns tracked using ImageJ software. Scale bars, 10 μm. D, Box-whisker plot of velocity (μm/sec) and distance traveled (μm) of NIS-GFP in HeLa cells transfected with ARF4 (n = 475) or VO (n = 339). E, RAI uptake in TPC-1 (NIS+) and MDA-MB-231 (NIS+) cells transfected with ARF4 and treated with dynasore for 1 hour prior to addition of 125I. F, Western blot analysis of NIS expression levels in TPC-1 (NIS+) cells as described in E. G, RAI uptake in TPC-1 cells transfected with ARF4, as well as WT NIS, 574AAAK577-mutant NIS, or 475ALAS478-mutant NIS. H, Representative co-IP assay for ARF4 with WT or mutant NIS. NS, not significant; *, P < 0.05; ***, P < 0.001.

Article Snippet: Untagged-ARF4 plasmid was purchased from Origene (#SC119092), whereas wild-type (WT) and mutant (QQ) VCP (rat) plasmid were kindly provided by Dr. Yihong Ye (National Institutes of Health, Bethesda, MD; refs. 11 , 12 ).

Techniques: Microscopy, Software, Whisker Assay, Transfection, Western Blot, Expressing, Mutagenesis, Co-Immunoprecipitation Assay

Journal: Cancer research

Article Title: Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake

doi: 10.1158/0008-5472.CAN-19-1957

Figure Lengend Snippet: ARF4 and VCP bind NIS in vitro and modulate PM NIS. A, Co-IP assays in MDA-MB-231 (NIS+) cells showing specific interaction between NIS and ARF4 (left) or VCP (right). B, Same as A, but in TPC-1 (NIS+) cells. C, PLA demonstrating specific interaction between NIS-MYC and ARF4 (top) or VCP (bottom) in MDA-MB-231, TPC-1, and HeLa cells. Red fluorescent spots, specific interactions. Blue, DAPI nuclear staining. Magnification, ×100. Scale bars, 10 μm. D and E, Western blot analysis of NIS protein levels at the PM relative to Na+/K+ ATPase following the CSBA in TPC-1 (NIS+) cells (D) and MDA-MB-231 (NIS+) cells (E) after ARF4 transfection. F and G, Same as D and E, but after VCP transfection. *, P < 0.05; ***, P < 0.001.

Article Snippet: Untagged-ARF4 plasmid was purchased from Origene (#SC119092), whereas wild-type (WT) and mutant (QQ) VCP (rat) plasmid were kindly provided by Dr. Yihong Ye (National Institutes of Health, Bethesda, MD; refs. 11 , 12 ).

Techniques: In Vitro, Co-Immunoprecipitation Assay, Staining, Western Blot, Transfection

Journal: Cancer research

Article Title: Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake

doi: 10.1158/0008-5472.CAN-19-1957

Figure Lengend Snippet: VCP and ARF4 expression is associated with poorer survival and response to RAI. A, ARF4 (left) and VCP (right) expression in normal thyroid and PTC in the THCA TCGA data set. B, VCP expression in normal thyroid (n = 12), PDTC (n = 17), and ATC (n = 20). C, ARF4 (left) and VCP (right) expression in PTC with BRAF-like (n = 272) or RAS-like genetic signatures (n = 119). D, ARF4 expression in PTC with indicated genetic alterations. E, Frequency (%) of indicated genetic alterations in PTC with low (Q1Q2) versus high ARF4 (Q3Q4) expression. F and G, Same as D and E, but for VCP expression. H–J, DFS for THCA with high (Q3Q4; >12.71) versus low (Q1Q2; <12.71) VCP expression for the entire PTC cohort (H), RAI-treated patients (I) and non–RAI-treated patients (J). K, Hazard ratios ±95% CI for patients stratified on median VCP and ARF4 tumoral expression in THCA with the indicated treatment and genetic signature or alteration. L and M, DFS for THCA with high (Q3Q4; >11.84) versus low (Q1Q2; <11.84) ARF4 expression for RAI-treated PTC patients (L) and non–RAI-treated PTC patients (M). NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Untagged-ARF4 plasmid was purchased from Origene (#SC119092), whereas wild-type (WT) and mutant (QQ) VCP (rat) plasmid were kindly provided by Dr. Yihong Ye (National Institutes of Health, Bethesda, MD; refs. 11 , 12 ).

