Journal: bioRxiv

Article Title: Identification of Human Transferrin Receptor as an Entry Co-receptor for Parvovirus B19 Infection of Human Erythroid Progenitor Cells

doi: 10.64898/2026.04.02.715920

Figure Lengend Snippet: (A) Protein diagram. VP1u-APEX2 consists of APEX2 fused to the C-terminus of the unique region of B19V VP1 (VP1u) via a seven-residue glycine-serine linker (GGSGGSG), followed by a Flag tag and a 6 × Histidine (His) tag. APEX2 has a linker-Flag-His tag fused at the C-terminus. (B) Analysis of purified proteins. VP1u-APEX2 and APEX2 proteins were expressed in bacteria and purified. Approximately (∼) 1 µg of each protein was separated by SDS-PAGE, followed by Coomassie blue staining. M, molecular weight marker. (C) Confocal microscopy of VP1u-APEX2 entry. 1 × 10 6 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C for 2 h. The cells were then immunostained with α-Flag to visualize internalized proteins under a Leica STED confocal microscope. Scale bar = 10 μm. Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole). (D) Western blotting of APEX2-biotinylated proteins. 1 × 10 7 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C. After 2 h, APEX2-mediated biotinylation was then performed as described in the Materials and Methods and Figure S1 . Biotinylated host proteins were purified with streptavidin-conjugated magnetic beads. The supernatant was collected as the flow-through (FT), and the beads were further washed several times and eluted as the pull-down (PD). Both FT and PD samples were analyzed by SDS-PAGE and immunoblotting using Alexa Fluor 680-conjugated streptavidin. (E) Analysis of VP1u-APEX2-biotinylated/associated proteins using quantitative mass spectrometry (qMS). Three independent PD samples prepared from VP1u-APEX2 and APEX2 (control) treated cells were analyzed by on-bead digestion and qMS. MS data were processed and analyzed as described in the Materials and Methods. The bubble plot shows protein enrichment (log 2 fold change) in the VP1u-APEX2 group relative to the APEX control, with color indicating subcellular localization based on Gene Ontology (GO) annotation. TFRC denotes human transferrin receptor 1 (hTfR).

Article Snippet: Purified proteins: Recombinant hTfR ECD protein tagged with a His-tag at the C-terminus (#11020-H07H) and recombinant human ferritin heavy chain 1/FTH1 (#13217-HNAE) were purchased from SinoBiological (Paoli, PA).

Techniques: Residue, FLAG-tag, Purification, Bacteria, SDS Page, Staining, Molecular Weight, Marker, Confocal Microscopy, Incubation, Microscopy, Western Blot, Magnetic Beads, Mass Spectrometry, Control, Protein Enrichment

Journal: Virchows Archiv : an international journal of pathology

Article Title: Toll-like receptors 1, 2, 4, 5, and 6 in gastric cancer.

doi: 10.1007/s00428-023-03635-1

Figure Lengend Snippet: Fig. 1 Representative images of cytoplasmic TLR2 and TLR6 expression immunostaining in gastric adenocarcinoma. Low cytoplasmic TLR2 expression (A), high cytoplasmic TLR2 expression (B), low cytoplasmic TLR6 expression (C), and high cytoplasmic TLR6 expression (D)

Article Snippet: Samples were rinsed in distilled water and phosphate-buffered saline with Tween (PBS-T) and endogenous peroxidase was then neutralized in peroxidase blocking solution (Dako S2023) for 5 min. After a wash in PBS-T, sections were incubated with antibodies (Dako S2022); TLR1 (diluted 1:300, ROCKLAND 800-656-7625), TLR2 (diluted 1:500, ROCKLAND 600-401-956), TLR4 (diluted 1:1000, abonova 214-291), TLR5 (1:75, NOVUS NBP-2-24787), and TLR6 (diluted 1:750, abonova PAB 3555).

