Journal: Nature communications

Article Title: A biosensor-based framework to measure latent proteostasis capacity.

doi: 10.1038/s41467-017-02562-5

Figure Lengend Snippet: Fig. 6 Probing changes in proteostasis through selective activation of different chaperone systems. a Investigation of the effect of induction of the heat shock response by HSF1 overexpression or selective overexpression of HSP90. Shown on left are western blots of the HEK293T cells matched for total protein via a BCA assay. These cells were co-transfected with HSF1 (or myc-tagged HSP90 or control of (non-fluorescent) Y66L Emerald fluorescent protein) and the L89G barnase biosensor (which also contains a myc tag). The right graphs show the Lower-slope and A50% analyses of these treatments and one or three of the biosensor variants as indicated vs. the wild-type* variant. Bars indicate means ± SEM. The right panels were analysed via a two-way ANOVA subjected to a Dunnett’s post-hoc test, results coded as ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = > 0.05. b Same paradigm as panel a. In this case, BAG1 was cotransfected with the barnase biosensor, HSP40 (DNAJB1) and HSP70 (HSPA1A). The right panels show the proportional dosage of each construct (by mass of DNA) in the transfection. Bars indicate means ± SEM. The right panel Lower-slope graph was analysed via a two-way ANOVA subjected to Dunnett’s post hoc test and the A50% graph was analysed via a one-way ANOVA subjected to a Tukey’s post-hoc test. Results coded as ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = > 0.05

Article Snippet: Plasmids expressing HSPA1A, DNAJB1 and mCherry were prepared as described previously35,36. pCMV6-AC BAG1 (cat# SC319483), pCMV6-AC HSF1 (cat# SC321225) and pCMV6-Entry myc-HSP70AA1 (cat# RC212496) were purchased from Origene.

Techniques: Activation Assay, Over Expression, Western Blot, BIA-KA, Transfection, Control, Variant Assay, Construct

Journal: Cancer gene therapy

Article Title: Functionality of hypoxia-induced BAX expression in a human glioblastoma xenograft model.

doi: 10.1038/sj.cgt.7700814

Figure Lengend Snippet: Figure 2 HIF-1a, mBAX, and hBAX expression in parent U-251 MG cells and HRE-Bax Clone D cells following 4–24 hours of (a) anoxic (N2) or oxic (O2) treatments; (b) 0.3 or 1% O2 treatments.

Article Snippet: Detection of protein on the immunoblots was performed using anti-human HIF-1a antibody raised in mouse (1:250 dilution; BD Biosciences Clontech, Palo Alto, CA), followed by peroxidaseconjugated horse anti-mouse IgG (1:30,000 dilution; Vector Laboratories, Burlingame, CA), as well as antimurine BAX monoclonal antibody and anti-human BAX monoclonal antibody (1:5000 dilution; R&D System, Inc., Minneapolis, MN) followed by goat anti-mouse IgG (1:50,000 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Techniques: Expressing

Journal: Annals of the Rheumatic Diseases

Article Title: Unveiling inflammatory and prehypertrophic cell populations as key contributors to knee cartilage degeneration in osteoarthritis using multi-omics data integration

doi: 10.1136/ard-2023-224420

Figure Lengend Snippet: The OA-associated changes between patients with OA and non-OA controls. (A) The Manhattan-like plot demonstrates the population-specific DE genes between patients with OA and non-OA controls. The numbers on the top panel denote the number of DE genes for each population. The dot size represents the value of −log10 (adjusted p). (B) The subnetworks identified by PhenomeExpress. The DE genes of preHTC were enriched in a pMAPK38 cascade subnetwork. (C) The visualisation of subpopulations of preHTC using UMAP projections, that is, preHTC-1 (blue), preHTC-2 (green), preHTC-3 (brown) and preHTC-4 (red), where a total of 25 778 chondrocytes were analysed by the integrative analysis of all 19 samples. (D) The bar plot shows the significant GO terms for each subpopulation. (E) The Manhattan-like plot shows the zone-specific DE genes between patients with OA and non-OA controls, that is, AS (red), SZ (yellow), MZ (blue) and DZ (green). The numbers on the top panel or bottom panel represent the number of significant upregulated or downregulated DE genes for each zone. (F) The bar plot shows the significant GO terms that were enriched by the zone-specific DE genes. Notably, the MZ-specific and DZ-specific DE genes were not enriched in any biologically meaningful pathways. (G) The subnetworks identified by PhenomeExpress. The DE genes of AS were enriched in response to the chemokine subnetwork and those of SZ were enriched in the regulation of the transcription subnetwork. (H) The top-ranked DE gene of AS or SZ between patients with OA and non-OA controls, BAG1, was validated by IHC staining. (I) The top-ranked DE gene of AS or SZ between patients with OA and non-OA controls, CD9, was validated by IHC staining. The quantification of positive cells from different zones (ie, SZ, MZ and DZ) is displayed by bar plots (replicates n=6), where the AS and SZ were hardly distinguished during IHC staining, therefore, the AS and SZ in Geo-seq results were merged together to only present as SZ in IHC staining. The scale bar: left, 500 μm; right, 50 μm. AS, articular surface; DZ, deep zone; GO, Gene Ontology; IHC, immunohistochemistry; InfC, inflammatory chondrocyte; MZ, middle zone; NS, no significant; OA, osteoarthritis; preInfC, pre-inflammatory chondrocyte; preFC, prefibrocartilage chondrocyte; preHTC, prehypertrophic chondrocyte; SZ, superficial zone; UMAP, uniform manifold approximation and projection. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After rinsing, sections were sealed with goat serum (ZSGB-BIO, China) for 20 min at room temperature and incubated with rabbit antihuman CD74 (1:50, ab181470, Abcam, UK), mouse antihuman BMP2 (1:100, 66383-1-Ig, Proteintech, China), rabbit antihuman IBSP (1:2000, ab270605, Abcam), rabbit antihuman CHI3L1 (1:200, ab255297, Abcam), rabbit antihuman GPR183 (1:200, ab150625, Abcam), rabbit antihuman BAG1 (1:100, 19064-1-AP, Proteintech), rrabbit antihuman CD9 (1:2000, 20597-1-AP, Proteintech) overnight at 4°C.

Techniques: Immunohistochemistry