Techniques: Expressing

Journal: Cancer research

Article Title: Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake

doi: 10.1158/0008-5472.CAN-19-1957

Figure Lengend Snippet: Putative model of NIS trafficking. NIS maintains a delicate balance between protein synthesis, folding, assembly, trafficking, and degradation. (i) We propose NIS is glycosylated in the ER and upon correct folding transported to the Golgi. (ii) Protein surveillance pathways exist that target NIS for ERAD. As VCP does not require ATPase activity to inhibit NIS function, it is likely VCP acts to unfold NIS (iii) prior to proteasomal degradation. (iv) ARF4 recognizes the VAPK motif in the NIS C-terminus and promotes vesicular trafficking to the PM, where NIS is active (v). (vi) PBF has a YARF endocytosis motif and acts to bind and internalize NIS away from the PM in a clathrin-dependent process. (vii) Although inhibition of recycling by dynasore suggests that ARF4 shuttles NIS to the PM, other proteins must promote recycling of NIS to the PM as with most PM transporters.

Article Snippet: Untagged-ARF4 plasmid was purchased from Origene (#SC119092), whereas wild-type (WT) and mutant (QQ) VCP (rat) plasmid were kindly provided by Dr. Yihong Ye (National Institutes of Health, Bethesda, MD; refs. 11 , 12 ).

Techniques: Activity Assay, Inhibition

Journal:

Article Title: Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

doi: 10.1073/pnas.0500095102

Figure Lengend Snippet: The rhodopsin C-terminal peptide and the GST fusion protein containing the C terminus of rhodopsin specifically bind a 20-kDa protein, a member of the ARF family of small GTPases. (A) RTCs move vectorially (dashed arrow) from the Golgi (G) and the TGN, through the ellipsoid region filled with mitochondria (M), to the connecting cilium (C), where they fuse with the plasma membrane of the RIS. Newly synthesized rhodopsin is then delivered to the ROS. N, nucleus; Sy, synapse. (B) Sequence of the frog rhodopsin C-terminal peptide (peptide C) folded as predicted from the crystal structure of rhodopsin (29). The truncated peptide (peptide CΔ5) models the adRP mutation Q344ter. The UV-activatable crosslinker Br-DAP was attached to the N-terminally biotinylated peptide C, and CΔ5, as indicated. (C) Retinal PNS was incubated with the biotinylated Br-DAP peptides. After UV illumination, proteins crosslinked to the peptides were either (i) separated by SDS/PAGE, blotted and probed with SA-HRP followed by the chemiluminescent immunodetection system (Left)or(ii) bound to SA-Sepharose and eluted (SA-Seph. B.), detected as above (Right). A 20-kDa protein (*) specifically crosslinked to peptide C. The band detected in the sample with peptide C and CΔ5 after SA-Sepharose binding (♦) is SA in complex with biotin (biotin-SA; see below). (D) Sequence analysis by microcapillary reverse-phase HPLC nanoelectrospray tandem MS of the 20-kDa band (*), excised from the silver-stained gel of the sample crosslinked to peptide C, bound, and eluted from SA-Sepharose, showed the presence of ARF4 (peptide 1) and potentially another GTPase that belongs to the class I ARFs (peptide 2). Peptide 3 is highly conserved among all ARFs. Sequence analysis of the band present in samples with peptide C and CΔ5(♦) revealed three tryptic fragments originating from SA in complex with biotin (biotin-SA), which was likely released from the SA-Sepharose column during elution. (E) GST fusion proteins were separated by SDS/PAGE and stained with SilverStain (Left). The GST fusion protein containing the rhodopsin C-terminal sequence (C) is a 30-kDa protein, the truncated protein lacking the regulatory sequence (CΔ5) migrates at 29 kDa and GST alone (-) at 28 kDa. The 26-kDa band present in all lanes is a breakdown product of GST. Immunoblotting of GST-fusion proteins with anti-rhodopsin C-terminal mAb 11D5 (Center) reveals that the fusion protein C contains the mAb 11D5 antigenic site, whereas CΔ5 does not. In GST pull-down assays (Right), GST-C specifically bound a 20-kDa protein (*). The higher-Mr bands are GST-fusion proteins and their breakdown products, as in Left.