Techniques: Expressing, Immunostaining

Journal: Advanced Science

Article Title: Melanoma Derived Exosomes Amplify Radiotherapy Induced Abscopal Effect via IRF7/I‐IFN Axis in Macrophages

doi: 10.1002/advs.202304991

Figure Lengend Snippet: CircPIK3R3/IRF7/I‐IFN axis participates in the combination of radiotherapy and anti‐PD1 mediated abscopal effect. A) The treatment model involving RT, anti‐PD1, and RO8191: C57/BL6 mice were subcutaneously inoculated with 1 × 106 sh‐NC B16F10 cells or sh‐circ‐0011074 B16F10 cells. On day 5, C57/BL6 mice were intravenously injected with 1 × 106 B16F10‐luc cells. Starting from day 6, mice were administered the IFN receptor agonist RO8191 via daily intraperitoneal injections at a dose of 1 mg kg −1 . On day 7, radiotherapy was initiated, with a daily dose of 8 Gy administered for 3 consecutive days. On day 7, mice were also administered anti‐PD1 via intraperitoneal injection every 2 days at a dose of 100 µg/mouse until the endpoint of observation. B,C) Measurement of subcutaneous tumor weight in each group to assess treatment efficacy ( n = 3). D,E) Evaluation of fluorescent intensity in lung metastatic foci using bioluminescence imaging to assess treatment efficacy ( n = 6). F,G) Immunohistochemical examination of CD8 + T cell infiltration in subcutaneous tumors and lung metastatic foci ( n = 3). H,I) Immunofluorescence detection of IRF7 + macrophage infiltration in subcutaneous tumors and lung metastatic foci (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two‐tailed unpaired Student t‐test.

Article Snippet: Anti‐mouse PD‐1 mAb (Bio X Cell, USA) was administered intraperitoneally (200 mg per mouse) every 2 days during treatment.

Techniques: Injection, Imaging, Immunohistochemical staining, Immunofluorescence, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Curcumol Induces Necroptosis of Hepatic Stellate Cells by Targeting KAT8 to Suppress HK2 Lactylation and Promote HUWE1-Dependent Ubiquitination

doi: 10.7150/ijbs.125009

Figure Lengend Snippet: Curcumol induces HSC necroptosis. A. LX2 cells were treated with different concentrations of Curcumol (0-80 μM) for 24 h, and cell viability was assessed to determine the optimal intervention concentration (n = 5). B-C. Cell viability was measured in LX2 cells, human normal liver cells Thle-2, and mouse normal liver cells AML12 after treatment with Curcumol (0-45 μM) for 24 h (CCK-8 assay, n = 5). D. LX2 cells were treated with or without Curcumol (30 μM) and the necrotic necroptosis inhibitor Nec-1 (50 μM) for 24 h, and the ultrastructure of the cells was observed using a transmission electron microscope; the right image is an enlarged view of the area framed in the left image (scale bar: 5 μm). E. Under the same treatment conditions, the proportion of necroptotic cells was detected by flow cytometry, and the results were quantitatively analyzed (n = 3). F-G. Detection of LDH release levels in LX2 cells treated with different concentrations of Curcumol (0-45 μM) for 24 h (n = 5); simultaneous comparison of the effects of Nec-1 (50 μM) treatment on Curcumol (30 μM)-induced LDH release, with quantitative analysis (n = 3-5). H. Western blot was used to detect the expression levels of RIPK1, RIPK3, MLKL, and their phosphorylated proteins (p-RIPK1, p-RIPK3, p-MLKL) in LX2 cells after 24 h of treatment with different concentrations of Curcumol (0-45 μM), and quantitative analysis was performed using grayscale analysis (n = 3). I. Co-IP assay to detect the interaction between RIPK1 and RIPK3 in LX2 cells after treatment with Curcumol (30 μM) for 24 h. J. Immunofluorescence assay to detect the expression of Collagen I and α-SMA in LX2 cells after treatment with Curcumol (30 μM) for 24 h (n = 3; scale bar: 10 μm). K-L. Western blotting was used to detect the protein levels of Collagen I and α-SMA in LX2 cells and mouse primary HSCs after 24 h of treatment with different concentrations of Curcumol (0-45 μM), and quantitative analysis was performed using grayscale analysis (n = 3). Data are shown as mean ± SD, and statistical differences were analyzed by one-way ANOVA. ns indicates no significance; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies employed in this study include: RIPK1 (17519-1-AP), RIPK3 (17563-1-AP), P-RIPK1 (66854-1-Ig), MLKL (21066-1-AP), P-MLKL (82090-2-RR), HK2 (22029-1-AP), PKM2 (15822-1-AP), LDHA (19987-1-AP), PFKM (55028-1-AP), HUWE1 (19430-1-AP), Ubiquitin (10201-2-AP), HA (51064-2-AP), His (66005-1-Ig), EP300 (20695-1-AP) and P62 (18420-1-AP) from proteintech; KAT8 (sc-271691) from Santa Cruz Biotechnology; KAT6A from Bioswamp; Collagen I (ab26003), β-actin (ab8226), Tubulin (Ab721), anti-mouse IgG (ab190475), anti-rabbit IgG (ab288151) and LC3B (ab192890) from Abcam; Pan-Kla (AB_2868521) from PTM-Bio laboratory; KAT6B(A17116), P-RIPK3(AP1260), AARS(A15017), and AARS2 (A7826) from Abclonal.

