Journal: Molecular Cell

Article Title: Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination

doi: 10.1016/j.molcel.2016.11.039

Figure Lengend Snippet:

Article Snippet: GST-UBE1 (human) , Boston Biochem , Cat. #E-306.

Techniques: Recombinant, Protease Inhibitor, Methylation, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Sequencing, Negative Control, Software

Journal: Journal of Biomedical Science

Article Title: Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions

doi: 10.1186/s12929-023-00990-8

Figure Lengend Snippet: UBA1 conveys Rab34 action on FABP5 stability to regulate lipid metabolism. A Transfected 3T3-L1 cells with the indicated plasmids (GFP-Rab34 or FABP5-c-Myc) were treated with MG132 (10 μmol/L, 12 h) and lysed under denaturing conditions. c-Myc-tagged FABP5 was purified by anti-c-Myc immunoprecipitation and ubiquitinated FABP5 was detected by Western Blot. An expression vector coding for hemagglutinin (HA)-tagged ubiquitin (HA-Ubiquitin) was employed for cotransfection of cells expressing FABP5-c-Myc, alone or in combination with GFP-Rab34. The graph shows the ratio of Ubiquitinated-FABP5-c-Myc immunosignal to Ponceau S immunosignal. Data are referred to values in non-transfected cells (100%) and expressed as mean ± SEM (n = 3 biological replicates). **P < 0.01; ***P < 0.001. B Co-immunoprecipitation analysis in cells expressing GFP-Rab34 and vectors coding for LD-associated proteins related to ubiquitination/deubiquitination processes: UBA1-c-Myc (top panel), UCHL3-c-Myc (middle panel), or ISG15-c-Myc (bottom panel). In each experimental setting, proteins were purified by anti-c-Myc immunoprecipitation and detected by Western Blot using anti-c-Myc or anti-GFP antibodies. C Representative immunoblots and quantification of FABP5 levels in 3T3-L1 cells transfected with GFP-Rab34 (+ , 0.8 µg/µL), UBA1-c-Myc (+ , 0.8 µg/µL; + + , 1.6 µg/µL) or both expression vectors in the absence or presence of MG132 (10 µmol/L, 12 h). Data represent the ratio of FABP5 immunosignal to β-actin immunosignal and referred to values in non-transfected cells (100%). Data are expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-transfected cells. $$ P < 0.01; $$$ P < 0.001 vs. their respective condition treated with MG132. # P < 0.05; ## P < 0.01. D, E Rescue experiments of FABP5 in 3T3-L1 cells expressing GFP-Rab34 and UBA1 siRNA (siUBA1), alone or in combination. At the end of the experiments, cells were processed for immunoblotting studies ( D ) (see also Fig. S4) and for measurement of TGs (lipogenesis) and glycerol content (lipolysis) ( E ). Data are referred to values in control cells (100%; Scr), and expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Plasmid coding for E1 Ubiquitin-Activating Enzyme 1 (UBA1) (pCMV3-UBA1-c-Myc) was purchased from SiNo Biological (Düsseldorfer, Eschborn, Germany).

Techniques: Transfection, Purification, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Cotransfection

Journal: Cell Death Discovery

Article Title: TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA

doi: 10.1038/cddiscovery.2017.52

Figure Lengend Snippet: The effects of SAHA and TRAIL DR5 on the expressions of autophagy-related genes in cancer cells. RNA was isolated from MDA-MB-231 or MCF-7 cells using high pure RNA isolation kit following manufacturer’s instructions. To obtain the first-strand cDNA, transcriptor first strand cDNA synthesis kit was used and cDNA was as a template in real-time PCR reactions with power SYBR green PCR master mix. Exprofile human autophagy Gene qPCR array kit was employed to describe related mRNA expression. Relative changes of gene expression in the array were calculated using the 2 −ΔΔCt (threshold cycle) method. ( a – c ). MDA-MB-231 cells, ( d – f ). MCF-7 cells, ( g ). Related data analysis. Relative quantitative values represent mean±S.E.M.; * represents statistical significance of P <0.05 comparing with Basal, ** represents statistical significance of P <0.01 comparing with Basal.

Article Snippet: Exprofile human autophagy gene qPCR array kit was obtained from Genecopoeia (Rockville, MD.