Article Snippet: The ARF4 peptide IQEAAEELQKMLQ was synthesized and conjugated to keyhole limpet hemocyanin by SynPep (Dublin, CA).

Techniques: Clinical Proteomics, Membrane, Synthesized, Sequencing, Mutagenesis, Incubation, SDS Page, Immunodetection, Binding Assay, Staining, Western Blot

Journal:

Article Title: Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

doi: 10.1073/pnas.0500095102

Figure Lengend Snippet: Interaction of the rhodopsin C terminus with the small GTPase ARF4. (A) Affinity-purified anti-ARF4 antibody recognizes a 20-kDa protein (*) inthe retinal cytosol. (B) Retinal cytosolic proteins crosslinked to the biotinylated Br-DAP peptides separated by SDS/PAGE and immunoblotted with anti-ARF4 or with SA-HRP. Anti-ARF4 detects a 20-kDa protein (*), indicating that ARF4 is present in all cytosolic samples (Left). A duplicate blot probed with SA-HRP (Right) reveals that, in UV-illuminated samples (+UV), the 20-kDa band (*) also contains biotinylated Br-DAP peptide C. Br-DAP peptide CΔ5 does not crosslink to ARF4. (C) Retinal cytosolic proteins crosslinked to the biotinylated Br-DAP peptides, purified with SA-Sepharose, immunoblotted with anti-ARF4 or SA-HRP. ARF4 is readily detectable with anti-ARF4 in the starting material (S) and the unbound fraction (UB) in the samples containing both peptides. However, ARF4 is detected exclusively, albeit in a small quantity, in the bound fraction containing peptide C (peptide C, lane B) but not peptide CΔ5. SA-HRP detects biotinylated Br-DAP peptide C in all three fractions (S, UB, B) but only a ≈20-kDa (♦) and lower-Mr proteins in the bound fraction containing peptide CΔ5 (lane B). Because peptide CΔ5 does not specifically crosslink to any cytosolic proteins in the 20-kDa range (see Fig. 1C), there are no crosslinked products detectable with SA-HRP in the starting or unbound material (peptide CΔ5, lanes S and UB) that could be the source of this material. This protein is biotin-SA and its breakdown products released in abundance from SA-Sepharose during elution (see also Fig. 1 C and D). Biotin-SA eluted with biotinylated Br-DAP peptide C (♦) interferes with the mobility of the specific crosslinked protein containing ARF4 and peptide C (*), which appears to be migrating more slowly in the bound fraction than in the starting material (compare lanes S and B, probed either with anti-ARF4 or with SA-HRP).

Article Snippet: The ARF4 peptide IQEAAEELQKMLQ was synthesized and conjugated to keyhole limpet hemocyanin by SynPep (Dublin, CA).

Techniques: Affinity Purification, SDS Page, Purification

Journal:

Article Title: Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

doi: 10.1073/pnas.0500095102

Figure Lengend Snippet: Newly synthesized rhodopsin is crosslinked to ARF4. (A) Sulfosuccinimidyl-3-[(4-azidophenyl) dithio] propionate (sSADP) was added (+) or not (-) to radiolabeled retinal PNS for 20 min before crosslinking was initiated by UV illumination. Crosslinked proteins were separated on nonreducing (-βME) 8% SDS/PAGE. The autoradiogram of the Commassie stained gel (Left Center), detecting mostly [35S]-rhodopsin, revealed, in addition to the rhodopsin monomer (Rh), a 50-kDa crosslinked product (*) containing newly synthesized rhodopsin and a ≈20-kDa protein. Anti-ARF4 (Right Center) recognized a doublet in the 20-kDa range and a 50-kDa crosslinked protein (*) on the nonreducing gels. The ARF4 doublet was also recognized by anti-glutathione–protein complex antibody (Right), suggesting that both bands contain glutathionated ARF4. Because the 50-kDa crosslinked product is detected in the [35S]-rhodopsin autoradiogram and is recognized by anti-ARF4, the protein crosslinked to rhodopsin is identified as ARF4. (B) The 50-kDa crosslinked product was excised from a nonreducing gel (-βME) and separated on reducing (+βME) 12% SDS/PAGE (Left). The autoradiogram of this gel (Center) shows that cleavage by βME released radiolabeled rhodopsin (Rh), whereas the anti-ARF4 immunoblot (Right) shows that βME also released ARF4 (*) from the 50-kDa crosslinked product.

