Journal: Stem cells translational medicine

Article Title: Sustained knockdown of a disease-causing gene in patient-specific induced pluripotent stem cells using lentiviral vector-based gene therapy.

doi: 10.5966/sctm.2013-0017

Figure Lengend Snippet: Figure 4. Analysis of PiZ knockdown in vivo. (A): Embryonic day (E) 13.5 chimeric fetuses generated from eGFP-short hairpin RNA (shRNA) transduced induced pluripotent stem cells (iPSCs). Fetal mice showed ubiquitous and strong eGFP expression, indicating high contribution of iPSCs to chimeras and sustained transgene expression (left and middle). Right: Enlarged fluorescence image of manually isolated E13.5 fetal liver. (B): Fluorescence-activated cell sorting (FACS) for the CD45/eGFP fraction containing fetal hepatoblasts. (C): Quantitative reverse transcription-polymerase chain reaction analysis for Afp, Ck18, Ttr, and hA1AT in E13.5 CD45/eGFP cells. The scr-shRNA-treated cell line and bulk population are shown in light blue and turquoise, respectively, and the PiZ-shRNA-treated cell line and bulk population are shown in light pink and dark pink, respectively. Cells from at least six fetal livers were pooled for each sample. PiZ-shRNA-treated cells showed reduced hA1AT expression compared with scr-shRNA-treated cells, whereas Afp, Ck18, and Ttr were at comparable levels in all samples. Values were normalized to albumin. (D, E): Western blot (D) and enzyme-linked immunosorbent assay (ELISA) (E) for human A1AT in lysates of E15.5 chimeric livers. Percentages of chimerism of respective samples are indicated in parentheses. The PiZ-shRNA sample showed a strong reduction in both assays Western blot and ELISA compared with scr-shRNA samples. Vinculin served as loading control in Western blot. (F): Immunohistochemical analysis of cryosections from E15.5 chimeric livers. Sections were stained using an antibody specific for human A1AT and analyzed using fluorescence microscopy. Pictures were taken with two seconds exposure time for Alexa 568 at a 40 magnification. eGFP-positive cells stained strongly positive for hA1AT in the scr-shRNA-treated sample and weakly positive in the PiZ-shRNA-treated sample. Abbreviations: Alb, albumin; Ck18, cytokeratin 18; DAPI, 4,6-diamidino-2-phenylindole; eGFP, enhanced green fluorescence protein; hA1AT, human -1-antitrypsin; scr, scramble; sh, short hairpin RNA; Ttr, transthyretin; vinc, vinculin.

Article Snippet: Sections were blocked with blocking solution (PBS, 0.3% Triton X-100, 5% filtered donkey serum) for 1 hour at room temperature followed by incubation at 4°C overnight with goat anti-human A1AT (A80–122A; Bethyl, Montgomery, TX, http:// www.bethyl.com) 1:500 in blocking solution.

Techniques: Knockdown, In Vivo, Generated, shRNA, Expressing, Fluorescence, Isolation, FACS, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Immunohistochemical staining, Staining, Microscopy

Journal: Stem cells translational medicine

Article Title: Sustained knockdown of a disease-causing gene in patient-specific induced pluripotent stem cells using lentiviral vector-based gene therapy.

