Journal: Autophagy

Article Title: Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases

doi: 10.1080/15548627.2017.1308985

Figure Lengend Snippet: BAG1 upregulation reduces ARpolyQ accumulation. (A to C) NSC34 cells treated with DMSO or 100 μM EHNA for 48 h. The bar graphs represent Hspb8 (A), Bag3 (B) and Bag1 (C) mRNA levels normalized with Rplp0 mRNA levels. Four independent biological samples for each condition were analyzed (n = 4) ± SD (*** = P < 0.001; One-Way ANOVA followed by the Uncorrected Fisher LSD test). (D) NSC34 cells overexpressing AR.Q46 alone or coexpressing BAG1 and treated with T for last 48 h. FRA shows insoluble AR-polyQ levels (upper panel) and bar graph represents the FRA mean relative optical density computed over 3 independent biological samples for each condition (n = 3) ± SD (* = P < 0.05, *** = P < 0.001; Two-Way ANOVA followed by the Uncorrected Fisher LSD test). WB (lower panel) shows AR.Q46 and BAG1 protein levels. (E) NSC34 cells expressing AR.Q46 alone or coexpressing BAG1 and treated with 10 nM T for last 48 h and/or 10 μM MG132 for last 16 h. FRA shows insoluble AR-polyQ levels, bar graph represents the FRA mean relative optical density computed over 3 independent biological samples for each condition (n = 3) ± SD (* = P < 0.05, *** = P < 0.001; Two-Way ANOVA followed by the Uncorrected Fisher LSD test and one-tailed unpaired Student t test for control condition). (F) WB analysis of NSC34 cells transfected with Bag1 siRNA shows the levels of BAG1; TUBA was used as loading control. Bar graph represents the WB mean relative optical density computed over 3 independent biological samples for each condition (n = 3) ± SD (** = P < 0.01, one-tailed unpaired Student t test). (G) FRA and WB of NSC34 transfected with AR.Q46 and with Bag1 siRNA shows AR.Q46 insoluble fraction; TUBA was used as loading control. Bar graph represents the FRA densitometric analysis of 3 independent biological samples for each condition (n = 3) ± SD (** = P < 0.01; *** = P < 0.001; one-tailed unpaired Student t test).

Article Snippet: The membranes were treated with a blocking solution containing 5% nonfat dried milk powder (Euroclone, EMR180500) in Tris-buffered saline with Tween 20 (0.01%; Sigma, P1379) (TBS-T; Tris base 20 mM, NaCl 140 mM, pH 7.6) for 1 h and then incubated with one of the following primary antibodies: (a) rabbit polyclonal AR-H280 (dilution 1:3,000) to detect wtAR and ARpolyQ (Santa Cruz Biotchnology, sc-13062); (b) rabbit polyclonal anti-LC3A/B (dilution 1:4,000; Sigma-Aldrich, L8918); (c) rabbit polyclonal anti-SQSTM1 (dilution 1:4,000; Abcam, ab91526); (d) rabbit polyclonal anti-dynein heavy chain (dilution 1:1,000; Santa Cruz Biotchnology, sc-9115); (e) rabbit polyclonal anti-GFP HRP conjugated (dilution 1:15,000; Vector Laboratories, MB-0712); (f) rabbit polyclonal anti-HA (dilution 1:1,000; Santa Cruz Biotchnology, sc-7392) to detect overexpressed BAG1; (g) rabbit polyclonal anti-BAG1 (dilution 1:1,000; Santa Cruz Biotchnology, sc-939) to detect endogenous BAG1; (h) rabbit polyclonal Cu/Zn SOD (1:1,000; Enzo Life Science, ADI-SOD-100) to detect SOD1; (i) mouse polyclonal anti-flag M2 (dilution 1:1,000; Sigma-Aldrich, F1804) to detect TARDBP; (j) mouse monoclonal anti-TUBA(dilution 1:4,000; Sigma-Aldrich, T6199); (k) rabbit polyclonal anti-GAPDH (1:4,000; Santa Cruz Biotchnology, sc-25778).

