Journal: Cell Death & Disease

Article Title: ISGylation prevents autophagic degradation of STING and promotes antitumor immunity in lung cancer

doi: 10.1038/s41419-026-08527-1

Figure Lengend Snippet: A The inhibitory activity of TST against USP18 was measured using the peptide RLRGG-AMC as a substrate. IC 50 was presented as mean ± SEM, n = 3 independent experiments. The chemical structure of TST is shown. B STD-NMR binding analysis for TST and USP18. The top spectrum is the 1 H NMR of USP18 (10 μM) in a mixture with TST (400 μM), while the bottom spectrum displays the STD-NMR of the same sample. C USP18-mediated de-ISGylation of STING examined by Western blot. Flag-STING, UBE1L-Myc-His (E1), UBCH8-Myc-His (E2), HERC5-Myc-His (E3), and Myc-ISG15 were co-transfected with GFP-USP18-WT into HEK-293T cells. Twenty-four hours post-transfection, cells were treated with TST (5, 25,125 μM) for 12 h, followed by an ISGylation assay. ISGylation experiments were performed as described in (Fig. ). D CHX chase assay was performed to analyze the effect of TST on STING protein stability in both USP18-WT (Negative Control, si-NC) and USP18-KD cells. LLC cells were treated with 100 µg/mL cycloheximide (CHX) for the indicated time points. The graph represents quantification of the STING protein levels. E CHX chase assay was used to analyze the effect of TST on STING protein stability in cells expressing either STING-WT or STING-4KR. The LLC cells were treated with 100 µg/mL cycloheximide (CHX) for the indicated time points. The graph represents quantification of the STING protein levels. F–G The synergistic antitumor effect of combined treatment of TST (10 mg/kg) and diABZi (3 mg/kg) in a mouse model. Tumors were dissected, photographed F and weighed G . H–K Tumor-infiltrating CD8 + T cells and NK1.1 + cells among CD45 + cells were analyzed using flow cytometry. Representative scatter plots H, J and frequencies I, K are shown ( n = 5 per group). The data presented is from a representative experiment, selected from three independent experiments. Data were presented as mean ± SEM, comparisons were conducted using two-way ANOVA for multiple comparisons, with ** indicating p < 0.01, *** indicating p < 0.001, **** indicating p < 0.0001 and n.s indicating not significant.

Article Snippet: Cells were treated with TST (50 μM) for 12 h and then 50 μg/mL of CHX (T1225, Topscience) at specific time intervals.

Techniques: Activity Assay, Binding Assay, Western Blot, Transfection, Negative Control, Expressing, Flow Cytometry

Journal: Science Advances

Article Title: Targeting oncoproteins with a positive selection assay for protein degraders

doi: 10.1126/sciadv.abd6263

Figure Lengend Snippet: ( A ) Immunoblot and ( B ) RT-qPCR analysis of the NCI-H1876 SCLC cell line that endogenously expresses ASCL1 infected to express the indicated sgRNAs. n = 3 biological replicates. ( C and E ) Immunoblot and ( D and F ) RT-qPCR analysis of NCI-H1876 human SCLC cells (C and D) and 97-2 mouse SCLC cells (E and F) after treatment with the CDK2 PROTAC degraders (TMX-2138 and TMX-2172) or the indicated negative controls, all used at 500 nM for either 36 hours (C and D) or 8 hours (E and F). Neg Deg, negative control degrader ZXH-7035. n = 3 biological replicates. ( G ) Immunoblot analysis and ( H ) quantification of ASCL1 protein levels in 97-2 cells first treated with the CDK2 PROTAC degrader or negative control (500 nM) for 4 hours and then treated with cycloheximide (CHX) (150 μg/ml) for the indicated times. S.E., short exposure; L.E., long exposure. n = 4 biological replicates. In all experiments, error bars represent SD except in (H), where error bars represent SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following compounds were purchased: POM (Selleck, #S1567), LEN (Selleck, #S1029), MG132 ( N -carbobenzyloxy- l -leucyl- l -leucyl- l -leucinal; Thermo Fisher Scientific, #47479020MG), MLN4924 (Active Biochem, #A-1139), MLN7243 (Thermo Fisher Scientific, #NC1129906), Spautin-1 (BioTechne; #5197/10), cycloheximide (VWR, #97064-724), BVdU (Chem-Impex International Inc., catalog no. 27735), actinomycin D (Thermo Fisher Scientific, #11805017), and dinaciclib (Selleck, #S2768).