doi: 10.5966/sctm.2013-0017

Figure Lengend Snippet: Figure 6. Knockdown of PiZ human -1-antitrypsin (hA1AT) in patient-specific induced pluripotent stem cells (iPSCs) after differentiation. (A): Left: Quantitative reverse transcription-polymerase chain reaction expression analysis for TTR, CK18, CYP1A1, and -1-antitrypsin (A1AT) at day 18 of hepatic differentiation. All three clonal cell lines and the bulk population transduced with PiZ-short hairpin RNA (shRNA) showed significantly reduced expression of hA1AT compared with scr-shRNA clones and bulk. Values were normalized to albumin. Right: Significance and fold change were calculated for the group of PiZ-shRNA-treated samples compared with the group of scr-shRNA-treated samples. A1AT was significantly differentially expressed between the two groups, whereas CK18 and CYP1A1 were not. (B): Left: Western blot for A1AT in day 18 differentiated PiZZ iPSC and scr-shRNA and PiZ-shRNA transduced bulk populations. The upper band (56 kDa) shows fully glycosylated A1AT, and the lower band (52 kDa) shows partially glycosylated A1AT. Right: Densities measured for both bands separately were added together and divided by density of vinculin, and then divided by the average of untreated samples. The PiZ-shRNA-treated bulk population showed a significant 66% reduction of A1AT. Lysates from HepG2 and HEK293T cells served as positive and negative controls, respectively. (C, D): Enzyme-linked immunosorbent assay (ELISA) for secreted A1AT (C) and albumin (D) in supernatants collected from day 18 differentiated iPSCs. The PiZ-shRNA-treated bulk population showed significantly decreased secretion of A1AT compared with scr-shRNA-treated cells, whereas secretion whereas secretion of albumin was at a comparable level. (E): Ethoxyresorufin-O-deethylase (EROD) assay for CYP1A1 activity. Values were at comparable levels among different iPSC samples. All values for ELISAs and EROD were normalized to 10,000 cells and 24 hours, and supernatant collected from HepG2 hepatoma cell line served as a positive control. (F): Immunocytochemical analysis for polymeric PiZ A1AT in cell clusters of day 18 differentiated iPSCs. Cells were stained using an antibody specific for polymeric human A1AT and analyzed using fluorescence microscopy. Pictures were taken with 2 seconds of exposure time for Alexa 568 at a magnification of 40. Clusters stained strongly positive for polymeric hA1AT in scr-shRNA-treated cells and weakly positive in PiZ-shRNA-treated cells. Statistical analysis was performed using the t test. , p .05; , p .01. Abbreviations: ALB, albumin; CK18, cytokeratin 18; DAPI, 4,6-diamidino-2- phenylindole; ns, not significant; scr, scramble; sh, short hairpin RNA; TTR, transthyretin.

Article Snippet: Sections were blocked with blocking solution (PBS, 0.3% Triton X-100, 5% filtered donkey serum) for 1 hour at room temperature followed by incubation at 4°C overnight with goat anti-human A1AT (A80–122A; Bethyl, Montgomery, TX, http:// www.bethyl.com) 1:500 in blocking solution.

Techniques: Knockdown, Reverse Transcription, Polymerase Chain Reaction, Expressing, Transduction, shRNA, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Positive Control, Staining, Fluorescence, Microscopy

Journal: Proteome science

Article Title: Identification of noval diagnostic biomarker for HFpEF based on proteomics and machine learning.

doi: 10.1186/s12953-025-00242-7

Figure Lengend Snippet: Fig. 6 Expression analysis of hub proteins in immune cells. (A) Expression of ITIH 4 in immune cells. (B) Expression of SERPINA1 in immune cells

Article Snippet: Plasma SERPINA3 levels were measured by ELISA kit SERPINA3(Human SERPINA3 ELISA Kit YX-E11217, sinobestbio), ITIH4 (Human ITIH4 ELISA Kit EK1670 BOSTER), SERPINA1 (Human Alpha 1 Antitrypsin/SERPINA1 ELISA Kit EK1634 BOSTER), AFM (Human Afamin/AFM ELISA Kit (EK1487 BOSTER) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Proteome science

Article Title: Identification of noval diagnostic biomarker for HFpEF based on proteomics and machine learning.

doi: 10.1186/s12953-025-00242-7

Figure Lengend Snippet: Fig. 7 Expression of feature proteins in tissue-specific analysis. (A) Expression of SERPINA3 in tissue-specific analysis. (B) Expression of SERPINA1 in tissue- specific analysis. (C) Expression of AFM in tissue-specific analysis. (D) Expression of ITIH4 in tissue-specific analysis

Article Snippet: Plasma SERPINA3 levels were measured by ELISA kit SERPINA3(Human SERPINA3 ELISA Kit YX-E11217, sinobestbio), ITIH4 (Human ITIH4 ELISA Kit EK1670 BOSTER), SERPINA1 (Human Alpha 1 Antitrypsin/SERPINA1 ELISA Kit EK1634 BOSTER), AFM (Human Afamin/AFM ELISA Kit (EK1487 BOSTER) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Proteome science

Article Title: Identification of noval diagnostic biomarker for HFpEF based on proteomics and machine learning.

