Journal: PLOS Genetics

Article Title: ZCCHC3 is a stress granule zinc knuckle protein that strongly suppresses LINE-1 retrotransposition

doi: 10.1371/journal.pgen.1010795

Figure Lengend Snippet: (A,B) Both ZCCHC3-FL and the short ZAP isoform tagged with GFP (GFP-PARP-13.2) colocalize with tagged exosome component EXOSC5 in cytoplasmic granules of stressed 293T cells (see arrows). C) On the other hand, V5-TEV-DIS3L protein does not colocalize with ZCCHC3-FL in cytoplasmic granules of stressed U2OS-WT cells, (D) nor does endogenous ZCCHC3 protein colocalize with endogenous DIS3L nuclear homolog DIS3 in granules of 293T cells. (E) V5-TEV-ZCCHC3 does not colocalize with endogenous EXOSC3 in SGs of stressed 2U2OS-WT cells. Cell nuclei were stained with Hoechst 33342 (right-most panels). Size bars are 10 μm. (F) HEK 293T cells expressing ZCCHC3-FL were lysed, and lysates were fractionated by sucrose velocity gradient centrifugation. Aliquots of each 900 μl fraction were subjected to SDS-PAGE and Western blotting with antibodies against endogenous MOV10, ZAP, and exosome components. The ~42 kD band above the expected 30 kD EXOSC3 band (marked by an arrow, lower panel) appears more in HMW fractions and may reflect post-translational modification, possibly ubiquitination . (G) Untransfected HEK 293T cell lysates were analyzed as in (F). (H) In 293T cells, protein expression from the V5-TEV-EXOSC3 construct is strongly inhibited by cotransfection of the shEXOSC3 shRNA construct, but not in untransfected or scrambled shRNA (shN3)-transfected controls . (I) HEK 293T cells expressing ZCCHC3-FL and shN3 control (top) or shEXOSC3 (bottom) shRNA constructs were lysed and subjected to sucrose velocity gradient centrifugation as above and analyzed by α-FLAG-M2 antibody. Cotransfection of shEXOSC3 caused redistribution of ZCCHC3 protein throughout the gradient suggesting its dissociation from HMW complexes.

Article Snippet: Commercial antibodies included rabbit (rb) α-SKIV2L2 (MTR4) (A13258), rb α-ZCCHC3 (A17235; α-ZCCHC3-Ab) (Abclonal), rb α-ZCCHC3 (ARP50730_P050; α-ZCCHC3-Av) (Aviva Systems Biology), mouse (ms) α-DYKDDDDK (binds FLAG-tag, 9A3), rb α-DYKDDDDK (D6W5B), ms α-HA-tag (6E2), rb α-HSP90 (#4874), and rb α-ZCCHC3 (#65321; α-ZCCHC3-CS) (Cell Signaling Technology), ms α-FLAG-M2 (F1804/F3165) (MilliporeSigma), ms α-DIS3 (PCRP-DIS3-1A7), ms α-G3BP2 (PCRP-G3BP2-1C7) (Deveopmental Studies Hybridoma Bank), rb α-EXOSC3 (15062-1-AP), rb α-MOV10 (10370-1-AP), rb α-alpha tubulin (11224-1-AP), rb α-ZC3HAV1 (16820-1-AP, α-ZAP) (ProteinTech), ms α-V5-tag (Thermo Fisher Scientific), goat (gt) α-eIF3η (N-20), and gt α-TIA1 (C-20) (Santa Cruz Biotechnology).

Techniques: Staining, Expressing, Gradient Centrifugation, SDS Page, Western Blot, Modification, Ubiquitin Proteomics, Construct, Cotransfection, shRNA, Transfection, Control

