Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , General morphology of livers 2 days after PHx. b , H&E staining of liver sections 2 days after PHx. c , d , Ki67 + percentages in HNF4α + hepatocytes ( c ) and HNF4α − NPCs ( d ) 2 days after PHx. Means ± SEM are shown, n=3 per group, **P < 0.01 (two-tailed unpaired t test). n.s., not significant. e , EpCAM was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). Stellate cells (GFAP) were not altered around the colony. f , CK19 was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). CD133 was highly expressed in the colony. g , Sox9 and CD44 were highly expressed in biliary epithelial cells (arrowhead), but was not upregulated in the colony (dashed line) compared to surrounding hepatocytes. h , AFP showed no difference between the colony (dashed line) and the surrounding tissue. i , Liver/body weight ratios after PHx. Means ± SEM from 3 or more mice analyzed for each time point and group are shown. j , k , Immunofluorescent staining of SKO liver sections 3 weeks after PHx. Colonies were at various sizes and locations as shown by arrows in j . Macroscopic colonies were found as shown by arrows and dashed lines in k . Immunofluorescent image in k corresponds to the magenta arrow in the liver image. PV, portal vein; CV, central vein. l , Vasculature shown by PECAM did not distinguish the colony (arrow). m , HGF expressed by NPCs was not concentrated in the colony (dashed line). Scale bars, 1 cm ( a ) 100 μm ( b , e - g , i - l ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Staining, Two Tailed Test

Journal: EBioMedicine

Article Title: Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice.

doi: 10.1016/j.ebiom.2024.105178

Figure Lengend Snippet: Fig. 1: Intravenously injected iRGD increases blood AFP levels in HCC-bearing mice. (a) Experimental setup to study the effect of iRGD on blood AFP levels in Huh-7 xenografted nude mice. (b and c) Blood AFP levels before and after intravenous injection of iRGD (b, c, n = 12), RGD control peptide (c (n = 6)), and PBS (c (n = 6)) in mice with HepG2 xenografts. Human AFP was not detectable in the blood of mice without tumours. In (c) the fold increase of AFP with pre-injection level set to 1; lines and error bars represent geometric means and 95% CI. (d) Experimental setup to study the effect of iRGD on blood AFP levels in TGFα/c-myc HCC mice. (e and f) iRGD specifically increased the blood AFP levels in TGFα/c-myc HCC mice. TGFα/c-myc mice (20–24 weeks old) with HCC according to MRI or without HCC (f) were intravenously injected with iRGD (e, f), RGD control peptide (f) or PBS (f). Data are fold changes of blood AFP due to the treatments (n = 48: iRGD; RGD control peptide: n = 34; PBS: n = 15); lines and error bars indicate medians and 95% CI. Dashed line: upper 95% CI increase in blood AFP in the PBS-injected HCC mice. (g) AFP expression in HCCs of TGFα/c-myc mice. HCCs and liver tissues were excised from TGFα/c-myc mice. Pairs of the tissue lysates were analysed for AFP and β-actin content by immunoblotting. Band densities were measured densitometrically. The ratio of AFP/β-actin in the livers was set to 1. (h) iRGD-induced accumulation of Evans blue in HCCs in TGFα/c-myc mice with HCC. TGFα/c-myc mice with HCCs were co- injected with iRGD or PBS (n = 12 per group) and Evans blue (EB). The dye content of the tumours was related to that in the livers; lines and error bars indicate geometric means (b, c, and h) or medians (e and f) and 95% CI; dashed line: upper 95% CI of the measured EB content of a HCC from the PBS-injected animals. (b): paired t test for log-transformed data; (c) One-way ANOVA with multiple comparison post-hoc test for log-transformed data; (e): Wilcoxon signed-rank test for log-transformed data; (f) Kruskal–Wallis test with Dunn’s multiple comparison post- hoc test; (h): two-sample t test. The indicated fold increase in (b, e and h) is the ratio of the geometric means with 95% CI.