Techniques: Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing, Gene Expression

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Expression levels of ALDH1A1 and HLTF predict sensitivity to HCQ in cancer cell lines. (A) MTT (72 h) in colon and lung cancer cells. (B) Differentially expressed genes in HCQ-S (HT29) and HCQ-R (HCT15) colon cancer cells. (C) HCQ IC50 and Hill Slope for 33 human cancer cell lines. Blue dots indicate sensitive cell lines (<16 µM IC50); green indicates intermediate resistant cell lines (IC50 > 16 µM, Slope > −2.1); red indicates resistant cell lines (IC50 > 16, slope < − 2.1) (D) Protein expression detected by western blot of the 2 most upregulated (ALDH1A1, LYZ) and the 2 most downregulated (ABCB1, HLTF) genes in HCQ-sensitive (Sen), HCQ-intermediate resistant (Int Res), and HCQ-resistant (Res) cells. ANOVA indicates no single gene predicts sensitivity or resistance. (E) CART analysis of expression level of 4 genes (ALDH1A1, LYZ, HLTF, ABCB1) identifies a 2-gene signature that is sufficient to predict all sensitive cell lines. Exp.: expression as detected by fluorescence intensity of the band/ control.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Expressing, Western Blot, Fluorescence

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: ALDH1A1 levels control entry and activity of chloroquine derivatives in cancer cells. (A-B) Aldeflour assay shows (A) CQ derivatives produce no impairment of ALDH1 enzyme function, (B) siRNA against ALDH1A1 impairs enzymatic function. (C) DC341-C3 strucutre. (D) Fluorescence microscopy of DC340-Cy3 (red fluorescence) in A375 cells treated with nontargeting siRNA (NT) or siALDH1A1; or vehicle and DEAB for 24 h. Mean +/− SD from multiple experiments. (E) DC340-Cy3 fluorescence following SiNT or SiALDH1A1 in HT29 in the presence or absence of verapamil. (F) CD340-Cy3 in A375P cells transiently transfected with control or ALDH1A1-expressing vector. (G) Immunoblotting against autophagy markers in HCT15 cells transfected with control or ADLH1A1-expressing vector +- HCQ. Mean +/− SD for band quantification from multiple experiments shown. (H) LysoSensor fluorescence (green) of HCT15 and HT29 cells transfected with control or ALDH1A1-expressing vector, or siNT or siALDHA1, respectively. (I-K) 72-h MTT mean +/− SD. (I) Knockdown of ALDH1A1 promotes resistance to HCQ. (J) DEAB co-treatment promotes resistance to HCQ. (K) Overexpression of ALDH1A1 promotes HCQ-sensitivity. *p < 0.05.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Activity Assay, Fluorescence, Microscopy, Transfection, Expressing, Plasmid Preparation, Western Blot, Over Expression

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Modulation of HCQ efficacy by ALDH1A1 and HLTF. (A) ROS levels in HCT15 and HT29 cells with overexpression or knockdown of ALHD1A1. (B) HCT15 cells transiently transfected with Control or ALDH1A1-expressing vector with or without HCQ (20 µM) for 12 h. (C) HT29 cells transfected with siNT (Non-Target) or siALDH1A1 with or without HCQ (20 µM) for 12 h. (D) Immunoblotting in lysates from the indicated cells treated with retinoic acid (RA 5 µM) +/− EZH2 inhibitor EPZ005687 2 µM (24 h). (E) Immunoblotting of lysates from HCT 15 cells transfected with control or ALDH1A1-expressing vector +/- EPZ005687 (2 µM), 24 h. (F) Immunoblotting from lysates from HCT15 cells treated with HCQ (20 µM) +/− Tiron (10 µM). (G) Immunoblotting of HCT15 cells transiently transfected with control or ALDH1A1-expressing vector +/− HCQ (20 µM) for 12 h. (H) ALDH1A1 facilitates entry of HCQ into the cell, where it accumulates in the lysosome. Lysosomal impairment leads to autophagy inhibition and the generation of ROS. ROS-associated DNA damage produces single-strand breaks with stalled replication forks. If HLTF is expressed, recruitment of the low fidelity DNA polymerase POLH allows translesion synthesis to occur promoting resistance to therapy. In the absence of HLTF-associated TLS, stalled replication forks collapse into double-strand breaks triggering cell death. Both RA and ROS can regulate KDM5D-PRC2-driven degradation of DNMT1 and upregulation of HLTF.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Inhibition, Translesion Synthesis

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Validation set and TCGA analysis of 2-gene profile. (A) HCQ IC50 and Hill Slope for 16 human cancer cell lines. Blue dots indicate sensitive cell lines (<16 µM IC50); green indicates intermediate resistant cell lines (IC50 > 16 µM, Slope > −2.1); red indicates resistant cell lines (IC50 > 16, slope < −2.1). (B) Protein expression of ALDH1A1 and HLTF, and percentage of HCQ-sensitive cell lines in the validation set based on the CART analysis in Fig. 1. (C) Percentage of stage IV cancer patients in The Cancer Genome Atlas (TCGA) with the indicated malignancies that had the indicated profile of ALDH1A1 and HLTF expression predicted by RNA sequencing. Blue, HCQ-S profile; red, HCQ-R profile; white, unknown. HNSCC, head and neck squamous cell carcinoma. (D) Distribution of 4 ALDH1A1 HLTF RNA-Seq profiles in stage I-IV melanoma tumors from the TCGA. WT, wild type; Mu, mutant.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Expressing, RNA Sequencing Assay, Mutagenesis