Journal: Journal of Human Immunity

Article Title: A novel heterozygous pathogenic AIRE variant causing autoimmunity but not infectious susceptibility

doi: 10.70962/jhi.20250151

Figure Lengend Snippet: DN impact of AIRE C337F on WT AIRE-mediated gene transcription. HEK-293T cell lines were transfected with plasmids encoding either WT AIRE or AIRE R257X , AIRE C311Y or AIRE C337F variants alone, or equal amounts WT AIRE together with AIRE R257X , AIRE C311Y , or AIRE C337F . (A and B) Transcriptional activity was assessed by measuring expression levels of (A) known AIRE -regulated genes KRT14 , IGFL1 , S100A8 , APOA4 , and INS or (B) non- AIRE -regulated genes CCHN and PRMT3 . Data are presented as mean fold-change in expression relative to cells transfected with WT 100% used as the calibrator sample (dotted line). Error bars represent the standard error of the mean from three independent experiments.

Article Snippet: Genes previously shown to be regulated by AIRE were analyzed by quantitative PCR using the following primers and probes (Applied Biosystems): S100A8 (Hs0037444264_g1), KRT14 (Hs00265033-m1), IGFL1 (Hs01651089-g1), APOA4 (Hs00166636_m1), and INS (Hs02741908_m1) ( , ).

Techniques: Transfection, Activity Assay, Expressing

Journal: bioRxiv

Article Title: CD300LG is a receptor for triglyceride-rich lipoproteins that facilitates postprandial lipid clearance

doi: 10.1101/2025.08.08.669356

Figure Lengend Snippet: CD300LG directly binds TRLs and ApoA4. A-B) Representative images (A) and quantification (B) from high-content fluorescence microscopy of Dil-labeled chylomicrons (Dil-CMs) binding to TIME cells transfected with human CD300LG or empty vector. C) Quantification of Dil-labeled VLDL, LDL, and HDL (human) binding to human CD300LG or empty vector- transfected TIME cells by high-content imaging. D) Dose-dependent competition of DiI-CM binding to CD300LG by recombinant human CD300LG-Fc quantified by high-content imaging. E) Dil-CMs directly bind recombinant human CD300LG-Fc, but not control-Fc, immobilized on ELISA plates. Total fluorescence intensity was measured across entire wells. F) Binding of Dil-CMs following a pre-incubation of TIME cells overexpressing CD300LG with the indicated concentrations of unlabeled VLDL, LDL, or HDL. Binding was quantified by high-content imaging and normalized to cells incubated with Dil-CMs alone. G) Binding of hCD300LG-Fc or control Fc to purified apolipoproteins immobilized to ELISA plates. Error bars denote S.D. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA. All data are representative of a minimum of two independent experiments.

Article Snippet: A total of 25 pmol of each of the recombinant human apolipoproteins ApoC2 (LSBio, LS-G20488), ApoC3 (LSBio, LS-G20489), ApoA4 (Sino Biological, 16082-H08H), and ApoA5 (Prospec-Tany, Cyt-025) were attached to a 96-well polystyrene plate similar to as described for CD300LG-Fc.

Techniques: Fluorescence, Microscopy, Labeling, Binding Assay, Transfection, Plasmid Preparation, Imaging, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Incubation, Purification

Journal: bioRxiv

Article Title: CD300LG is a receptor for triglyceride-rich lipoproteins that facilitates postprandial lipid clearance