Techniques: Western Blot, Quantitative RT-PCR, Infection, Negative Control

Journal: Cells

Article Title: Dynamics of T-Cell Intracellular Antigen 1-Dependent Stress Granules in Proteostasis and Welander Distal Myopathy under Oxidative Stress

doi: 10.3390/cells11050884

Figure Lengend Snippet: TIA1a WT/WDM expression does not alter phosphorylation/dephosphorylation dynamics in an oxidative stress model. ( A , B ) Western blot of total cell extracts obtained from FT293 GFP-TIA1a WT ( A ) and FT293 GFP-TIA1a WDM ( B ) lines subjected to different treatments indicated in the legend at the top. Analyses were performed with anti-TIA1, anti-eIF2α-phosphorylated (P), anti-eIF2α (Total), anti-HuR, and anti-tubulin-α (TUBA) antibodies from three independent experiments. Molecular weight markers (kDa) and identification of each band are shown. The + and − signs indicate whether the sample contains the reagent (Tet, NaAsO 2 , CHX) or not. The +/− sign indicates that NaAsO 2 is added and removed after 1 h. Abbreviations: Tet, tetracy-cline; NaAsO 2 , sodium arsenite; CHX, cycloheximide.

Article Snippet: Oxidative stress was induced as above, and cycloheximide (CHX; 5 μg/mL; Merck, Darmstadt, Germany) was also used, as a control.

Techniques: Expressing, Phospho-proteomics, De-Phosphorylation Assay, Western Blot, Molecular Weight

Journal: Journal of Cancer Prevention

Article Title: Overcoming the Intrinsic Gefitinib-resistance via Downregulation of AXL in Non-small Cell Lung Cancer

doi: 10.15430/JCP.2019.24.4.217

Figure Lengend Snippet: Enhancement of AXL degradation by yuanhuadine (YD) in H1299 cells. (A, C) Cells were treated with 25 μg/mL cycloheximide (CHX) and 10 nM YD for the indicated times. Cell lysates were analyzed by Western blot with an antibody against AXL. β-actin was used as a loading control. (B, D) AXL expression levels were quantifies by densitometry using ImageJ.

Article Snippet: Cycloheximide (CHX) was purchased from A.G. Scientific (San Diego, CA, USA).

Techniques: Western Blot, Control, Expressing

Journal: Molecular Oncology

Article Title: Endothelin‐converting enzyme‐1c promotes stem cell traits and aggressiveness in colorectal cancer cells

doi: 10.1002/1878-0261.12609

Figure Lengend Snippet: Mutation of Lys‐6 to Arg enhances stability of ECE1c. (A) DLD‐1 clone cells expressing Flag‐tagged ECE1c WT or ECE1c K6R proteins were incubated with 20 μg·mL −1 cycloheximide (CHX) in the absence (for 36 h) or presence (for 12 h) of 25 μ m silmitasertib. ECE1c proteins were detected by western blot with an anti‐Flag antibody, using β‐actin as loading control. Representative blots are shown (upper). Relative levels (%) of Flag‐ECE1c proteins from three independent experiments were calculated (lower). (B) DLD‐1 clones described in A were grown in medium without FBS for 48 h. Culture supernatants were used to measure ET‐1 levels using ELISA according to manufacturer’s instructions (Thermo Fisher). Data represent average ± SEM ( n = 3). ANOVA and Tukey tests were used. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: Cells (5 × 10 5 ) were seeded into P60 plates and cultured for 36 h in complete medium under normal conditions, with 20 μg·mL −1 cycloheximide (CHX) in the absence or presence of 25 μ m silmitasertib (ApexBio Technology LLC, Houston, TX, USA).

