Journal: PLoS ONE

Article Title: Dimethyl Sulfoxide Promotes the Multiple Functions of the Tumor Suppressor HLJ1 through Activator Protein-1 Activation in NSCLC Cells

doi: 10.1371/journal.pone.0033772

Figure Lengend Snippet: (A) Real-time quantitative RT-PCR reveals that HLJ1 mRNA expression was induced by DMSO in a concentration-dependent manner (0.1–2%, v/v) after 48 h incubation. (B) Concentration-dependent DMSO-induced HLJ1 expression at the protein level was confirmed by Western blot analysis. α-tubulin was a control for protein loading and transfer. (C) Time-dependent DMSO-induced HLJ1 expression at the mRNA level was verified by real-time quantitative RT-PCR. The results indicated that the mRNA level of HLJ1 was significantly increased after 2% DMSO treatment for 2 h. * P < 0.05, significantly different from the vehicle-treated control. (D) The protein expression level of HLJ1 after 2% DMSO treatment at the indicated time was detected by Western blot analysis. The result indicated that HLJ1 protein expression was time-dependent and was sustained with DMSO treatment for 48 h. α-tubulin was a control for protein loading and transfer.

Article Snippet: The primary antibodies for JunD, JunB, Fra-1, and HLJ1 were purchased from Santa Cruz Biotechnology, Inc. β-actin or α-tubulin was used as the internal control for gel loading.

Techniques: Quantitative RT-PCR, Expressing, Concentration Assay, Incubation, Western Blot, Control

Journal: Cell Reports Methods

Article Title: Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging

doi: 10.1016/j.crmeth.2024.100875

Figure Lengend Snippet: Generation of compartment-targeted, misfolding-prone Fluc DM variants to perturb and sense subcellular proteostasis in Drosophila (A) Generation of transgenic organelle-targeted sensors of protein quality control based on a misfolding-prone mutant firefly luciferase (Fluc DM ) fused to EGFP. The mitochondrial targeting sequence from the human mitochondrial COX VIII protein was utilized to generate the mito-Fluc DM variant, whereas a standard nuclear targeting sequence was used to generate the NLS-Fluc DM . General (untargeted) sensors and reporters for mitochondria and the nucleus were site integrated and are expressed ubiquitously (downstream of a tubulin promoter) and at similar levels, as indicated by qRT-PCR with 3 batches of flies and the mean ± SD (no significant changes, one-way ANOVA). (B) Immunostaining and confocal microscopy of enterocytes indicate that Fluc DM variants exhibit the expected specificity in subcellular localization. General (untargeted) Fluc DM is detected in the cytoplasm (but also in the nucleus and plasma membrane), mito-Fluc DM is detected in ATP5A-stained mitochondria, and NLS-Fluc DM is found in the nucleus. Scale bars represent 20 and 15 μm, as indicated. (C) Immunostaining and confocal microscopy of brain cells from the antennal lobe indicates a similar localization. The untargeted Fluc DM is detected in the cytoplasm, mito-Fluc DM is detected in ATP5A-positive mitochondria, and NLS-Fluc DM is found in the nucleus. Scale bars represent 10 and 5 μm. (D) Immunostaining of enterocytes from heat-shocked and control flies identifies Fluc DM -GFP aggregates that accumulate in the cytoplasm in response to thermal stress compared to non-heat-shocked controls. Similar heat-induced cytoplasmic aggregates are also found in heat-shocked NLS-Fluc DM -EGFP and mito-Fluc DM -EGFP cells. In the case of mito-Fluc DM -EGFP, these aggregates are recognizable because they produce larger puncta than the staining that corresponds to mito-Fluc DM -EGFP-positive mitochondria (B). Scale bar, 10 μm.

Article Snippet: Ponceau S staining (ThermoFisher #A40000279) and anti-β-actin and/or anti-α-tubulin antibodies (Cell Signaling Technologies, #8457 and #2125) were used as loading controls.

Techniques: Transgenic Assay, Control, Mutagenesis, Luciferase, Sequencing, Variant Assay, Quantitative RT-PCR, Immunostaining, Confocal Microscopy, Clinical Proteomics, Membrane, Staining

Journal: Cell Reports Methods

Article Title: Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging

doi: 10.1016/j.crmeth.2024.100875

Figure Lengend Snippet: Distinct aggregation-prone proteins differentially impact subcellular proteostasis (A–C) Analyses of detergent-soluble and insoluble fractions from skeletal muscle from 10-day-old flies that express a toxic, aggregation-prone protein with 49(GGGGCC) repeats (orange) compared to non-toxic controls, 8(GGGGCC)-containing proteins (gray), and/or mCherry (white). Western blots with anti-GFP antibodies detect the levels of Fluc DM -EGFP, whereas Ponceau staining and α-tubulin are used as normalization controls. The toxic 49(GGGGCC) protein increases the detergent-insoluble levels of Fluc DM variants that localize to the cytoplasm (A), mitochondria (B), and the nucleus (C), indicating that aggregation-prone proteins with 49(GGGGCC) repeats generally disrupt proteostasis across multiple cell compartments. n = 3 (biological replicates) with the mean ± SD indicated; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (one-way ANOVA). (D–F) Western blots of detergent-soluble and -insoluble fractions from skeletal muscles of flies that express pathogenic ataxin-3 with poly-glutamine tract expansion (hATXN3.tr-Q78; red) versus a non-pathogenic ataxin-3 (hATXN3.tr-Q27, gray) and mCherry controls (white). There is no modulation of detergent-soluble and -insoluble Fluc DM levels (D), whereas the effects of hATXN3.tr-Q78 on mito-Fluc DM (E) and NLS-Fluc DM (F) levels are inconsistent when compared to the hATXN3.tr-Q27 versus the mCherry control. n = 3 (biological replicates) with the mean ± SD indicated; ∗ p < 0.05, ∗∗ p < 0.01 (one-way ANOVA). Altogether, these findings indicate that distinct aggregation-prone proteins have strikingly different impacts on subcellular proteostasis.

Article Snippet: Ponceau S staining (ThermoFisher #A40000279) and anti-β-actin and/or anti-α-tubulin antibodies (Cell Signaling Technologies, #8457 and #2125) were used as loading controls.

Techniques: Western Blot, Staining, Muscles, Control

Journal: Cell Reports Methods

Article Title: Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging

doi: 10.1016/j.crmeth.2024.100875

Figure Lengend Snippet:

Article Snippet: Ponceau S staining (ThermoFisher #A40000279) and anti-β-actin and/or anti-α-tubulin antibodies (Cell Signaling Technologies, #8457 and #2125) were used as loading controls.

Techniques: Recombinant, SYBR Green Assay, Membrane, Electron Microscopy, Lysis, Bicinchoninic Acid Protein Assay, Luciferase, Reverse Transcription, Mass Spectrometry, Solubility, Gene Expression, Knock-In, Software