doi: 10.1101/2025.08.08.669356

Figure Lengend Snippet: The c-terminus of ApoA4 contributes to CD300LG binding to TRLs. A) Sequence alignment of the conserved EQXQ motif in the C-terminal region of ApoA4 across species. B) Helical wheel diagram of the c-terminus of ApoA4 showing the one- sided charge distribution of glutamate residues created by the repeating EQXQ motif. C) Helical wheel diagram showing the redistribution of the charged glutamate residues within the “scrambled” control peptides. D-E) Direct binding of peptides corresponding to the human (D) or mouse (E) ApoA4 C-terminus (hA4-Cterm, mA4-Cterm) or scrambled controls (hA4-Scr, mA4- Scr) to recombinant human CD300LG-Fc. F) hA4-Cterm peptide inhibits the binding between human CD300LG-Fc and Dil-CMs. CD300LG-Fc coated plates were pre-incubated with peptides for 2 hours prior to the addition of CMs. G) hA4-Cterm peptide dose-dependently inhibits the binding of Dil-CM to CD300LG expressing TIME cells. TIME cells were pre-incubated with peptides for 30 minutes on ice prior to the addition of Dil-CMs. H) Oral fat tolerance test (OFTT) for WT mice treated with saline or 40 µg of mA4-Cterm or mA4-Scr 15 minutes before an oral gavage of olive oil (N = 9-11). AUC is quantified on the right. *p<0.05, **p<0.01, ****p<0.0001 by one-way or two-way ANOVA as appropriate. All data are representative of a minimum of two independent experiments. Data points in panels D and E represent the average of 2 independent experiments.

Article Snippet: A total of 25 pmol of each of the recombinant human apolipoproteins ApoC2 (LSBio, LS-G20488), ApoC3 (LSBio, LS-G20489), ApoA4 (Sino Biological, 16082-H08H), and ApoA5 (Prospec-Tany, Cyt-025) were attached to a 96-well polystyrene plate similar to as described for CD300LG-Fc.

Techniques: Binding Assay, Sequencing, Control, Recombinant, Incubation, Expressing, Saline

Journal: Cell death & disease

Article Title: miR-181a-5p mediates the effects of BMP4 on intestinal cell proliferation and differentiation.

doi: 10.1038/s41419-025-07730-w

Figure Lengend Snippet: Fig. 1 BMP4 increased enterocyte marker expression and inhibited intestinal stemness in human duodenum and mouse SI organoids. A–D Human duodenum organoids were cultured in the presence of BMP4 (500 ng/ml) or control PBS for 4 d. Total RNA was extracted, and KRT20, APOA4, FABP1, and VILLIN mRNA expression was assessed by qPCR (A). Data are normalized to GAPDH; n = 3 biological repeats. Representative image of organoids from control PBS and BMP4 treatment groups is shown in B with quantification of the organoid area shown in (C) (98 organoids per PBS group and 105 organoids per BMP4 group were measured). qPCR analysis of OLFM4 expression (D). E–H Mouse SI organoids were cultured in the presence of BMP4 (500 ng/ml) or control PBS for 4 d. qPCR analysis of selected enterocyte markers in mouse SI organoids differentiated in the absence or presence of BMP4 (E). Data are normalized to GAPDH; n = 3 biological repeats. Representative image of organoids from control PBS and BMP4 treatment is shown in (F). Quantification of the organoid area is shown in (G) (21 organoids per PBS group and 25 organoids per BMP4 group were measured). OLFM4, Ki67, and CCND1 mRNA expression were assessed by qPCR (H). Data are normalized to GAPDH; n = 3 biological repeats. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005.

Article Snippet: Antibodies to Cyclin D1 (ab134175) and KRT20 (ab854) from Abcam (Cambridge, UK), OLFM4 (#39141), APOA4 (#5700), FABP1 (#13368), HK1 (#2024), Na-K ATPase (#3010) (all from Cell Signaling, Danvers, MA) and HK2 (GeneTex, Irvine, CA; GTX124375), VILLIN (sc-7672, Santa Cruz Biotechnology, Dallas, TX), and β-actin (A1978, Sigma) were used, and following blotting with a horseradish peroxidase-conjugated secondary antibody, protein expression was visualized using an enhanced chemiluminescence (ECL) detection system.

