Journal: BMC Complementary Medicine and Therapies

Article Title: Ammidin ameliorates myocardial hypoxia/reoxygenation injury by inhibiting the ACSL4/AMPK/mTOR-mediated ferroptosis pathway

doi: 10.1186/s12906-023-04289-x

Figure Lengend Snippet: Effect of 2 μM Fer-1 on ROS levels in H/R-induced cardiomyocytes. ( A ) Cardiomyocyte viability. ( B ) LDH release. ( C ) MDA content. ( D ) ROS fluorescence image, Merge: A combination of ROS (green fluorescence) and nucleus (blue fluorescence). ( E ) ROS quantitative analysis. ( F ) SOD content. ( G ) JC-1 staining showed that JC-1 aggregated in the mitochondria of normal cells emitted red fluorescence, while JC-1 in dead cells emitted green fluorescence due to its presence in the cytoplasm as a monomer. Merge: A combination of aggregates (red fluorescence), monomers (green fluorescence) and nucleus (blue fluorescence). ( H ) Red/green fluorescence ratio at the MMP level. ( I-K ) Western blotting revealing the expression levels of ACSL4 and GPX4 in cardiomyocytes ( * P < 0.05 vs. control; # P < 0.05 vs. H/R; scale bars, 100 μm; n = 3). Proteins were then transferred onto PVDF membranes. After cutting according to the molecular size, the primary antibody was incubated. Fer-1, ferrostatin-1; ROS, reactive oxygen species; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; MDA, malondialdehyde; SOD, superoxide dismutase; JC-1, 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide; MMP, mitochondrial membrane potential; ACSL4, Acyl-CoA synthetase long chain family member 4; GPX4, glutathione peroxidase 4

Article Snippet: Anti-β-actin (cat. no. sc-47,778; dilution, 1:1000) and goat anti-rabbit IgG (cat. no. sc-2004; dilution, 1:20,000) were purchased from Santa Cruz Biotechnology, Inc. Primary antibodies against proteins, including ACSL4 (cat. no. ab155282; dilution, 1:1000), GPX4 (cat. no. ab125066; dilution, 1:1000), AMPK (cat. no. ab32047; dilution, 1:1000), phosphorylated (p-AMPK) (cat. no. ab133448; dilution, 1:1000), mTOR (cat. no. ab134903; dilution, 1:500) and p-mTOR (cat. no. ab109 268; dilution, 1:500), were purchased from Abcam.

Techniques: Fluorescence, Staining, Western Blot, Expressing, Control, Incubation, Membrane

Journal: Separations

Article Title: Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations

doi: 10.3390/separations8110204

Figure Lengend Snippet: Figure 6. The protein pattern and biomarker of the exosomes isolated from different sample volumes. A protein ladder reference (Vivantis Technologies, Shah Alam, Selangor Darul Ehsan, Malaysia, cat no. PR0623) had 2 reference bands (25 and 72 kDa) coupled with blue chromophore for easy identification (A). SDS-PAGE stained with Coomassie blue G250 showed multiple bands with the same patterns (B). The Western blot results showed a single band of TSG101 at about 48.9 kDa exhibiting the exosome biomarker (C). Thirty micrograms of protein were loaded for both SDS-PAGE and the Western blot. All experiments above were run with a reducing agent. M = Marker of protein molecular weight.

Article Snippet: The primary antibodies, human ApoA-1 antibody (cat no. MAB 36641-SP) and human TSG101 antibody (cat no. orb337267), were supplied by R&D System, Inc., Minneapolis, MN, USA and Biorbyt, Cambridge, Cambridgeshire, UK, respectively.