Article Snippet: The ARF4 peptide IQEAAEELQKMLQ was synthesized and conjugated to keyhole limpet hemocyanin by SynPep (Dublin, CA).

Techniques: Synthesized, SDS Page, Staining, Western Blot

Journal:

Article Title: Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

doi: 10.1073/pnas.0500095102

Figure Lengend Snippet: ARF4 is concentrated in the vicinity of the Golgi/TGN, where it regulates budding of post-Golgi RTCs. (A and D) ARF4 (green) is a retinal cytosolic protein. It is highly concentrated around the photoreceptor Golgi (G) in the RIS (arrows in A) but completely absent from the ROS. Dashed lines outline the RIS and the base of the ROS of a single photoreceptor. M, mitochondria; N, nucleus (blue); retinal layers: ONL, outer nuclear; OPL, outer plexiform; INL, inner nuclear; IPL, inner plexiform. (B) Rhodopsin (red) is abundant in the ROS. In the RIS it is detectable, with mAb 11D5, mostly in the Golgi (arrows), and the RTCs (star). (C and F) EM images detailing photoreceptor structure and the localization of biosynthetic organelles. RPE, retinal pigment epithelium; COS, cone outer segments; asterisk, oil droplet. (Bar, 5 μm in A–C; 10 μm in D; and 0.5 μm in F.) (E) Anti-ARF4 and the ARF4 peptide were added to the in vitro trafficking assay at concentrations inhibitory for mAb 11D5 and the rhodopsin C-terminal peptide but not for the control antibody or peptide (4, 33). (Upper) A representative autoradiogram. (Lower) Budding of RTCs in the control +ATP was set at 100%. The data are presented as the means ± SE of three separate experiments. Like mAb 1D5, anti-ARF4 and the ARF4 peptide profoundly inhibited RTC budding (**, P < 0.005).

Article Snippet: The ARF4 peptide IQEAAEELQKMLQ was synthesized and conjugated to keyhole limpet hemocyanin by SynPep (Dublin, CA).

Techniques: In Vitro, Control

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: ARF4 binds to USP7 and is downregulated by USP7 inhibition A-C Proteins bound to USP7 were identified in SHG-140 by Co-IP and MS, and the screened proteins were analyzed for the KEGG pathway. D Heat map showing the comparative results of TMT proteomics analysis after 48 h treatment using DMSO or P5091 (2µM) in SHG-140. E Venn diagram showing down-regulated proteins in response to P5091 (yellow), USP7 interaction candidates identified by Co-IP (purple), and overlapping proteins. F Volcano plot showing the differential proteins from TMT proteomics analysis and the location of ARF4

Article Snippet: GeneChem (China) designed two shRNAs lentivirus against USP7, shUSP7-1: 5’ -UGUAUCUAUUGACUGCCCUTT- 3’ and shUSP7-2: 5’ -UGGAUUUGUGGUUACGUUACUC-3’, and an ARF4 overexpression lentivirus.

Techniques: Inhibition, Co-Immunoprecipitation Assay

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: ARF4 is an anti-apoptotic substrate for USP7 A Interaction between USP7 and ARF4 in SHG-140 was determined using the Co-IP assay. B SHG-140 cells were transfected with different shUSP7 for 24 h and ARF4 expression was determined by western blotting, n = 3. C. Expression of ARF4 was determined by western blotting after treatment of SHG-140 with different concentrations of P5091 for 48 h, n = 3. D , E Changes in apoptosis after overexpression of ARF4 in SHG-140 and T98G cells treated with P5091 (2 µM) for 48 h or transfected with shUSP7 for 24 h were detected by flow cytometry, n = 3. Q3 (Annexin V-FITC + PI-) subpopulation was considered early-stage apoptosis and Q2 (Annexin V-FITC + PI+) was late-stage apoptosis or necrosis. Cell proportion comparisons between groups were done in both early- and late-stage subpopulation separately and in total. F-I Changes in apoptotic proteins after overexpression of ARF4 in SHG-140 and T98G cells treated with P5091 (2 µM) for 48 h or transfected with shUSP7 for 24 h were observed by western blotting analysis, n = 3. Statistics are expressed as mean ± S.E.M., # P = NS, *P < 0.05,**P < 0.01,***P < 0.001, or ****P < 0.0001

Article Snippet: GeneChem (China) designed two shRNAs lentivirus against USP7, shUSP7-1: 5’ -UGUAUCUAUUGACUGCCCUTT- 3’ and shUSP7-2: 5’ -UGGAUUUGUGGUUACGUUACUC-3’, and an ARF4 overexpression lentivirus.