Article Snippet: Immunoblotting of lysates obtained from pairs of liver and HCC tissue from TGFα/c-myc mice was performed as described previously.38 Gel-resolved proteins were electrotransferred to nitrocellulose membranes and incubated with antibodies raised against mouse-AFP (#AF5369, R&D Systems, Minneapolis, MN, RRID:AB_2258018) and anti-β-actin (#A2066, Sigma– Aldrich/Merck, RRID:AB_476693).

Techniques: Injection, Control, Expressing, Western Blot, Transformation Assay, Comparison

Journal: EBioMedicine

Article Title: Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice.

doi: 10.1016/j.ebiom.2024.105178

Figure Lengend Snippet: Fig. 3: iRGD-induced elevation of the blood AFP concentration depends on NRP-1 and the tumour blood concentration gradient of AFP. (a) Anti-NRP-1 prevented iRGD-induced increase in the blood AFP concentration. TGFα/c-myc tumour mice that displayed a robust iRGD-induced increase in blood AFP level one week earlier were injected with anti-NRP-1 and the effect of iRGD on blood AFP level was determined (n = 3 per group). Fold increase of AFP with pre-injection level at the first time point was set to 1. Lines and error bars represent medians and 95% CI. (b– e) iRGD-induced elevation of the blood AFP level correlated negatively with the pre-injection blood AFP level in TGFα/c-myc mice (b), DEN- CCl4-HCC mice (c), mice with Huh-7 (d) or HepG2 xenografts (e). Spearman correlation r (b and c) and Pearson correlation r (d and e) with 95% CI, two tailed p-values and the log–log regression lines. (f) iRGD increased the blood AFP levels in HepG2 xenografted nude mice and low basal AFP (<67 ng/ml, n = 36, left panel), but not in animals with high basal AFP (>67 ng/ml, n = 12, right panel). Lines and error bars indicate geometric means and 95% CI. [(g) iRGD increased the blood AFP levels in TGFα/c-myc mice with HCC and normal basal AFP (<67 ng/ml, n = 36, left panel), but not in mice with elevated basal AFP (>67 ng, n = 12, right panel). Lines and error bars represent medians (left) or geometric means (right) with 95% CI. Significance was calculated with one sample t test (a, left) and the unpaired t test (a, right), paired t test (f and g, right) and Wilcoxon matched-pairs signed-rank test (g, left). The indicated fold increase in (f) is the geometric mean ratio with 95% CI. The indicated fold increase in (g) is the median of the ratios with 95% CI.

Article Snippet: Immunoblotting of lysates obtained from pairs of liver and HCC tissue from TGFα/c-myc mice was performed as described previously.38 Gel-resolved proteins were electrotransferred to nitrocellulose membranes and incubated with antibodies raised against mouse-AFP (#AF5369, R&D Systems, Minneapolis, MN, RRID:AB_2258018) and anti-β-actin (#A2066, Sigma– Aldrich/Merck, RRID:AB_476693).

Techniques: Concentration Assay, Injection, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Isolation and characterisation of AFP + /DLK1 + double-positive hepatic stem/progenitor-like cells from fibrotic partial hepatectomy model

doi: 10.1186/s13287-025-04728-1

Figure Lengend Snippet: Schematic presentation of the strategy for isolating AFP + /DLK1 + double-positive cells from the fibrotic PHx model