Journal: Cell Death Discovery

Article Title: USP39 regulates pyruvate handling in non-small cell lung cancer

doi: 10.1038/s41420-024-02264-0

Figure Lengend Snippet: A LC–MS/MS analysis of immunoconjugates pulled down with USP39 antibody. Number of peptides and coverage of USP39, DLAT, and PDHA. B Western blot analysis of immunoconjugates from ( A ). C Cellular fractionation of NCI-H1975 cells. Lamin B1, TOMM40 and Tubulin serve as markers for nucleic, membrane, and cytosolic fractions respectively. D Protein levels of PDH complex components after 48 h of USP39 siRNA-mediated silencing with quantification ( n = 3). E Protein levels of PDHA after 48 h of USP39 siRNA-mediated silencing and treatment with 5 µM of MG132 for 4 h ( n = 3). F Enzymatic activity of the PDH complex ( n = 3) after 48 h of USP39 knockdown. G mRNA expression levels of PDHA and DLAT upon 48 h of USP39 silencing with siRNAs ( n = 3). H In vitro deubiquitination assay showing the cleavage of Lys 48 and Lys 63 U 1-7 polyubiquitin chains after incubation with recombinant USP39 for 16 h. I Ubiquitination level of PDHA in USP39-overexpressing cells and after treatment with MG132 for 4 h ( n = 3). Quantification of ubiquitination levels were normalized to total PDHA levels and to the control. J Lys 63 -ubiquitination level of PDHA in USP39-knockdown cells and after treatment with MG132 for 4 h ( n = 3). Quantification of Lys 63 -ubiquitination levels were normalized to total PDHA levels and to the control. K Protein level of PDHA after 72 h of USP39 overexpression with quantification ( n = 3). Error bars ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The following antibodies were used: DDK (TA50011-100, OriGene, Rockville, MD, USA), DLAT (68303-1-Ig, ProteinTech Group, Chicago, IL, USA), Lamin B1 (12586S, Cell Signaling Technology, Danvers, MS, USA), PDHA (3205S, Cell Signalling), TOMM40 (sc-11414, Santa Cruz Biotech, Dallas, TX, USA), Tubulin (T8203, Sigma-Aldrich, St. Louis, MO, USA), Ubiquitin (3933S, Cell Signalling), Lys 63 -Ubiquitin (5621S, Cell Signalling), USP39 (23865-1-AP, ProteinTech Group), Vinculin (ab129002, Abcam, Cambridge, UK).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Cell Fractionation, Membrane, Activity Assay, Knockdown, Expressing, In Vitro, Incubation, Recombinant, Ubiquitin Proteomics, Control, Over Expression

Journal: Bone marrow transplantation

Article Title: G-CSF downregulates natural killer cell-mediated cytotoxicity in donors for hematopoietic SCT.

doi: 10.1038/bmt.2011.22

Figure Lengend Snippet: Figure 4 The role of PI3K expression in G-CSF-treated NK-92MI cells. (a) Expression of PI3K by western blot analysis and the relative values of PI3K were normalized to the internal glyceraldehyde-3-phosphate dehydrogenase control. (b) Effect of wortmannin (100 nM) and G-CSF (4 ng/mL) on NK cell- mediated cytotoxicity. This analysis was performed on three independent experiments with similar results. The data were expressed as mean±s.d. *Po0.05 was considered significantly different from the control data.

Article Snippet: For in vitro cytotoxicity assay, 1 106 NK-92MI cells per mL were pretreated with recombinant human G-CSF (2 and 4ng/mL; CytoLab Ltd, Rehovot, Israel) and/or inhibitors, such as 10 mM PD98059 (BioSource, Camarillo, CA, USA) or 100 nM wortmannin (Cell Signaling, Boston, MA, USA), for 16 h and then adjusted for the following cytotoxicity method.

Techniques: Expressing, Western Blot, Control

Journal: Bone marrow transplantation

Article Title: G-CSF downregulates natural killer cell-mediated cytotoxicity in donors for hematopoietic SCT.

doi: 10.1038/bmt.2011.22

Figure Lengend Snippet: Figure 6 Expression of cytotoxic-related genes in NK-92MI cells with and without G-CSF treatment. (a) RT-PCR assay of mRNA levels of ubiquitin, calreticulin and TPI genes in G-CSF (0 and 4 ng/mL)-treated NK-92MI cells. (b) Effect of inhibitors wortmannin (100 nM) and PD98059 (10 mM) on TPI expression in NK-92MI cells with and without G-CSF treatment. The relative values were normalized to the internal glyceraldehyde-3-phosphate dehydrogenase control and were calculated from at least three independent experiments. All results were expressed as mean±s.d. *Po0.05 was considered significantly different from the control data.

Article Snippet: For in vitro cytotoxicity assay, 1 106 NK-92MI cells per mL were pretreated with recombinant human G-CSF (2 and 4ng/mL; CytoLab Ltd, Rehovot, Israel) and/or inhibitors, such as 10 mM PD98059 (BioSource, Camarillo, CA, USA) or 100 nM wortmannin (Cell Signaling, Boston, MA, USA), for 16 h and then adjusted for the following cytotoxicity method.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics, Control