Techniques: Marker, Expressing, Cell Culture, Control

Journal: Cell death & disease

Article Title: miR-181a-5p mediates the effects of BMP4 on intestinal cell proliferation and differentiation.

doi: 10.1038/s41419-025-07730-w

Figure Lengend Snippet: Fig. 3 Inhibition of miR-181a-5p increased enterocyte marker expression in mouse SI organoids. Mouse SI organoids were cultured in the presence of LNA miR-181a-5p inhibitor or control oligos (0.75 μM) for 5 d. A qPCR analysis of selected enterocyte markers; n = 3 biological repeats. B Mouse organoid lysates were extracted, and IAP activity determined; n = 3 biological repeats. C Treatment with LNA miR-181a-5p inhibitor increased enterocyte marker protein expression. The images are representative of three independent experiments. VILLIN, APOA4, and FABP1 signals from three separate experiments were quantitated by densitometer and expressed as fold change with respect to β-actin. D LNA miR-181a-5p inhibitor decreased endogenous miR-181a-5p expression; n = 3 biological repeats. E–G Microscopy analysis of enterocytes in mouse SI organoids. The relative fluorescence intensities of KRT20 (control oligos n = 14 organoids; miR-181a-5p inhibitor n = 11 organoids), Na,K-ATPase (control oligos n = 14 organoids; miR-181a-5p inhibitor n = 12 organoids), and VILLIN (control oligos n = 10 organoids; miR-181a-5p inhibitor n = 10 organoids) staining were quantified using ImageJ. H–J C57BL/6 mice were injected IP with control oligos (n = 5 mice) or miR-181a-5p inhibitor (n = 5 mice). Seven d after injection, mouse SI were harvested and analyzed. Western blot analysis (H). Image signals from five mice were quantitated by densitometer and expressed as fold change with respect to β-actin. Representative Fast Red staining of the small intestine revealed an increase in IAP activity (J). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005.

Article Snippet: Antibodies to Cyclin D1 (ab134175) and KRT20 (ab854) from Abcam (Cambridge, UK), OLFM4 (#39141), APOA4 (#5700), FABP1 (#13368), HK1 (#2024), Na-K ATPase (#3010) (all from Cell Signaling, Danvers, MA) and HK2 (GeneTex, Irvine, CA; GTX124375), VILLIN (sc-7672, Santa Cruz Biotechnology, Dallas, TX), and β-actin (A1978, Sigma) were used, and following blotting with a horseradish peroxidase-conjugated secondary antibody, protein expression was visualized using an enhanced chemiluminescence (ECL) detection system.

Techniques: Inhibition, Marker, Expressing, Cell Culture, Control, Activity Assay, Microscopy, Staining, Injection, Western Blot

Journal: Cell death & disease

Article Title: miR-181a-5p mediates the effects of BMP4 on intestinal cell proliferation and differentiation.

doi: 10.1038/s41419-025-07730-w

Figure Lengend Snippet: Fig. 5 Inhibition of miR-181a-5p increased enterocyte marker expression in human duodenum organoids. Human duodenum organoids were cultured in the presence of LNA miR-181a-5p inhibitor or control oligos (1 μM) for 7 d. A qPCR analysis of enterocyte markers IAP, APOA4, FABP1, VILLIN, and Na,K-ATPase expression; n = 3 biological repeats. B Treatment with LNA miR-181a-5p inhibitor decreased endogenous miR- 181a-5p expression; n = 3 biological repeats. C–E IF staining of organoids for KRT20 (C), Na,K-ATPase (D), and VILLIN (E). The relative fluorescence intensity of KRT20 (control oligos n = 10 organoids; miR-181a-5p inhibitor n = 10 organoids), Na,K-ATPase (control oligos n = 14 organoids; miR-181a-5p inhibitor n = 11 organoids), and VILLIN (control oligos n = 17 organoids; miR-181a-5p inhibitor n = 9 organoids) staining was quantified using ImageJ fluorescence analyzer. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005.

Article Snippet: Antibodies to Cyclin D1 (ab134175) and KRT20 (ab854) from Abcam (Cambridge, UK), OLFM4 (#39141), APOA4 (#5700), FABP1 (#13368), HK1 (#2024), Na-K ATPase (#3010) (all from Cell Signaling, Danvers, MA) and HK2 (GeneTex, Irvine, CA; GTX124375), VILLIN (sc-7672, Santa Cruz Biotechnology, Dallas, TX), and β-actin (A1978, Sigma) were used, and following blotting with a horseradish peroxidase-conjugated secondary antibody, protein expression was visualized using an enhanced chemiluminescence (ECL) detection system.