Article Snippet: Cells were then blocked with 2.5% BSA for 1 h. The cells were subsequently incubated with primary antibodies against AFP (1:200; Proteintech-cn, Wuhan, 14550-1-AP) and DLK1 (1:200; MBL, Beijing, D187-3) at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at room temperature in the dark (1:1000, Abcam, USA).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Isolation and characterisation of AFP + /DLK1 + double-positive hepatic stem/progenitor-like cells from fibrotic partial hepatectomy model

doi: 10.1186/s13287-025-04728-1

Figure Lengend Snippet: Pre-injection of CCl 4 promotes liver regeneration after 2/3 PHx. a Schematic drawing of mouse liver anatomy and positioning of silk threads for knots. b Liver /Body Weight Ratio (%) after PHx. n = 2 per group. c Level of serum ALT and AST after PHx. n = 3 per group. d Relative mRNA expression levels of Afp , Dlk1 , Lgr5 , Ki67 , HNF4α , Alb , Krt-8 , Krt-18 , Krt7 ,and Krt-19 in regenerating livers after PHx. Values are normalised to Actb. n = 3 per group. e AFP (green), DLK1 (red), and DAPI (blue) immunofluorescence analyses in regenerating livers after PHx on the fibrotic model. Scale bars: 100 μm. f Relative fluorescence intensity (%). Statistical analysis was undertaken with Two-way ANOVA; (* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001.)

Article Snippet: Cells were then blocked with 2.5% BSA for 1 h. The cells were subsequently incubated with primary antibodies against AFP (1:200; Proteintech-cn, Wuhan, 14550-1-AP) and DLK1 (1:200; MBL, Beijing, D187-3) at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at room temperature in the dark (1:1000, Abcam, USA).

Techniques: Injection, Expressing, Immunofluorescence, Fluorescence

Journal: Stem Cell Research & Therapy

Article Title: Isolation and characterisation of AFP + /DLK1 + double-positive hepatic stem/progenitor-like cells from fibrotic partial hepatectomy model

doi: 10.1186/s13287-025-04728-1

Figure Lengend Snippet: Isolation of AFP + /DLK1 + Double-Positive Cells from regenerating liver by density gradient differential centrifugation. a Program diagram of density gradient differential centrifugation. Whole liver cells obtained by enzymatic digestion (as shown in ①). By lowspeed centrifugation (50× g , 5 min), liver parenchymal cells are deposited at the bottom of the centrifuge tube, and liver nonparenchymal cells are suspended in the supernatant (as shown in ②). The supernatant is collected and centrifuged at medium speed (200× g , 5 min) to obtain nonparenchymal cell masses (as shown in ③). The nonparenchymal cells are placed in the top layer of the gradient centrifuge solution (as shown in ④). By differential centrifugation (600× g , 10 min, sped up by 9, and sped down by 1), all kinds of cells are separated into different Percoll layers (as shown in ⑤). Cells are collected from 50%–70% Percoll layer and HSPClike cells obtained by centrifugation (250× g , 5 min) (as shown in ⑥). b Flow cytometry analysis of AFP + /DLK1 + double-positive cells in each Percoll layer. c Percentage of AFP + /DLK1 + double-positive cells (by flow cytometry). d Relative mRNA expression levels of Afp , Dlk1 , Lgr5 , and Ki67 in each Percoll layer cells. Values are normalised to Actb. n = 3 per group. e AFP (green), DLK1 (red), and DAPI (blue) immunofluorescence analyses in the 50%– 70% layer. Scale bars:20 μm. f Relative fluorescence intensity (%). Statistical analysis was undertaken with Two-way ANOVA; (* p < 0.05; **** p < 0.0001.)

Article Snippet: Cells were then blocked with 2.5% BSA for 1 h. The cells were subsequently incubated with primary antibodies against AFP (1:200; Proteintech-cn, Wuhan, 14550-1-AP) and DLK1 (1:200; MBL, Beijing, D187-3) at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at room temperature in the dark (1:1000, Abcam, USA).

