Journal: Pharmaceutics

Article Title: Nanoparticle-Mediated Therapy with miR-198 Sensitizes Pancreatic Cancer to Gemcitabine Treatment through Downregulation of VCP-Mediated Autophagy.

doi: 10.3390/pharmaceutics15082038

Figure Lengend Snippet: Figure 2. In vitro administration of miR-198 impairs the autophagy maturation process and reverses when VCP is overexpressed. (a) AsPC-1-miR-198 cells were transfected with either LPNP-Ctrl or LPNP-pVCP and then treated with different concentrations of gemcitabine (0, 0.001, 0.01, 0.1, 1, 10, and 100 µg/mL) for 48 hrs. Cell viability was determined with the MTT assay (n = 5, * p < 0.05). AsPC-1-miR-CTL and AsPC-1-miR-198 were transfected with LPNP-Ctrl or LPNP-pVCP together with mRFP-GFP-LC3 and cultured in complete medium with gemcitabine (20 µg/mL). Cells were also cultured in the presence or absence of CQ (10 µM). (b,c) LC3 expression was then visualized with fluorescence microscopy under green channel, red channel, and both channels overlapped. These are representative pictures taken from at least three different replica experiments and, for quantification of LC3-RFP-GFP expression via red and green puncta colocalization, Pearson’s correlation coefficient was used. Ten independent fields of cells with more than thirty cells in each field were counted for each panel of cells (n = 5, * p < 0.05).

Article Snippet: VCP expression plasmid (pVCP) was purchased from OriGene, Rockville, MD, USA, and the mRFP-GFP-LC3 (pLC3) plasmid was furnished by Dr. Mary K. Estes at Baylor College of Medicine, Houston, TX, USA.

Techniques: In Vitro, Transfection, MTT Assay, Cell Culture, Expressing, Microscopy

Journal: Brain

Article Title: Reply: Hereditary spastic paraplegia caused by a mutation in the VCP gene VCP: A Jack of all trades in neuro- and myodegeneration?

doi: 10.1093/brain/aws202

Figure Lengend Snippet: Figure 1 VCP and WASH complex expression pattern in a VCP haploinsufficient mouse strain. Left: Note the decreased VCP protein expression (rabbit polyclonal, Novus Biologicals NB100-1557) in conjunction with increased strumpellin protein expression (rabbit poly- clonal EP085378/SY1593) (Clemen et al., 2010) in total protein extract preparations from skeletal muscle tissue derived from wild-type (WT) and VCP haploinsufficient (HEM) littermates. GAPDH was used as an internal loading control (not shown). Right: Quantitative real-time PCR analyses of VCP and WASH complex subunit messenger RNA levels in skeletal muscle tissue derived from the same mice. Note that the messenger RNA levels of 6 out of 7 WASH complex components are upregulated. GAPDH messenger RNA levels were used for normalization and ratios of HEM to WT were calculated. Mean values and standard errors were obtained from two experiments (four measurements each) of two animals per genotype.

Article Snippet: Left: Note the decreased VCP protein expression (rabbit polyclonal, Novus Biologicals NB100-1557) in conjunction with increased strumpellin protein expression (rabbit polyclonal EP085378/SY1593) (Clemen et al., 2010) in total protein extract preparations from skeletal muscle tissue derived from wild-type (WT) and VCP haploinsufficient (HEM) littermates.

Techniques: Expressing, Derivative Assay, Control, Real-time Polymerase Chain Reaction

Journal: Human Molecular Genetics

Article Title: Alteration of the unfolded protein response modifies neurodegeneration in a mouse model of Marinesco–Sjögren syndrome

doi: 10.1093/hmg/ddp464

Figure Lengend Snippet: Ubiquitin-positive protein inclusions in Purkinje cells. ( A – I ) ER and nuclear localization of protein inclusions. Cerebellar sections from 3-month-old Sil1 −/− (A–C), 2-month-old Sil1 −/− ; Hyou1 +/− (D–F) and 3-month-old Sil1 −/− ; Dnajc3 −/− (G–I) mice were immunostained with antibodies against BiP (A, D, G) and ubiquitin (Ub; B, E, H). Merged images are shown in C, F, I. ( J – L ) ERAD chaperone p97/VCP partially co-localizes with ubiquitylated protein inclusions in Sil1 −/− Purkinje cells. Brain sections from 80-day-old Sil1 −/− mice were subjected to immunohistochemistry with antibodies against p97/VCP (J) and ubiquitin (Ub, K). Merged image is shown in L. Scale bar = 5 µm.

Article Snippet: Mouse antibody to p97/VCP (Novus Biologicals) and purified rabbit antiserum to HYOU1 was a used at 1:200 and 1:500 dilutions, respectively ( ).

Techniques: Ubiquitin Proteomics, Immunohistochemistry

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A–D) Luciferase assay of SREBP-1c activity by AAA-ATPase p97/VCP. HEK293 cells were transfected with Gal4-RE-Luc, pRL-SV40, and the indicated expression plasmids. After transfection, cells were treated with 50 μM EPA (E). After 24 hr, cell extracts were examined using luciferase assays. Quantification was performed on four samples. (F and G) HepG2 cells were transfected with the indicated siRNA. After 24 hr transfection, cells were incubated with 10% DLS containing 3 μM 25-hydroxycholesterol for 24 hr. (F) Immunoblot analysis of the indicated proteins in cytosolic extracts (CE) and nuclear extracts (NE). Cont, control; P, precursor; N, nuclear. (G) qRT-PCR analysis of SREBP-1 and related genes was performed. Quantification was performed on six samples. Data are represented as means ± SEM. * P < 0.05, ** P < 0.01. See also .

Article Snippet: Human p97/VCP (NM_007126, SC125280), human UFD1L (NM_005659, SC320168), and human NPLOC4 (NM_017921, SC113845) expression plasmids were purchased from OriGene.

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Incubation, Western Blot, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A) qRT-PCR analysis of SREBP-1 related genes. HepG2 cells were transfected with the indicated siRNA. 24 hr after transfection, cells were incubated with 10% DLS containing 3 μM 25-hydroxycholesterol for 24 hr. Data are represented as means ± SEM. Quantification was performed on six samples. *P < 0.05, ** P < 0.01. (B) Luciferase assay of SREBP-1c regulated by p97. HEK293A cells were transfected with Gal4-RE-Luc, pRL-SV40, and the indicated expression plasmids. After 24 hr, cell extracts were examined using luciferase assays. Data are represented as means ± SEM. Quantification was performed on six samples. *P < 0.05, ** P < 0.01. (C) The RHBDL4 with mutation of ubiquitin interaction motif (UIM) show weaker cleavage activity for SREBP-1c. HEK293 cells were transfected with HSV-SREBP-1c and either RHBDL4 WT or mutant RHBDL4 (ΔUIM) plasmid. The indicated proteins in cytosolic extracts (CE) and nuclear extracts (NE) were immunoblotted. HSV-SREBPs were immunoblotted with HSV antibodies. P, precursor; N, nuclear.

Article Snippet: Human p97/VCP (NM_007126, SC125280), human UFD1L (NM_005659, SC320168), and human NPLOC4 (NM_017921, SC113845) expression plasmids were purchased from OriGene.

Techniques: Quantitative RT-PCR, Transfection, Incubation, Luciferase, Expressing, Mutagenesis, Ubiquitin Proteomics, Activity Assay, Plasmid Preparation