Techniques: Inhibition, Marker, Expressing, Cell Culture, Control, Staining

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) Volcano plot of differentially expressed genes from RNA sequencing, showing upregulation of PCSK9 and downregulation of APOA4 in the LD+TMAO group compared to the LD group. (B) qRT-qPCR analysis of APOA4 mRNA expression levels across groups (n = 3 per group). (C) qRT-qPCR analysis of PCSK9 mRNA levels across groups (n = 3 per group). (D) Western blot analysis of APOA4 and PCSK9 protein levels across groups (n = 3 per group). (E–F). Quantification results of the Western blot analysis using ImageJ. (G, H). Serum APOA4 and PCSK9 levels in patients with cholelithiasis and the control group detected by ELISA. ** p < 0.01, *** p < 0.001. LD, lithogenic diet; TMAO, trimethylamine-N-oxide; NC, negative control; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; GSD, gallstone disease; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) Lithogenesis in mice from the LD and LD+DMB groups. (B) Weights of solid contents in the gallbladders of mice from each group. (C) Serum TMAO levels in the LD and LD+DMB groups detected by ELISA. (D) Immunofluorescence staining of APOA4 and PCSK9 in hepatic tissues from the LD and LD+DMB groups. (E, F) qRT-qPCR analysis of APOA4 and PCSK9 mRNA expression levels in hepatic tissues from the LD and LD+DMB groups. * p < 0.05, ** p < 0.01, **** p < 0.0001. LD, lithogenic diet; DMB, 3,3-dimethyl-1-butanol; TMAO, trimethylamine-N-oxide; ELISA, enzyme-linked immunosorbent assay; APOA4, apolipoprotein A4; PCSK9 proprotein convertase subtilisin/kexin type 9.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A–E) APOA4 , PCSK9 , HMGCR, ABCG5 , and ABCG8 mRNA expression levels in TMAO-treated and control AML12 cells (n = 3 per group). (F) Immunofluorescence staining for APOA4, PCSK9, HMGCR, ABCG5, and ABCG8 in cells from each group. ** p < 0.01, *** p < 0.001. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Expressing, Control, Immunofluorescence, Staining, Binding Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) WB analysis of PCSK9, HMGCR, ABCG5, and ABCG8 in NC, TMAO-treated, and APOA4-overexpressed cells. (B–E) Changes in PCSK9 , HMGCR , ABCG5 , and ABCG8 mRNA levels after APOA4 overexpression (n = 3 per group). (F) Immunofluorescence staining for intracellular PCSK9, HMGCR, ABCG5, and ABCG8 after APOA4 overexpression. (G) WB analysis of APOA4, HMGCR, ABCG5, and ABCG8 in NC, TMAO-treated, and PCSK9-knockdown cells. (H–K) Changes in mRNA levels of PCSK9 , HMGCR , ABCG5 , and ABCG8 after PCSK9 knockdown (n = 3 per group). (L) Immunofluorescence staining for PCSK9, HMGCR, ABCG5, and ABCG8 in cells after PCSK9 knockdown. * p < 0.05, ** p < 0.01. +, positive expression; -, negative expression; TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Over Expression, Immunofluorescence, Staining, Knockdown, Expressing, Binding Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: Gut microbiota produce trimethylamine, which enters the liver via the portal circulation and is primarily oxidized by FMO3 to produce TMAO. TMAO upregulates hepatic PCSK9 gene expression while downregulating APOA4 expression. PCSK9 overexpression inhibits APOA4 expression, while low APOA4 expression further promotes PCSK9 expression, forming a feedback loop that dysregulates cholesterol metabolism. This upregulates cholesterol synthesis by HMGCR and cholesterol efflux by ABCG5/8. Consequently, biliary concentrations of cholesterol and bile acids increase and decrease, respectively, thereby promoting cholesterol gallstone formation. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; FMO3, flavin containing monooxygenase 3; TMA, Trimethylamine.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Gene Expression, Expressing, Over Expression, Binding Assay