Techniques: Mutagenesis, Expressing, Incubation, Western Blot, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Microbiota-reprogrammed phosphatidylcholine inactivates cytotoxic CD8 T cells through UFMylation via exosomal SerpinB9 in multiple myeloma

doi: 10.1038/s41467-025-57966-5

Figure Lengend Snippet: a Representative image of Co-IP using a UFM1 antibody in CD8 T cells. Rabbit IgG was used as a negative control. Mean± SD, n = 3 biological replicates, data represent two independent experiments. b TP53 protein level in CD8 T cells transfected with or without UFM1 shRNAs. Mean± SD, n = 3 biological replicates, data represent two independent experiments. c TP53 protein level detected in CD8 T cells transfected with or without UFM1 shRNAs after cycloheximide treatment. Mean± SD, n = 3 biological replicates, data represent two independent experiments. d UFM1 protein level in CD8 T cells transfected with or without the Sb9 expression vector. e Representative images of Co-IP using TP53 antibody in scrambled and Sb9 expression vector (0.5, 1, and 2 μg)-transfected CD8 T cells. Rabbit IgG was used as a negative control. Mean± SD, n = 3 biological replicates, data represent two independent experiments. f TP53 protein level in CD8 T cells transfected with or without UFC1 shRNAs. Mean± SD, n = 3 biological replicates, data represent two independent experiments. g Representative image of Co-IP using a UFM1 antibody in CD8 T cells. Mean± SD, n = 3 biological replicates, data represent two independent experiments. Rabbit IgG was used as a negative control. Sb9 SerpinB9, NC negative control, OE overexpression, UFC1, ubiquitin-fold modifier conjugating enzyme 1. All statistical tests were two-sided. Statistical significance was assessed using one-way ANOVA followed by post-hoc Tukey’s test for ( b , c , e , and f ), which was performed utilizing unpaired Student’s t test for a , d , and g . Adjustments were made for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: When grown to ~80% confluence, CD8 T cells were treated with 50 μg/mL cycloheximide (IC0720, Solarbio) for 3 h, 6 h, and 12 h. Then, CD8 T cells were collected, followed by the detection of TP53 levels using WB.

Techniques: Co-Immunoprecipitation Assay, Negative Control, Transfection, Expressing, Plasmid Preparation, Over Expression, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control

doi: 10.1101/2023.01.31.526456

Figure Lengend Snippet: (A) Immunoblot showing the protein levels of GCN1 and eRF1 in HEKR4 PTC reporter cells treated with negative control (Ctrl) or GCN1-targeting siRNA (GCN1 KD) in combination with a 6-hour incubation of 2.5 μM NVS1.1 or DMSO as control treatment. Vinculin served as a loading control. (B) Representative immunoblot showing protein levels of eRF1 and GAPDH (loading control) of HEKR4 PTC reporter cells treated with 2.5 μM NVS1.1 for 6 hours in the absence or presence of the translation elongation inhibitor cycloheximide (CHX). (C) (Top) Polysome profile showing A 260 readout of lysate deriving from HEKR4 PTC reporter cells treated with 25 μM NVS1.1 for 30 min that was separated over a 15%-50% sucrose gradient and fractionated. (Bottom) The proteins in every odd-numbered fraction of the gradient were precipitated and analyzed by immunoblotting for the indicated proteins. The fractions 3 and 5 were diluted 1/15 and 1/3, respectively. (D). Heavy polysome fractions from (C) were pooled and the proteins were analyzed by label-free mass spectrometry. Detected diGly events on lysine residues indicative of ubiquitination were normalized to the corresponding protein abundance. The fold change of the diGly frequency (log 2 diGly (NVS1.1/DMSO)) is shown for the protein eRF1 and for ribosomal proteins that showed a statistically significant difference (pVal < 0.05) upon NVS1.1 treatment

Article Snippet: The NVS compounds, Bortezomib (BTZ, Merck, Cat# 504314), MLN4924 (Selleckchem, Cat# S7109) and Cycloheximide (CHX, Focus Biomolecules, Cat# 10-117) were all dissolved in DMSO, which also served as vehicle control.

