Journal: The Journal of General Virology

Article Title: Hepatitis B virus is a stealth virus that minimizes proteomic and secretomic changes in primary human hepatocytes

doi: 10.1099/jgv.0.002170

Figure Lengend Snippet: Quantification of APOB in culture supernatants of HBV-infected PHH. PHHs were infected with HBV at an MOI of 1,000 VGE/cell, and culture supernatants were collected after the final 3 days of incubation (8 dpi). APOB levels were measured by ELISA. Data represent mean± sd from a single experiment; individual points represent four replicates.

Article Snippet: Secreted apolipoprotein B (APOB) levels in culture supernatants were measured using an ELISA kit (#KE00158-96T, Proteintech).

Techniques: Infection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) ELISA of culture medium for Apolipoprotein B (APOB), taken from iHeps 2 days after the end of the hepatic differentiation protocol, and from PHH culture media 2 days after plating. (B) Brightfield microscopy of 3D tubular branching strictures observed when Laminin 411 concentrations are increased. Scale bar = 100μm. (C) Alkaline Phsophatase (ALP) activity measured by colourimetric assay on culture medium taken 2 days the end of the hepatic differentiation protocol.

Article Snippet: APOB secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, # 3715-1H-6) according to product literature.

Techniques: Enzyme-linked Immunosorbent Assay, Microscopy, Activity Assay

Journal: Journal of Extracellular Biology

Article Title: Scalable purification of extracellular vesicles with high yield and purity using multimodal flowthrough chromatography

doi: 10.1002/jex2.138

Figure Lengend Snippet: MFC exhibits a higher recovery rate of EVs from human serum than SEC while maintaining a similar level of purity to SEC. (a) Protein yields as determined by a microBCA protein assay. (b) Particle yields determined by NTA. (c) Particle/protein ratios of EVs. (d) Normalised mean particle size distributions determined with NTA. (e) TEM images of serum isolates from SEC. (f) TEM images of serum isolates from MFC. Red arrows indicate small particles of 60 nm or less. The larger blue arrows indicate typical EV shaped particles. (g) Silver stain analysis of crude serum, serum EVs isolated by SEC, and serum EVs isolated by MFC. Equal protein amounts (2 µg) were loaded. (h) Western blotting of EV‐associated proteins alix, TSG101, CD81, flotillin and syntenin in both SEC and MFC isolated samples loaded to equal volume. (i) Apolipoprotein B detected with ELISA. (j) ApoB/particle ratio. Results in A, B, C, D, I and J represent biological replicates ( n = 6). Significance levels are indicated with asterisks (* p < 0.05, *** p < 0.001).

Article Snippet: ApoB concentration of samples was quantified using an Apolipoprotein B quantikine ELISA (DAPB00, R&D Systems) following the manufacturer's protocol.

Techniques: Silver Staining, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Lipid Research

Article Title: ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism

doi: 10.1016/j.jlr.2024.100500

Figure Lengend Snippet: ANGPTL3 deficiency decreases ApoB100 secretion and promotes chaperone-mediated degradation of ApoB100 in HepG2 cells. A: Cells were pulsed (150 μCi/ml) for 20 min in media supplemented with 200 μM oleic acid and chased with cold media replete with cysteine and methionine (also containing 200 μM oleic acid). ApoB100 was pulled down from media collected 3 h after the initial 20 min pulse period. Samples were run on a 3%–7% gel, fixed, treated with E3HANCE, dried, and exposed to radiographic film. n = 3 wells per genotype. B: Radiolabeled ApoB100 immunoprecipitated from media collected after 3 h. Counts were normalized to total TCA precipitable protein. n = 3 wells per genotype. P -value as indicated by unpaired Student’s t test. C: Radiolabeled ApoB100 immunoprecipitated from media collected at the indicated time points was counted and normalized to total TCA precipitable protein. n = 6 wells per group per genotype. P -value as indicated by ordinary two-way ANOVA with Sidak’s correction for multiple comparisons. D: Radiolabeled ApoB100 immunoprecipitated from lysates collected at the indicated time points was counted and normalized to total TCA precipitable protein. n = 6 wells per group per genotype. E: Radiolabeled ApoB was immunoprecipitated from media and lysates collected 3 h after the initial 20 min pulse period. ApoB distribution at 3 h was calculated as a percentage based on ApoB100 present after the initial 20 min pulse period. Counts were normalized to total TCA precipitable protein. n = 6 wells per genotype. P -values as determined by two-way ANOVA with Sidak correction performed on full dataset presented in Fig. 6 . F: ApoB100 immunoprecipitated from unlabeled lysates and blotted for Grp78. G: Quantification of fold-change of Grp78 normalized to ApoB100. n = 5 samples per group. P -value as indicated by unpaired Student’s t test. ANGPTL3, angiopoietin-like protein 3; ApoB100, apolipoprotein B100; TCA, trichloroacetic acid.