Techniques: Biomarker Discovery, Isolation, SDS Page, Staining, Western Blot, Marker, Molecular Weight

Journal: Nature Communications

Article Title: Plk1 overexpression induces chromosomal instability and suppresses tumor development

doi: 10.1038/s41467-018-05429-5

Figure Lengend Snippet: Overexpression of Plk1 impairs Cep55 and ESCRT loading into the cytokinesis midbody. a (+/Plk1);(rtTA/rtTA) MEFs were untreated (–Dox) or treated for 24 h with doxycycline (+Dox), fixed and stained for α-tubulin (red) and DAPI (DNA, green). Cells in cytokinesis ( n > 100 per condition; n = 3 replicates) were evaluated for aberrant cytokinesis, considering the midbody formation and shape, and correct distribution of DNA into the two daughter cells. Scale bars, 10 μm. b The length of the cytokinesis bridge was evaluated by measuring the distance in between the two daughter nuclei in MEFs untreated or after 24 h of Dox treatment (–Dox, n = 17 cells; +Dox, 23 cells). Scale bars, 10 μm. In a , b *, p < 0.05; Student’s t -test. c MEFs expressing a CEP55-EGFP fusion (green) were treated for 24 h with Dox (+Dox), in the absence or presence of 1 μM of the Plk1 inhibitor BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated as control (–Dox). α-tubulin is in red. Data represent the percentage of cells with positive CEP55-EGFP signal at the midbody (–Dox, n = 134; +Dox, n = 94; Dox + BI, n = 47 cells; n = 3 replicates (–Dox, +Dox) or 2 replicates (Dox+BI)). Scale bar, 5 μm. d MEFs expressing a Tsg101-mCherry fusion (red) were treated for 24 h with Dox (+Dox), in the absence or presence of 1 μM of BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated as control (–Dox). α-tubulin is in green. Data represent Tsg101-mCherry mean intensity at the midbody (–Dox, n = 81; +Dox, n = 60; Dox + BI, n = 37 cells; n = 2 replicates). Scale bars, 5 μm. e MEFs were treated for 24 h with Dox (+Dox), in the absence or presence of 1 μM of BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated (–Dox). Cells were stained for α-tubulin (red) and DAPI (DNA, green) and binucleation index was quantified from more than 600 cells in each sample ( n = 5 replicates). Scale bars, 50 μm. In c – e , n.s. not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; one-way ANOVA

Article Snippet: MEFs were transfected either with cDNA encoding for MKLP1 fused to GFP (Addgene #70145), CEP55 (obtained from the Mammalian Gene Collection) fused to GFP, or the ESCRT-I member TSG101 fused to the mCherry reporter (Addgene #38318).

Techniques: Over Expression, Staining, Expressing, Control

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: Exosomes derived from lung cancer cells are internalized by THP-1-M0 macrophages and mediate EHF delivery. Exosomes were isolated from A549 and H520 cells. (A) TEM images showed the typical cup-shaped morphology of exosomes isolated from A549 and H520 cell supernatants (scale bar: 100 nm). (B) NTA of exosomes from A549 and H520 cells. (C) Western blot analysis of exosome markers (CD9 and TSG101) and the endoplasmic reticulum marker Calnexin in exosome lysates and parental A549/H520 cell lysates. (D-E) THP-1-M0 macrophages (differentiated from THP-1 cells with 100 ng/mL PMA for 24 h) were co-incubated with DiI-labeled exosomes isolated from A549 and H520 cells. (D) Fluorescence microscopy images of THP-1-M0 macrophages after co-incubation with PKH67-labeled exosomes (scale bar: 20 μm). (E-F) Western blot and quantification of EHF protein levels in THP-1-M0 macrophages after incubation with exosomes from A549 or H520 cells. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Derivative Assay, Isolation, Western Blot, Marker, Incubation, Labeling, Fluorescence, Microscopy

Journal: Oncogene

Article Title: Overexpression of tumor susceptibility gene TSG101 in human papillary thyroid carcinomas.

doi: 10.1038/sj.onc.1205612

Figure Lengend Snippet: Figure 1 Endogenous TSG101 protein expression in SK-Hep-1 cells. (a) A diagrammatic illustration of the functional domains of TSG101 protein and epitopes of antisera used in this study. The numbers refer to the amino acid residues of TSG101. The in- active ubiquitin-conjugating domain (UBC), the proline-rich region (PRR), coiled-coil domain (CC) and steadiness box (SB) are indicated. (b) Endogenously expressed TSG101 in 30 mg of SK-Hep-1 cell lysate was separated and blotted onto nitrocellu- lose membrane. It was then detected by sc-6037, a commercial antiserum against mouse tsg101 protein (lane 1) or rabbit preim- mune serum (lane 2); antiserum #654 (lane 3); rabbit antiserum #654 that was preabsorbed by GST protein (lane 4); and rabbit antiserum #654 that was preabsorbed by GST-TSG (1 – 291) fusion protein (lane 5)