Techniques: Concentration Assay, CCK-8 Assay, Transmission Assay, Microscopy, Flow Cytometry, Comparison, Western Blot, Expressing, Co-Immunoprecipitation Assay, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Curcumol Induces Necroptosis of Hepatic Stellate Cells by Targeting KAT8 to Suppress HK2 Lactylation and Promote HUWE1-Dependent Ubiquitination

doi: 10.7150/ijbs.125009

Figure Lengend Snippet: Curcumol induces HSCs necroptosis by suppressing HK2-mediated ubiquitination of RIPK1. A. Transmission electron microscopy images of LX2 cells treated with Curcumol (30 μM) for 24 h in the presence or absence of 2-DG (5 mM) (right: magnified region; scale bar: 5 μm). B, E. Western blot analysis of RIPK1, RIPK3, p-RIPK1, and p-RIPK3 expression in LX2 cells treated with Curcumol (30 μM) and 2-DG (5 mM) for 24 h, with densitometric quantification (n = 3). C, F. Western blot analysis of RIPK1, RIPK3, p-RIPK1, and p-RIPK3 expression in LX2 cells transfected with HK2 overexpression plasmid and treated with Curcumol (30 μM) for 24 h, with densitometric quantification (n = 3). D. Co-immunoprecipitation (Co-IP) analysis of RIPK1-RIPK3 interaction in LX2 cells transfected with HK2 overexpression plasmid and treated with Curcumol (30 μM) plus 2-DG (5 mM) for 24 h. G. Western blot analysis of RIPK1 expression in HK2 siRNA-transfected HK2-overexpressing LX2 cells, with densitometric quantification (n = 3). H. Immunofluorescence staining showing colocalization of HK2 and RIPK1 in LX2 cells treated with Curcumol (30 μM) for 24 h (n = 3; scale bar: 2.5 μm). I. RT-qPCR analysis of RIPK1 mRNA expression in LX2 cells treated with increasing concentrations of Curcumol (0-45 μM) for 24 h (n = 5). J. Immunoprecipitation analysis of RIPK1 ubiquitination in LX2 cells transfected with HK2 overexpression plasmid and treated with Curcumol (30 μM) for 24 h (n = 3). K. Western blot analysis of RIPK1 protein stability in LX2 cells treated with Curcumol (30 μM), cycloheximide (CHX, 20 μg/mL) for 24 h in the presence or absence of protein synthesis inhibitor, with quantification of half-life (n = 3). L. Western blot analysis of RIPK1 protein expression in LX2 cells treated with Curcumol (30 μM), cycloheximide (CHX, 20 μg/mL), proteasome inhibitor MG132 (10 μM), alone or in combination, with quantification (n = 3). M,N. Western blot analysis of RIPK1 protein levels in LX2 cells co-transfected with HK2 siRNA and HK2 overexpression plasmid, followed by Curcumol (30 μM) treatment for 24 h, with quantification (n = 3). O. Western blot analysis of RIPK1 expression in LX2 cells transfected with HK2 overexpression plasmid and treated with autophagy inhibitor chloroquine (CQ, 20 μg/mL), MG132 (10 μM), alone or in combination, for 24 h, with quantification (n = 3). Data are shown as mean ± SD, and statistical differences were analyzed by one-way ANOVA. ns indicates no significance; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies employed in this study include: RIPK1 (17519-1-AP), RIPK3 (17563-1-AP), P-RIPK1 (66854-1-Ig), MLKL (21066-1-AP), P-MLKL (82090-2-RR), HK2 (22029-1-AP), PKM2 (15822-1-AP), LDHA (19987-1-AP), PFKM (55028-1-AP), HUWE1 (19430-1-AP), Ubiquitin (10201-2-AP), HA (51064-2-AP), His (66005-1-Ig), EP300 (20695-1-AP) and P62 (18420-1-AP) from proteintech; KAT8 (sc-271691) from Santa Cruz Biotechnology; KAT6A from Bioswamp; Collagen I (ab26003), β-actin (ab8226), Tubulin (Ab721), anti-mouse IgG (ab190475), anti-rabbit IgG (ab288151) and LC3B (ab192890) from Abcam; Pan-Kla (AB_2868521) from PTM-Bio laboratory; KAT6B(A17116), P-RIPK3(AP1260), AARS(A15017), and AARS2 (A7826) from Abclonal.