Techniques: Western Blot, Negative Control, Incubation, Control, Mass Spectrometry, Ubiquitin Proteomics, Quantitative Proteomics

Journal: Genes & Diseases

Article Title: The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs

doi: 10.1016/j.gendis.2018.02.001

Figure Lengend Snippet: Fusion proteins of eGFP and Gaussia luciferase (GLuc) with multiple copies of the MODC PEST domain exhibit varied instabilities . ( A ) Schematic representation of the construction of eGFP-3modc C-terminal fusion and its control eGFP vectors. Both constructs were generated on the basis of our homemade pSEH retroviral vector, yielding pSEH-eGFP and pSEH-eGFP-3modc. LTR, long terminal repeat for MSCV retrovirus; Hygro, hygromycin B resistance gene; hEFH, a hybrid promoter consisting of human EF1α promoter and HIV enhancer; eGFP, enhanced green fluorescent protein; stop, stop codon; 3modc, three copies of the PEST-containing degradation domain of mouse ornithine decarboxylase. ( B ) The fluorescence signal of the eGFP-3modc fusion after cycloheximide (CHX) treatment. The retroviral vectors were used to make stable cell lines of HCT116 cells after hygromycin selection. The stable lines were seeded at subconfluency and treated with cycloheximide (100 μg/ml). Fluorescence signals were recorded at the indicated time points. Representative images are shown. ( C ) Quantitative analysis of the fluorescence intensity in the samples prepared in (B). “*” p < 0.05 compared with GFP signal between 0 h and 12 h. ( D ) Schematic representation of the construction of GLuc-3modc fusion and its control GLuc vectors. Both constructs were retroviral vectors, namely pSEH-GLuc and pSEH-GLuc-3modc. ( E ). The retroviral vectors were used to make stable HCT116 cell lines, as described in (B). Basal GLuc activity was measured. “**” p < 0.01, compared with non-fusion GLuc control line. ( F ). GLuc activity retained after CHX treatment. The GLuc and GLuc-3modc stable lines were treated with CHX (100 μg/ml). GLuc activities were measured at the indicated time points. “*p < 0.05, “**”p < 0.01, compared with the GLuc activity at 0 h time point.

Article Snippet: 293pTP and RAPA cells derived from HEK-293 cells as previously characterized., All cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA), containing 100 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C in 5% CO 2 as described., , Cycloheximide (CHX) and hygromycin B were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Luciferase, Construct, Generated, Plasmid Preparation, Fluorescence, Stable Transfection, Selection, Activity Assay

Journal: Genes & Diseases

Article Title: The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs

doi: 10.1016/j.gendis.2018.02.001

Figure Lengend Snippet: Characterization of the degradability of BiFP-modc C-terminal fusion proteins . ( A ) Schematic representation of destabilized BiFP constructs with zero to three copies of MODC PEST domain at the C-terminus of BiFP. The fusion constructs were generated and cloned in the pSEH retroviral vector, yielding pSEH-BiFP, pSEH-BiFP-modc, pSEH-BiFP-2modc, and pSEH-BiFP-3modc, respectively. ( B ) BiFP fusion constructs were used for retrovirus packaging and generating stable lines in HCT116 cells after hygromycin selection. Subconfluent stable lines were treated with CHX (100 μg/ml). Fluorescence signals were recorded at the indicated time points. Representative images are shown. ( C ) Quantitative analysis of the fluorescence signal for the CHX-treated stable lines shown in ( B ). “*” p < 0.05; “**” p < 0.01, compared with the BiFP signals at 0 h after CHX treatment of respective stable lines.

Article Snippet: 293pTP and RAPA cells derived from HEK-293 cells as previously characterized., All cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA), containing 100 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C in 5% CO 2 as described., , Cycloheximide (CHX) and hygromycin B were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Construct, Generated, Clone Assay, Plasmid Preparation, Selection, Fluorescence