Article Snippet: As shown in Figure 1b, antiserum #654 detected a prominent band in the same position as the band that could be detected by a commercial TSG101 antibody (sc-6037, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Figure 1b; lanes 1 and 3).

Techniques: Expressing, Functional Assay, Ubiquitin Proteomics, Membrane

Journal: Oncogene

Article Title: Overexpression of tumor susceptibility gene TSG101 in human papillary thyroid carcinomas.

doi: 10.1038/sj.onc.1205612

Figure Lengend Snippet: Figure 2 Immunohistochemical and in situ hybridization analyses of a classical PTC specimen. A classical PTC specimen was stained with (a) rabbit antiserum #654, the neoplastic epithelial cells (left side) revealing intense intracytoplasmic immunoreactivity; (b) rabbit antiserum #654, which was absorbed by GST protein; (c) a higher magnification of b showing negative staining of non- neoplastic follicular cells (right side); (d) preimmune serum; (e) in situ hybridization of same PTC specimen using TSG101 antisense riboprobe; (f) a higher magnification of e (Magnifications: a, b, d and e, 406; c and f, 2006)

Article Snippet: As shown in Figure 1b, antiserum #654 detected a prominent band in the same position as the band that could be detected by a commercial TSG101 antibody (sc-6037, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Figure 1b; lanes 1 and 3).

Techniques: Immunohistochemical staining, In Situ Hybridization, Staining, Negative Staining

Journal: Oncogene

Article Title: Overexpression of tumor susceptibility gene TSG101 in human papillary thyroid carcinomas.

doi: 10.1038/sj.onc.1205612

Figure Lengend Snippet: Figure 3 TSG101 gene expression in a PTC specimen of solid/trabecular type. Immunohistochemical staining of a solid/trabecular type PTC specimen using (a) rabbit antiserum #654; (b) rabbit antiserum #654, which was absorbed by GST protein; (c) preimmune serum; (d) a higher magnification of b showing non-neoplastic follicular cells. Arrows indicated cytoplasmic dot-staining. In situ hybridization of same PTC specimen using (e) TSG101 antisense riboprobe, (f) TSG101 sense riboprobe (Magnifications: a, b, c, d, e and f, 2006)

Article Snippet: As shown in Figure 1b, antiserum #654 detected a prominent band in the same position as the band that could be detected by a commercial TSG101 antibody (sc-6037, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Figure 1b; lanes 1 and 3).

Techniques: Gene Expression, Immunohistochemical staining, Staining, In Situ Hybridization

Journal: Oncogene

Article Title: Overexpression of tumor susceptibility gene TSG101 in human papillary thyroid carcinomas.

doi: 10.1038/sj.onc.1205612

Figure Lengend Snippet: Figure 4 Semi-quantitative RT – PCR analysis of TSG101 ex- pression in normal and PTC specimens. (a) Autoradiography of Southern analysis. RT – PCR products were blotted and hybri- dized with [32P]oligonucleotide probes specific for TSG101 and GADPH. Normal thyroid tissues: lanes 1 – 3; PTC tissues: lanes 4 – 6. (b) A bar graph illustrates the density ratio of TSG101/ GAPDH hybridization signals of three normal thyroid (bars 1 – 3) and PTC specimens (bars 4 – 6) that were calculated by densi- tometric scanning of autoradiogram shown in a

Article Snippet: As shown in Figure 1b, antiserum #654 detected a prominent band in the same position as the band that could be detected by a commercial TSG101 antibody (sc-6037, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Figure 1b; lanes 1 and 3).

Techniques: Quantitative RT-PCR, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Hybridization