Techniques: Ubiquitin Proteomics, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: Curcumol Induces Necroptosis of Hepatic Stellate Cells by Targeting KAT8 to Suppress HK2 Lactylation and Promote HUWE1-Dependent Ubiquitination

doi: 10.7150/ijbs.125009

Figure Lengend Snippet: Curcumol targets KAT8 to suppress glycolysis and induce necroptosis in hepatic stellate cells (HSCs) in vivo . A. Representative images of liver sections from vehicle, different doses of Curcumol, and CCl₄-induced fibrotic mice (n = 5), stained with hematoxylin-eosin (H&E), Masson, and Sirius Red, and analyzed by immunohistochemistry (IHC) for α-SMA, Collagen I, and KAT8. Scale bar = 200 μm. B. Liver-to-body weight ratios of mice in the CCl₄ model group, Curcumol treatment group (n = 5). C-E. Serum levels of aspartate aminotransferase (AST), laminin (LN), and hyaluronic acid (HA) in CCl₄-induced fibrotic mice treated with vehicle, measured by biochemical assays (n = 5). F, G. Immunofluorescence analysis of colocalization between α-SMA (green) and HK2 (red), or α-SMA (green) and RIPK1 (red), in liver tissues from normal and CCl₄-induced fibrotic mice (n = 3). Scale bar = 20 μm. H,J. Serum levels of ALT and collagen IV (Col IV) in CCl₄-induced fibrotic mice treated with vehicle, Curcumol, measured by ELISA (n = 5). I. Western blot analysis of KAT8 and HK2 protein expression in liver tissues from vehicle-, Curcumol-, and CCl₄-treated mice, with densitometric quantification (n = 5). K-L. Western blot analysis of α-SMA, Collagen I, PFK1, PKM2, HK2, LDHA, RIPK1, RIPK3, phosphorylated RIPK1 (p-RIPK1), and phosphorylated RIPK3 (p-RIPK3) in liver tissues from vehicle-, Curcumol-, and KAT8 OE + Curcumol-treated CCl₄-induced fibrotic mice (n=3), with densitometric quantification. M. Western blot analysis of α-SMA, Collagen I, PFK1, PKM2, HK2, LDHA, RIPK1, RIPK3, phosphorylated RIPK1 (p-RIPK1), and phosphorylated RIPK3 (p-RIPK3) in liver tissues from vehicle-, Curcumol-, and KAT8 OE + Curcumol-treated TGF-β induced primary HSCs (n=3), with densitometric quantification. N. Serum lactate levels in CCl₄-induced fibrotic mice treated with different doses of Curcumol, measured by lactate assay kits (n = 5). Data are shown as mean ± SD, and statistical differences were analyzed by one-way ANOVA. ns indicates no significance; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies employed in this study include: RIPK1 (17519-1-AP), RIPK3 (17563-1-AP), P-RIPK1 (66854-1-Ig), MLKL (21066-1-AP), P-MLKL (82090-2-RR), HK2 (22029-1-AP), PKM2 (15822-1-AP), LDHA (19987-1-AP), PFKM (55028-1-AP), HUWE1 (19430-1-AP), Ubiquitin (10201-2-AP), HA (51064-2-AP), His (66005-1-Ig), EP300 (20695-1-AP) and P62 (18420-1-AP) from proteintech; KAT8 (sc-271691) from Santa Cruz Biotechnology; KAT6A from Bioswamp; Collagen I (ab26003), β-actin (ab8226), Tubulin (Ab721), anti-mouse IgG (ab190475), anti-rabbit IgG (ab288151) and LC3B (ab192890) from Abcam; Pan-Kla (AB_2868521) from PTM-Bio laboratory; KAT6B(A17116), P-RIPK3(AP1260), AARS(A15017), and AARS2 (A7826) from Abclonal.