Techniques: Isolation, Centrifugation, Gradient Centrifugation, Flow Cytometry, Expressing, Immunofluorescence, Fluorescence

Journal: Frontiers in Neuroscience

Article Title: Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

doi: 10.3389/fnins.2023.1107436

Figure Lengend Snippet: Colocalization of Aβ protein along with astrocytes or microglia/macrophage markers in the wholemount neuroretinas. (A) Negative control omitted for primary antibodies; double immunolabeling of (B–D) 6E10 and GFAP markers, and (E–G) 6E10 and IBA1 markers across the three animal groups are illustrated. 6E10(+)ve neuritic type Aβ plaques (white arrowheads) or large deposits (yellow arrowheads), GFAP+(ve) phagocytic astrocytes/PLS (red box), IBA1+(ve) microglia (blue arrowheads) or blood-derived macrophages (yellow box) are indicted. (H) Aβ plaque counts were compared across the three animal groups and illustrated using scatter plots. (I–K) 6E10, GFAP, and IBA1 immunoreactivities, respectively were compared across the three animal groups and illustrated using scatter plots. Significance levels of p < 0.05 are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar, 20 μm; bright red, 6E10; bright green, IBA1; blue, DAPI. WT, C57BL/6J wildtype; SEM, standard error of mean.

Article Snippet: In addition to 6E10, GFAP, and IBA1, primary antibodies OCTN1 (rabbit polyclonal, Cat# ACT-014, Alomone Labs), a marker for Ergo transporter and AQP4 (rabbit polyclonal, Cat# AQP-004, Alomone Labs), a major membrane water channel in the CNS were used in the eye cross-sections.

Techniques: Negative Control, Immunolabeling, Derivative Assay

Journal: Frontiers in Neuroscience

Article Title: Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

doi: 10.3389/fnins.2023.1107436

Figure Lengend Snippet: Expression of 6E10, IBA1, and GFAP protein markers in the wholemount neuroretinas.

Article Snippet: In addition to 6E10, GFAP, and IBA1, primary antibodies OCTN1 (rabbit polyclonal, Cat# ACT-014, Alomone Labs), a marker for Ergo transporter and AQP4 (rabbit polyclonal, Cat# AQP-004, Alomone Labs), a major membrane water channel in the CNS were used in the eye cross-sections.

Techniques: Expressing

Journal: Frontiers in Neuroscience

Article Title: Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

doi: 10.3389/fnins.2023.1107436

Figure Lengend Snippet: Evidence for Aβ clearance by phagosome like structures on the surface of wholemount neuroretinas. (A–D) Negative control omitted for primary antibodies to distinguish debris or autofluorescence bodies (yellow arrowheads), (E–H) IBA1+(ve) phagocytic blood-derived macrophages or PLS (white arrowheads), and (I–L) GFAP+(ve) phagocytic astrocytes or PLS (white arrowheads) are illustrated on the surface of wholemount neuroretinas. (C) White circles in negative control show poor uptake of DAPI which distinguish the PLS from debris or autofluorescence based on DAPI(+)ve clumps of nuclei ( G,K , indicated by yellow and red boxes). Mean counts of GFAP(+)ve (M) and IBA1(+)ve (N) PLSs as indicated by red and yellow boxes, respectively were compared across the three animal groups and illustrated using scatter plots. Significance levels of p < 0.05 are indicated as * p < 0.05 and ** p < 0.01. Scale bar, 20 μm; bright red, 6E10; bright green, IBA1/GFAP; blue, DAPI. SEM, standard error of mean; PLS, phagosome like structure.

Article Snippet: In addition to 6E10, GFAP, and IBA1, primary antibodies OCTN1 (rabbit polyclonal, Cat# ACT-014, Alomone Labs), a marker for Ergo transporter and AQP4 (rabbit polyclonal, Cat# AQP-004, Alomone Labs), a major membrane water channel in the CNS were used in the eye cross-sections.