Article Snippet: The ApoB content in unconcentrated fractions was measured by human ApoB ELISA kit (Cat# 3715-1HP-2, Mabtech) following the manufacturer’s instructions.

Techniques: Immunoprecipitation

Journal: Journal of Lipid Research

Article Title: ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism

doi: 10.1016/j.jlr.2024.100500

Figure Lengend Snippet: ApoB100 degradation is enhanced in a post-Golgi compartment in ANGPTL3-deficient HepG2 cells lacking LDLR. A: Validation of LDLR knockout in CRISPR-edited HepG2 cells. DKO = ANGPTL3 −/− ;LDLR −/− HepG2 cells. B: Cells were pulsed (150 μCi/ml) for 20 min in media supplemented with 600 μM oleic acid. Cells were then chased with cold media replete with cysteine and methionine (also containing oleic acid). ApoB100 was immunoprecipitated from media and lysates collected 3 h after the initial 20 min pulse period. ApoB distribution was calculated as a percentage based on ApoB100 present at t = 1:20. Counts were normalized to total TCA precipitable protein. n = 6 wells per genotype. P -values as indicated by two-way ANOVA with Sidak correction. C: Cells were pulsed (150 μCi/ml) for 20 min in media supplemented with 600 μM oleic acid and 25 μM MG-132. Cells were then chased with cold media replete with cysteine and methionine (also containing oleic acid and 25 μM MG-132). D: ApoB100 was immunoprecipitated from media and lysates collected 3 h after the initial 20 min pulse period. ApoB distribution was calculated as a percentage based on ApoB100 present at t = 1:20. Counts were normalized to total TCA precipitable protein. n = 6 wells per genotype. P -values as indicated by two-way ANOVA with Sidak correction. E: Cells were pulsed (150 μCi/ml) for 20 min in media supplemented with 600 μM oleic acid and 10 μM brefeldin A. Cells were then chased with cold media replete with cysteine and methionine (also containing 600 μM oleic acid and 10 μM brefeldin A). F: ApoB100 was immunoprecipitated from media and lysates collected 6 h after the beginning of the experiment. ApoB distribution was calculated as a percentage based on ApoB100 present at t = 3:20. n = 4 wells per group per genotype. ANGPTL3, angiopoietin-like protein 3; ApoB100, apolipoprotein B100; LDLR, low-density lipoprotein receptor; TCA, trichloroacetic acid.

Article Snippet: The ApoB content in unconcentrated fractions was measured by human ApoB ELISA kit (Cat# 3715-1HP-2, Mabtech) following the manufacturer’s instructions.