Techniques: In Vivo, Staining, Immunohistochemistry, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Lactate Assay

Journal: International Journal of Biological Sciences

Article Title: Curcumol Induces Necroptosis of Hepatic Stellate Cells by Targeting KAT8 to Suppress HK2 Lactylation and Promote HUWE1-Dependent Ubiquitination

doi: 10.7150/ijbs.125009

Figure Lengend Snippet: KAT8 promotes hepatic fibrosis in vivo via HK2-mediated regulation of RIPK1-dependent necroptosis. A. Liver fibrosis staging was performed using the Ishak score. For histopathological analysis, H&E, Masson, and IHC were used to stain a-SMA, HK2 and KAT8 in 15 human liver samples (F0/1, 3; F2, 3; F3, 4; F4, 5). Representative images are shown. Scale bar: 200µm, n=6/group. B. Quantification of positive KAT8 and HK2 IHC staining (n= 6 / group). C-E. Liver tissues from CCl4-induced fibrotic mice (n=5) treated with vehicle, Curcumol, or KAT8 overexpression combined with Curcumol (KAT8 OE + Curcumol 30mg/kg) were subjected to hematoxylin and eosin (H&E) staining, Masson's trichrome staining, Sirius Red staining, and immunohistochemical (IHC) analysis for α-smooth muscle actin (α-SMA), collagen I, and KAT8. Scale bar = 200 μm. F. Protein expression of KAT8 and hexokinase 2 (HK2) in heart, liver, lung, kidney, and spleen tissues from CCl4-induced fibrotic mice (vehicle and KAT8 OE groups, n=5) was quantified by immunoblotting (n=3). G. Western blot analysis was performed to determine protein levels of α-SMA, collagen I, pyruvate kinase M2 (PKM2), phosphofructokinase-1 (PFK1), HK2, and lactate dehydrogenase A (LDHA) in liver tissues from vehicle, Curcumol, KAT8 OE, and KAT8 OE + Curcumol (30mg/kg) groups (n=5). H. Primary mouse hepatic stellate cells (HSCs) were subjected to KAT8 knockdown or overexpression, and immunoblotting was performed to assess collagen I and α-SMA expression after 24 h of Curcumol (30mg/kg) treatment (n=3; scale bar: 10 μm). I. Serum biochemical parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT)and alkaline phosphatase (ALP) were evaluated in vehicle, Curcumol (30mg/kg), KAT8 OE, and KAT8 OE + Curcumol (30mg/kg) groups (n=5). J. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum levels of hyaluronic acid (HA), laminin (LN), procollagen III (PCIII), and collagen IV (Col IV) in the same groups (n=5). K. Immunofluorescence analysis was performed to examine the co-localization of α-SMA (green) and HK2 (red) in liver tissues from CCl4-induced fibrotic mice (vehicle, KAT8 OE, and KAT8 OE + Curcumol (30mg/kg) groups, n=3). Scale bar = 20 μm. Data are shown as mean ± SD, and statistical differences were analyzed by one-way ANOVA. ns indicates no significance; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies employed in this study include: RIPK1 (17519-1-AP), RIPK3 (17563-1-AP), P-RIPK1 (66854-1-Ig), MLKL (21066-1-AP), P-MLKL (82090-2-RR), HK2 (22029-1-AP), PKM2 (15822-1-AP), LDHA (19987-1-AP), PFKM (55028-1-AP), HUWE1 (19430-1-AP), Ubiquitin (10201-2-AP), HA (51064-2-AP), His (66005-1-Ig), EP300 (20695-1-AP) and P62 (18420-1-AP) from proteintech; KAT8 (sc-271691) from Santa Cruz Biotechnology; KAT6A from Bioswamp; Collagen I (ab26003), β-actin (ab8226), Tubulin (Ab721), anti-mouse IgG (ab190475), anti-rabbit IgG (ab288151) and LC3B (ab192890) from Abcam; Pan-Kla (AB_2868521) from PTM-Bio laboratory; KAT6B(A17116), P-RIPK3(AP1260), AARS(A15017), and AARS2 (A7826) from Abclonal.

Techniques: In Vivo, Staining, Immunohistochemistry, Over Expression, Immunohistochemical staining, Expressing, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Immunofluorescence