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Western Blot, Over Expression, Flow Cytometry

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: USP7 regulates ARF4 stability through K48-linked deubiquitination A , B . After transfection of SHG-140 cells with shUSP7 for 24 h or treatment of cells with P5091 (2 µM) for 48 h, cells were treated with CHX (100 µg/ml) at different times and ARF4 expression was analyzed by western blotting, n = 3. C , D . SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with the proteasome inhibitor MG132 (10 µM) for 6 h. ARF4 expression was analyzed by western blotting, n = 3. E , F . SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with MG132 (10 µM) for 6 h. The protein extracts were immunoprecipitated with IgG beads of anti-ARF4, and then ubiquitin and ARF4 expression were detected by western blotting. G , H . SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with MG132 (10 µM) for 6 h. The protein extracts were immunoprecipitated with IgG beads of anti-ARF4, and then the expression of K48-ubiquitin, K63-ubiquitin and ARF4 was detected by western blotting. I A proposed mechanism for USP7 to regulate the ARF4 level in GBM. All statistics are expressed as mean ± S.E.M., # P = NS, *P < 0.05,**P < 0.01 or ***P < 0.001

Article Snippet: GeneChem (China) designed two shRNAs lentivirus against USP7, shUSP7-1: 5’ -UGUAUCUAUUGACUGCCCUTT- 3’ and shUSP7-2: 5’ -UGGAUUUGUGGUUACGUUACUC-3’, and an ARF4 overexpression lentivirus.

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: P5091 inhibits tumorigenicity in intracranial xenograft models Female BALB/c nude mice were injected intraperitoneally with PBS, P5091 (5 mg/kg/day, 10 mg/kg/day) and treated 2 days per week. Treatment started on day 7 after implantation and lasted for approximately 21 days. A Representative images of bioluminescence in mice on days 7, 14 and 28 after implantation. B Quantitative analysis of these bioluminescence images, n = 6. C Overall survival of the PBS and P5091 treatment groups, n = 6. D Representative H&E images of tumor sections. Scale bar, 300 μm. E Representative IHC images of tumor sections for anti-USP7, anti-ARF4, anti-BAX, anti-BCL-2, and anti-CLEAVED-CASPASE. Scale bar, 300 μm. Statistics are expressed as mean ± S.E.M., # P = NS, *P < 0.05,**P < 0.01 or ****P < 0.0001

Article Snippet: GeneChem (China) designed two shRNAs lentivirus against USP7, shUSP7-1: 5’ -UGUAUCUAUUGACUGCCCUTT- 3’ and shUSP7-2: 5’ -UGGAUUUGUGGUUACGUUACUC-3’, and an ARF4 overexpression lentivirus.

Techniques: Injection

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: ARF4 binds to USP7 and is downregulated by USP7 inhibition A-C Proteins bound to USP7 were identified in SHG-140 by Co-IP and MS, and the screened proteins were analyzed for the KEGG pathway. D Heat map showing the comparative results of TMT proteomics analysis after 48 h treatment using DMSO or P5091 (2µM) in SHG-140. E Venn diagram showing down-regulated proteins in response to P5091 (yellow), USP7 interaction candidates identified by Co-IP (purple), and overlapping proteins. F Volcano plot showing the differential proteins from TMT proteomics analysis and the location of ARF4

Article Snippet: Sangon Biotech (China) designed two siRNAs against ARF4, siARF4-1: 5’ -GCAAGACAACCAUUCUGUAUATT- 3’ and siARF4-2: 5’ - CCAUCAGUGAAAUGACAGAUATT- 3’.