Techniques: Biomarker Discovery, Knock-Out, CRISPR, Immunoprecipitation

Journal: Journal of Lipid Research

Article Title: ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism

doi: 10.1016/j.jlr.2024.100500

Figure Lengend Snippet: ApoB100 degradation is enhanced in a pre-Golgi compartment in ANGPTL3-deficient HepG2 cells. A: Cells were treated with 600 μM oleic acid, homogenized, and fractionated by ultracentrifugation across a continuous iodixanol gradient. Proteins in concentrated fractions were separated on a 4%–20% SDS-PAGE gradient gel. B: Cells were pulsed (150 μCi/ml) for 20 min in media supplemented with 600 μM oleic acid and 10 μM brefeldin A. Cells were then chased with cold media replete with cysteine and methionine (also containing 600 μM oleic acid and 10 μM brefeldin A). ApoB100 was immunoprecipitated from media and lysates collected 6 h after the beginning of the experiment and after a washout period ending 12 h after the beginning of the experiment. C: ApoB distribution at t = 6 was calculated as a percentage based on ApoB100 present at t = 3:20. n = 4 wells per group per genotype. P -value as determined by two-way ANOVA with Sidak correction performed on full dataset presented in Fig. 6 . D: ApoB distribution at t = 12 was calculated as a percentage based on ApoB100 present at t = 3:20. n = 4 wells per group per genotype. P -value as indicated by unpaired Student’s t test. E: Cells were pulsed (150 μCi/ml) for 20 min in media supplemented with 600 μM oleic acid and 25 μM MG-132 where indicated. Cells were then chased with cold media replete with cysteine and methionine (also containing 600 μM oleic acid and 25 μM MG-132). F: ApoB100 was immunoprecipitated from lysates collected 3 h after the beginning of the experiment. ApoB distribution was calculated as a percentage based on ApoB100 present at t = 1:20. Counts were normalized to total TCA precipitable protein. n = 6 wells per genotype. P -values as determined by two-way ANOVA with Sidak correction performed on full dataset presented in Fig. 6 . G: Cells were pulsed (150 μCi/ml) for 20 min in media supplemented with 600 μM oleic acid and 100 nM bafilomycin A1 in the presence and absence of 25 μM MG-132. Cells were then chased with cold media replete with cysteine and methionine (also containing 600 μM oleic acid and 100 nM bafilomycin A1 ± MG-132). ApoB100 was immunoprecipitated from media and lysates collected 6 h after the beginning of the experiment and after a washout period ending 12 h after the beginning of the experiment. H: ApoB distribution at t = 3 was calculated as a percentage based on ApoB100 present at t = 1:20. n = 5 wells per group per genotype. P -value as indicated by unpaired Student’s t test. I: ApoB distribution at t = 3 was calculated as a percentage based on ApoB100 present at t = 1:20. n = 5 wells per group per genotype. J: ApoB distribution at t = 3 was calculated as a percentage based on ApoB100 present at t = 1:20. n = 3 wells per group per genotype. ANGPTL3, angiopoietin-like protein 3; ApoB100, apolipoprotein B100.

Article Snippet: The ApoB content in unconcentrated fractions was measured by human ApoB ELISA kit (Cat# 3715-1HP-2, Mabtech) following the manufacturer’s instructions.

Techniques: SDS Page, Immunoprecipitation

Journal: JCI insight

Article Title: Localized T3 production modifies the transcriptome and promotes the hepatocyte-like lineage in iPSC-derived hepatic organoids.

doi: 10.1172/jci.insight.173780

Figure Lengend Snippet: Figure 3. Formation of hepatic organoids in the presence or absence of TH. (A) Graphic representation of the development of hepatic organoids in the presence or absence of TH. The yellow arrow indicates the addition of TH (from day 5 to day 50). The final concentration of T4-HOs (red) and T3-HOs (blue) was 1 nM T4 (free T4 = ~15 pM) and 200 pM T3 (free T3 = ~10 pM); V-HOs were grown in the absence of T4 or T3 (black). C1–C6 are the indicated differentiation cocktails used (see methods). (B) Bright-field images (3 conditions) of hepatoblast at day 22 and hepatic organoids at day 29. Scale bar: 200 μm. (C) Relative mRNA levels of HNF4A (hepatocyte marker) and CEBPA from day 26 to day 32 (n = 4). (D) Albumin levels in the medium from day 35 to day 50 (n = 4). (E) Relative mRNA levels of KRT7 at day 46 and day 50 (n = 4, except T4-HOs, n = 3). (F) ALB/KRT7 mRNA ratio during from day 38 to day 46 (n = 4 except T4-HOs and T3-HOs at day 42, n = 2; T4-HOs at day 46, n = 3). (G and H) Apolipoprotein B (APOB) and Apolipoprotein A1 (APOA1) levels from the medium from day 35 to day 50, comparing V-HOs (black) versus T4-HOs (red). Ten organoids per well (n = 4). Two-tailed Student’s t test for comparing V-HOs versus T4-HOs, and 1-way ANOVA and Tukey test were used for multiple comparisons. Data are the mean of duplicates, represented as aligned scatter dot plots and their mean. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The days of the differentiation are shown on the x axis (see legend Figure 1).

Article Snippet: Albumin, apolipoprotein B, and apolipoprotein A1 concentration in the medium was measured using human albumin ELISA kit (Bethyl Laboratories), Human Apolipoprotein B/ApoB Quantikine ELISA Kit (R&D systems), and Human Apolipoprotein A/ApoA1 Quantikine ELISA Kit (R&D systems), respectively.

Techniques: Concentration Assay, Marker, Two Tailed Test