Journal: Antioxidants (Basel, Switzerland)

Article Title: OTUB1-SLC7A11 Axis Mediates 4-Octyl Itaconate Protection Against Acetaminophen-Induced Ferroptotic Liver Injury.

doi: 10.3390/antiox14060698

Figure Lengend Snippet: Figure 3. 4-OI mitigates APAP-induced dysregulation of iron metabolism. (A) Pathways associated with genes/proteins interacting with differential metabolites were analyzed using the R package CePa. logP (x-axis): negative log of the enrichment p-value for drug bias-related metabolites. foldP (y-axis): ratio of enrichment p-values for efficacy-related vs. bias-related metabolites. The red circle highlights the ferroptosis pathway, which is identified as the most prominent among all analyzed pathways. (B) Hepatic iron content (n = 3). (C–I) Representative Western blot images and quantification of transferrin, TFR1, FTH1, FTL1, FPN1, and hepcidin protein levels in mouse liver tissues (n = 3). (J–L) A RT-qPCR assay determined the mRNA expression of FTH1, FTL1, and FPN1 (n = 6). (M) Representative immunohistochemical staining images showing FTH1, FTL1, and FPN1 expression. Scale bar: 100 µm, n = 3. GAPDH was used as internal loading control. * p < 0.05, ** p < 0.01, *** p < 0.001; ns: not significant.

Article Snippet: Primary antibodies for SLC7A11, OTUB1, CD44, ubiquitin, GPX4, Nrf2, transferrin, TFR1, FTH1, FTL1, FPN1, hepcidin, and GAPDH were obtained from the following sources: SLC7A11 and OTUB1 antibodies from Abcam (Cambridge, UK); hepcidin antibody from Affinity (Changzhou, China); CD44, ubiquitin, GPX4, Nrf2, transferrin, TFR1, FTH1, FTL1, FPN1, and GAPDH antibodies from Proteintech (Wuhan, China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Control

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: Design and characterization of cell type specific Wnt signaling enhancing mimetic molecules. ( A ) Scheme of designed molecules. Fragments of RSPO2 spanning the Fu1 and Fu2 domains (action module) were fused the C-terminus of cell type specific or control scFv (targeting module). Specific mutations used are indicated. ( B ) In vitro STF activity of four RSPO proteins. Furin domains of human RSPO1-4 were tested as fusions to an anti-GFP scFv. RSPO2 furin domain alone (N37-E143) was used as a control. Luciferase activity unit is arbitrary (RLU). ( C ) STF activity of RSPO mimetics on HEK293 ( top ) or HuH-7 ( bottom ) in the presence ( left ) or absence ( right ) of an exogenous Wnt source (30% Wnt3a conditioned media). ( D ) Quantitative PCR analysis of ASGR1 , ASGR2 and TFRC gene expression in HEK293, HuH-7 and A431 cells. The signals were relative to ACTB . ( E ) Western Blot analysis on LRP6 receptor and DVL2 phosphorylation in HuH-7 cells. Asterisk indicates a non-specific band that is detected in HuH-7 by the antibody used. The lower bands indicated by the arrow correspond to DVL2 with and without phosphorylation. Tubulin (TUB) is used as the loading control. 10% Wnt3a conditioned media was used as the exogenous Wnt source. Images were cropped for clarity. The uncropped images are presented in Fig. .

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: In Vitro, Activity Assay, Luciferase, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: Confirmation of receptor-specific activation of Wnt signaling. ( A ) STF activity in HEK293 cells transiently transfected with TFR1 (control, top ), ASGR1 ( middle ), and ASGR1 together with ASGR 2 cDNAs ( bottom ), either in the presence ( left ) or absence ( right ) of exogenous Wnt source (30% Wnt3a conditioned media). ( B ) STF activity in A431 cells with TFR1 or ASGR1 overexpression as specified. ( C ) Flow cytometry analysis of cell surface level of FZD proteins. HEK293 cells were transiently transfected with either ZNRF3 alone ( top ) or ASGR1 and ZNRF3 cDNAs ( bottom ), treated with RSPO mimetics as specified at 10 nM, then stained by the pan-FZD antibody 18R5.

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: Activation Assay, Activity Assay, Transfection, Over Expression, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: In vivo tissue-specific enhancement of Wnt signaling. ( A ) STF activity of the RSPO mimetic molecules in the appended-IgG format. ( B ) BLI analysis of the ASGR1 antibody (expressed as a Fab) on human and mouse ASGR1. The affinity (Kd) determined by steady-state fitting is indicated. ( C ) Axin2 and ( D ) Ki67 expression in liver ( upper panel ) and small intestine ( lower panel ) were analyzed by quantitative PCR, 48 h after i.p. injection of proteins as specified (n = 8 mice per group). Statistical analysis was performed using 1-way ANOVA: (ns) not significant, ** p < 0.01, **** p < 0.0001. ( E ) Immunofluorescence staining of liver samples for Ki67 and HNF4α from 10 mg/kg treatment groups. White arrows denote some double-positive cells as examples. DAPI was used to stain nuclei.