Techniques: Inhibition, Co-Immunoprecipitation Assay

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: ARF4 is an anti-apoptotic substrate for USP7 A Interaction between USP7 and ARF4 in SHG-140 was determined using the Co-IP assay. B SHG-140 cells were transfected with different shUSP7 for 24 h and ARF4 expression was determined by western blotting, n = 3. C. Expression of ARF4 was determined by western blotting after treatment of SHG-140 with different concentrations of P5091 for 48 h, n = 3. D , E Changes in apoptosis after overexpression of ARF4 in SHG-140 and T98G cells treated with P5091 (2 µM) for 48 h or transfected with shUSP7 for 24 h were detected by flow cytometry, n = 3. Q3 (Annexin V-FITC + PI-) subpopulation was considered early-stage apoptosis and Q2 (Annexin V-FITC + PI+) was late-stage apoptosis or necrosis. Cell proportion comparisons between groups were done in both early- and late-stage subpopulation separately and in total. F-I Changes in apoptotic proteins after overexpression of ARF4 in SHG-140 and T98G cells treated with P5091 (2 µM) for 48 h or transfected with shUSP7 for 24 h were observed by western blotting analysis, n = 3. Statistics are expressed as mean ± S.E.M., # P = NS, *P < 0.05,**P < 0.01,***P < 0.001, or ****P < 0.0001

Article Snippet: Sangon Biotech (China) designed two siRNAs against ARF4, siARF4-1: 5’ -GCAAGACAACCAUUCUGUAUATT- 3’ and siARF4-2: 5’ - CCAUCAGUGAAAUGACAGAUATT- 3’.

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Western Blot, Over Expression, Flow Cytometry

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: USP7 regulates ARF4 stability through K48-linked deubiquitination A , B . After transfection of SHG-140 cells with shUSP7 for 24 h or treatment of cells with P5091 (2 µM) for 48 h, cells were treated with CHX (100 µg/ml) at different times and ARF4 expression was analyzed by western blotting, n = 3. C , D . SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with the proteasome inhibitor MG132 (10 µM) for 6 h. ARF4 expression was analyzed by western blotting, n = 3. E , F . SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with MG132 (10 µM) for 6 h. The protein extracts were immunoprecipitated with IgG beads of anti-ARF4, and then ubiquitin and ARF4 expression were detected by western blotting. G , H . SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with MG132 (10 µM) for 6 h. The protein extracts were immunoprecipitated with IgG beads of anti-ARF4, and then the expression of K48-ubiquitin, K63-ubiquitin and ARF4 was detected by western blotting. I A proposed mechanism for USP7 to regulate the ARF4 level in GBM. All statistics are expressed as mean ± S.E.M., # P = NS, *P < 0.05,**P < 0.01 or ***P < 0.001

Article Snippet: Sangon Biotech (China) designed two siRNAs against ARF4, siARF4-1: 5’ -GCAAGACAACCAUUCUGUAUATT- 3’ and siARF4-2: 5’ - CCAUCAGUGAAAUGACAGAUATT- 3’.

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: Cancer Cell International

Article Title: USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

doi: 10.1186/s12935-021-02208-z

Figure Lengend Snippet: P5091 inhibits tumorigenicity in intracranial xenograft models Female BALB/c nude mice were injected intraperitoneally with PBS, P5091 (5 mg/kg/day, 10 mg/kg/day) and treated 2 days per week. Treatment started on day 7 after implantation and lasted for approximately 21 days. A Representative images of bioluminescence in mice on days 7, 14 and 28 after implantation. B Quantitative analysis of these bioluminescence images, n = 6. C Overall survival of the PBS and P5091 treatment groups, n = 6. D Representative H&E images of tumor sections. Scale bar, 300 μm. E Representative IHC images of tumor sections for anti-USP7, anti-ARF4, anti-BAX, anti-BCL-2, and anti-CLEAVED-CASPASE. Scale bar, 300 μm. Statistics are expressed as mean ± S.E.M., # P = NS, *P < 0.05,**P < 0.01 or ****P < 0.0001

Article Snippet: Sangon Biotech (China) designed two siRNAs against ARF4, siARF4-1: 5’ -GCAAGACAACCAUUCUGUAUATT- 3’ and siARF4-2: 5’ - CCAUCAGUGAAAUGACAGAUATT- 3’.

Techniques: Injection