Techniques: Expressing, One-tailed Test, Control, Transfection

Journal: Autophagy

Article Title: Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases

doi: 10.1080/15548627.2017.1308985

Figure Lengend Snippet: EHNA effects on neuronal and motoneuronal cells obtained by differentiation of human iPSCs generated from SBMA patients. (A) IF analysis of neuronal and motoneuronal cells obtained by differentiation of human iPSCs generated from SBMA patients and treated with ethanol or 10 nM T (40x magnification). TUBB3 (green) and SMI312 (red) (upper inset), AR (green) and MNX1/HB9 (red) (lower inset). Nuclei were stained with DAPI (blue); scale bars: 30 μm. (B) Bar graph represents the percentage of MNX1/HB9-positive cells; 6 fields for each condition were analyzed (n = 6) ± SD (one-tailed unpaired Student t test, not significant). (C to I) RealTime PCR analyses performed on iPSCs differentiated to neuronal cells treated with 10 nM T and/or 100 μM EHNA for last 48 h. The bar graphs represent HSPB8 (C), BAG3 (D), BAG1 (E), SQSTM1 (F), LC3 (G), TFEB (H) and BECN1 (I) mRNA levels normalized with RPLP0 mRNA levels. Four independent samples for each condition were analyzed (n = 4) ± SD (* = P < 0.05, ** = P < 0.01, *** = P < 0.001; Two-Way ANOVA followed by the Uncorrected Fisher LSD test).

Article Snippet: The membranes were treated with a blocking solution containing 5% nonfat dried milk powder (Euroclone, EMR180500) in Tris-buffered saline with Tween 20 (0.01%; Sigma, P1379) (TBS-T; Tris base 20 mM, NaCl 140 mM, pH 7.6) for 1 h and then incubated with one of the following primary antibodies: (a) rabbit polyclonal AR-H280 (dilution 1:3,000) to detect wtAR and ARpolyQ (Santa Cruz Biotchnology, sc-13062); (b) rabbit polyclonal anti-LC3A/B (dilution 1:4,000; Sigma-Aldrich, L8918); (c) rabbit polyclonal anti-SQSTM1 (dilution 1:4,000; Abcam, ab91526); (d) rabbit polyclonal anti-dynein heavy chain (dilution 1:1,000; Santa Cruz Biotchnology, sc-9115); (e) rabbit polyclonal anti-GFP HRP conjugated (dilution 1:15,000; Vector Laboratories, MB-0712); (f) rabbit polyclonal anti-HA (dilution 1:1,000; Santa Cruz Biotchnology, sc-7392) to detect overexpressed BAG1; (g) rabbit polyclonal anti-BAG1 (dilution 1:1,000; Santa Cruz Biotchnology, sc-939) to detect endogenous BAG1; (h) rabbit polyclonal Cu/Zn SOD (1:1,000; Enzo Life Science, ADI-SOD-100) to detect SOD1; (i) mouse polyclonal anti-flag M2 (dilution 1:1,000; Sigma-Aldrich, F1804) to detect TARDBP; (j) mouse monoclonal anti-TUBA(dilution 1:4,000; Sigma-Aldrich, T6199); (k) rabbit polyclonal anti-GAPDH (1:4,000; Santa Cruz Biotchnology, sc-25778).

Techniques: Generated, Staining, One-tailed Test

Journal: International Journal of Molecular Medicine

Article Title: G-protein-coupled receptor 30 mediates the effects of estrogen on endothelial cell tube formation in vitro

doi: 10.3892/ijmm.2017.2957

Figure Lengend Snippet: Western blot analysis demonstraing that the levels of (A) p-PI3K (p85) and (B) p-Akt (Ser 473 ) were significantly increased in the presence of E2 (A and B, * P=0.0001 and & P=0.0001, respectively) or G1 (A and B, * P=0.0001 and & P= 0.0027, respectively) in human umbilical vein endothelial cells (HUVECs) under hypoxia-reoxygenation (H/R) conditions. The effect was blocked by treatment with G15 (A and B, # P<0.0001 and $ P=0.0098, respectively). Western blot analysis also demonstrated that the levels of (C) p-eNOS (Ser 1177 ) were significantly decreased under H/R conditions in HUVECs compared to normoxic conditions ( * P= 0.0111). This decrease was significantly increased in the presence of E2 or G1 in HUVECs under H/R conditions ( # P<0.0043 and P= 0.0104). This effect was significantly blocked by treatment with wortmannin (Wort, 100 nM) ( ** P<0.0001, # # P<0.0001). eNOS, endothelial nitric oxide synthase; E2, 17-β-estradiol.

Article Snippet: Non-specific binding was blocked by incubating membranes in 5% non-fat milk for 1 h and the membranes were then incubated with monoclonal anti-GPR30 (1:1,000 dilution) or monoclonal anti-p-PI3K (p85) (1:500 dilution) or polyclonal anti-PI3K (p85) (1:500 dilution; sc-292114; Santa Cruz Biotechnology, Inc.) or monoclonal anti-p-Akt (p-Akt Ser 473 ) (1:1,000 dilution) or polyclonal anti-Akt (1:500 dilution; sc-5298; Santa Cruz Biotechnology, Inc.) or monoclonal anti-p-eNOS (Ser 1177 ) (1:1,000 dilution) or monoclonal anti-eNOS (1:1,000 dilution) antibodies.

Techniques: Western Blot

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