doi: 10.1186/s12953-025-00242-7

Figure Lengend Snippet: Fig. 8 The expression profile analysis of feature proteins. (A) SERPINA3 expression in clinical plasma samples. (B) SERPINA1 expression in clinical plasma samples. (C) AFM expression in clinical plasma samples. (D) ITIH4 expression in clinical plasma samples

Article Snippet: Plasma SERPINA3 levels were measured by ELISA kit SERPINA3(Human SERPINA3 ELISA Kit YX-E11217, sinobestbio), ITIH4 (Human ITIH4 ELISA Kit EK1670 BOSTER), SERPINA1 (Human Alpha 1 Antitrypsin/SERPINA1 ELISA Kit EK1634 BOSTER), AFM (Human Afamin/AFM ELISA Kit (EK1487 BOSTER) according to the manufacturer’s instructions.

Techniques: Expressing, Clinical Proteomics

Journal:

Article Title: Characterization of an ERAD pathway for non-glycosylated BiP substrates which requires Herp

doi: 10.1016/j.molcel.2007.09.012

Figure Lengend Snippet: NS1 κ LC expressed in P3U.1 cells (A), RE61 λ LC and BiP (C) or HA-γ V-CH1 (E) were transiently co-expressed with or without Herp-FLAG in 293T cells. At 24 hr post-transfection, cell extracts were prepared from cells treated with or without lactacystin and immunoprecipitated with anti-κ LC antiserum (A), anti-λ LC antiserum (C) or anti-HA antibody (E). Cell extracts and precipitated samples were subjected to immunoblot analyses as indicated. Non-secreted λ LC RE61 (B) or HA-γ V-CH1 (D) were transiently expressed in 293T cells. At 24 hr post-transfection, cells were labeled with 35S-methionine/cysteine for 15 min and chased for the indicated times in the presence or absence of lactacystin. Immunoprecipitated samples from cell extracts were subjected to SDS-PAGE, followed by autoradiography. The signals for λ LC RE61 (B) or HA-γ V-CH1 (D) were quantified as expressed as a percent of that present at t=0. The values are shown at the bottom of each lane. The α1-antitrypsin (AAT) NHK variant (F) or Z variant (G) was transiently co-expressed with or without Herp-FLAG in 293T cells. At 24 hr post-transfection, cell extracts were prepared after treatment with or without tunicamycin for 3 hr and subjected to immunoprecipitation with anti-α1-antitrypsin antiserum. Cell extracts and precipitated samples were subjected to immunoblot analysis as indicated.

Article Snippet: All other antibodies were purchased from companies; anti-mouse IgG (Igγ and κ) and anti-mouse IgM (Igμ and λ) (Southern Biotech), anti-actin, anti-Hsc70, and anti-FLAG D-8 (Santa Cruz), anti-ubiquitinated proteins FK2, anti-HC8, and anti-S1 (BIOMOL), and anti-α1 antitrypsin (MP Biomedicals).

Techniques: Transfection, Immunoprecipitation, Western Blot, Labeling, SDS Page, Autoradiography, Variant Assay

Journal: Cell reports

Article Title: Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity

doi: 10.1016/j.celrep.2022.111775

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-AAT , Proteintech , 16382-1-AP; RRID: AB_10641185.

Techniques: Recombinant, Electron Microscopy, Lysis, Lactate Assay, Pyruvate Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Sequencing, Mutagenesis, Software, Gentle, Saline, Modification

Journal: The American Journal of Tropical Medicine and Hygiene

Article Title: A Pilot Study of Enterade (VS001), an Oral Amino Acid Formulation, in Malnourished Bangladeshi Children with Environmental Enteric Dysfunction

doi: 10.4269/ajtmh.24-0402

Figure Lengend Snippet: Environmental enteric dysfunction biomarkers

Article Snippet: The biomarkers tested included lactoferrin (Eagle Biosciences Inc., Nashua, NH), myeloperoxidase (Eagle Biosciences Inc., Nashua, NH), alpha-1 antitrypsin (Eagle Biosciences Inc., Nashua, NH), and neopterin (Biomatik, Wilmington, DE).

Techniques: Biomarker Discovery, Control