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: In Vivo, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Injection, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence

doi: 10.1038/s41467-020-16337-y

Figure Lengend Snippet: a BCA (protein concentration) of circulating EVs from healthy donors ( h EVs) and P. vivax patients ( Pv EVs) ( n = 10, individual samples). Data show mean ± SD. Two-sided, Mann–Whitney test, * p = 0.0232 (GraphPad). b P. vivax proteins identified by different unique peptides (sequences below the description of corresponding proteins). UniProtKB accession numbers and gene name corresponding to the P. vivax PvP01 strain are shown. c Western blot analysis of Pv EVs obtained from different individual patients (Pv1-7 and Pv10) and human donors (H1 and H3) using anti P. vivax MSP3.1 (upper membrane) and PHISTc (bottom membrane) antibodies. MSP3.1 and PHISTc recombinant truncated-proteins fused to GST, were used as positive controls. Molecular weight in kDaltons (kDa) is shown to the left. M: molecular size marker. Image representative of three independent experiments. d Nanoparticle tracking analysis (NTA) profile (size [nm] versus concentration [particles/mL]) of pooled h EVs and Pv EVs was analysed using NanoSight LM10-12. Table shows mean of three measurement of pooled h EVs and Pv EVs quantified by NTA (particles concentration) and BCA (protein concentration). e Beads-based flow cytometry analysis of pooled h EVs and Pv EVs using six EV markers (CD9, CD63, CD81, GAL3, CD5L and CD71). Data show median fluorescence intensity (MFI) of each antibody and control antibodies (rabbit and mouse-isotype) ± SD (technical replicates, n = 3). Unpaired and two-sided, t -test ** p = 0.0032 (CD63), *** p = 0.0006 (CD81, CD71), **** p < 0.0001 (CD9) (GraphPad). Source data are provided as a Source data file and Supplementary Data , and .

Article Snippet: Further, cells were stained using the following antibodies: CD90-PE/Cy7 [5E10] (Biolegend, Cat#328124) 1/100; CD44-FITC [KM201] (Abcam Cat#ab25340) 1/100; CD54-Alexa Fluor® 488 [HCD54] (Biolegend, Cat#322714) 1/400; CD71-PE [AC102] (Miltenyi Biotec, Cat#130-091-728) 1/400; CD45-PerCP [2D1] (BD Biosciences, Cat#345809) 1/50.

Techniques: Protein Concentration, MANN-WHITNEY, Western Blot, Membrane, Recombinant, Molecular Weight, Marker, Concentration Assay, Flow Cytometry, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD71 , REA902 , 50 , 130-115-028 , FITC (PE) , Miltenyi Biotec.

Techniques: Imaging

Journal: Cell reports

Article Title: PI5P4Kα promotes glucose and iron acquisition to support metabolic fitness in pancreatic cancer

doi: 10.1016/j.celrep.2025.116199

Figure Lengend Snippet: (A) FerroOrange staining in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2. Immunofluorescence microscopy images of FerroOrange and DAPI indicating the representative effect of three independent experiments (scale bar, 20 μm) and quantitation of immunofluorescence microscopy images. Data are presented as signal intensity relative to the sh scr condition and are the average of three independent experiments. (B) Transferrin uptake in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2. Data are presented as signal intensity relative to the sh scr condition and are the average of three independent experiments. (C) Surface staining of CD71 quantified using flow cytometry. Data are presented as median intensity relative to the sh scr condition and are the average of three independent experiments. (D) FerroOrange staining in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2 in the presence of FAC. (E) Quantification of relative cell number in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2 in the absence or presence of ferrostatin, ferrostatin with ferric ammonium citrate (FAC), and FAC alone. Data are presented relative to the sh scr condition for each respective treatment and are the average of three independent experiments. (F) Correlation plots between the expression of PIP4K2A from TCGA, with the indicated gene signatures. (G) Kaplan-Meier overall survival curve of patients with PDAC stratified by the expression levels of genes in the indicated gene signatures. (H–J) The MIA PaCa-2 cells were transduced with sh scr, sh α#1, or sh α#2 in the absence or presence of FAC. (H) Glucose consumption. (I) Lactate production. Data are presented relative to the cell number for each respective treatment and are representative of three independent experiments. (J) Immunoblot assessing GLUT1 and PI5P4Kα levels. Tubulin was used as a loading control. Data are representative of three independent experiments. (B–E, H, and I) Data are presented as mean ± SD. Statistical significance was calculated using unpaired two-tailed Student’s t test without (E, H, and I) or with (B–D) Welch’s correction and with pairwise Wilcoxon rank-sum test (F). ns, not significant, * p < 0.05, ** p ≤ 0.001, and *** p ≤ 0.0001.

Article Snippet: Antibodies used were as follows: PI5P4Kα (5527, Cell Signaling), PARP (46D11, Cell Signaling), Glut1 (73015, Cell Signaling), and α-tubulin (T6199, Sigma), p62 (H00008878, Abnova), LC3 (12741, Cell Signaling), p-Histone H3 (9701, Cell Signaling), ASNS (14681–1-AP, Proteintech) and cleaved caspase 3 (9664, Cell Signaling) and PE-conjugated anti-CD71 antibody (130–115-029, Miltenyi Biotec).

Techniques: Staining, Transduction, Immunofluorescence, Microscopy, Quantitation Assay, Flow Cytometry, Expressing, Western Blot, Control, Two Tailed Test