Techniques: Negative Control, Derivative Assay

Journal: Frontiers in Neuroscience

Article Title: Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

doi: 10.3389/fnins.2023.1107436

Figure Lengend Snippet: Evidence for Aβ clearance through glymphatic and perivascular drainage systems on the surface of wholemount neuroretinas. 6E10 and GFAP colocalization revealed the existence of glymphatic drainages on the surface of wholemount neuroretinas. (A–D) Glymphatic drainage mediated by tightly attached astrocytic end-feet (white arrowheads) toward optic nerve (white asterisk), and the (E,F) 3D projections demonstrated its existence at surface level. (G–J) Aβ influx into the perivascular drainage by terminal end of the capillary bed (yellow arrowheads) on the surface of wholemount neuroretinas. (K) 3D projections demonstrated the existence of capillary bed involved in Aβ clearance at surface level. Scale bars, 50 μm and 20 μm; bright red, 6E10; bright green, GFAP; blue, DAPI. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer.

Article Snippet: In addition to 6E10, GFAP, and IBA1, primary antibodies OCTN1 (rabbit polyclonal, Cat# ACT-014, Alomone Labs), a marker for Ergo transporter and AQP4 (rabbit polyclonal, Cat# AQP-004, Alomone Labs), a major membrane water channel in the CNS were used in the eye cross-sections.

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

doi: 10.3389/fnins.2023.1107436

Figure Lengend Snippet: Expression of IBA1 and GFAP protein markers in the retinal cross-sections.

Article Snippet: In addition to 6E10, GFAP, and IBA1, primary antibodies OCTN1 (rabbit polyclonal, Cat# ACT-014, Alomone Labs), a marker for Ergo transporter and AQP4 (rabbit polyclonal, Cat# AQP-004, Alomone Labs), a major membrane water channel in the CNS were used in the eye cross-sections.

Techniques: Expressing

Journal: Frontiers in Neuroscience

Article Title: Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

doi: 10.3389/fnins.2023.1107436

Figure Lengend Snippet: Colocalization of 6E10 and GFAP protein markers in the retinal cross-sections. Expression pattern of 6E10 and GFAP markers in the central and peripheral regions of (A,D) WT (C57BL/6J) controls, (B,E) non-treated 5XFAD, and (C,F) Ergo-treated 5XFAD are illustrated. White arrowheads indicate intraneuronal accumulation of 6E10(+)ve Aβ proteins, and yellow arrowheads indicate GFAP(+)ve astrocytes, predominantly localized in NFL. GFAP immunoreactivity in the (G) central, and (H) peripheral retinas were compared across the three animal groups and illustrated using scatter plots. Significance levels of p < 0.05 are indicated as ** p < 0.01 and *** p < 0.001. Scale bar, 20 μm; bright red, 6E10; bright green, GFAP. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

Article Snippet: In addition to 6E10, GFAP, and IBA1, primary antibodies OCTN1 (rabbit polyclonal, Cat# ACT-014, Alomone Labs), a marker for Ergo transporter and AQP4 (rabbit polyclonal, Cat# AQP-004, Alomone Labs), a major membrane water channel in the CNS were used in the eye cross-sections.

Techniques: Expressing

Journal: Frontiers in Neuroscience

Article Title: Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

doi: 10.3389/fnins.2023.1107436

Figure Lengend Snippet: Correlation between 6E10 and IBA1 or GFAP or AQP4 protein markers in the wholemount neuroretinas and retinal cross-sections. Spearman’s rank-order correlation test was conducted to see the association between protein markers based on the plane of view (surface vs. cross-sections). The strength of association between 6E10 and IBA1 expressions in the hemi-retinal wholemounts (A–C) , and retinal cross-sections (G–I) ; between 6E10 and GFAP expressions in the hemi-retinal wholemounts (D–F) , and retinal cross-sections (J–L) ; and between 6E10 and AQP4 expressions in the retinal cross-sections (M–O) are illustrated. Significance levels of p < 0.05 are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: In addition to 6E10, GFAP, and IBA1, primary antibodies OCTN1 (rabbit polyclonal, Cat# ACT-014, Alomone Labs), a marker for Ergo transporter and AQP4 (rabbit polyclonal, Cat# AQP-004, Alomone Labs), a major membrane water channel in the CNS were used in the eye cross-sections.

Techniques: