Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: SPP1 promotes Th17 cell differentiation via interactions with ITGB1 and CD44. (A) Flow cytometry revealed a dose-dependent facilitation of Th17 differentiation by SPP1, with a concentration of 0.2 μg/mL emerging as optimal for ensuing experimental endeavors. Th17 cells were identified as CD3+CD4+IL-17A+ cells, and these results were represented as the percentage of Th17 cells among CD3+ T cells. (B) Flow cytometry indicated that ursolic acid dose-dependently inhibited SPP1-induced Th17 cell differentiation, with a concentration of 5 μM emerging as optimal for subsequent investigative pursuits. (C) Western blot analyses were conducted to detect the phosphorylation level of ERK protein. (D) SPP1 KD decreased the ERK phosphorylation level and substantially curtailed Th17 cell differentiation. (E) Co-immunoprecipitation analyses suggested strong interactions between SPP1 and both ITGB1 and CD44. (F) Both ITGB inhibitors and CD44 antagonists suppressed the SPP1-driven Th17 cell differentiation, with an even more pronounced inhibitory effect upon their combined application. SPP1, secreted phosphoprotein 1; ITGB1, integrin β1; CD44A, CD44 antagonists; ERK, extracellular signal-regulated kinase; SD, standard deviation. Data are represented as mean±SD. n=3. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: To assess the influence of ITGB1 and CD44 modulation on Th17 differentiation, cells were additionally co-incubated with ITGB1 inhibitor GLPG0187 (1 nM, HY-100506; MedChemExpress, Monmouth, NJ, USA) or CD44 antagonist (1 μg/ mL, 553131; BD Biosciences, San Jose, CA, USA) at specific concentrations recommended by preliminary dose-response studies.

Techniques: Cell Differentiation, Flow Cytometry, Concentration Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, Standard Deviation

Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: Ursolic acid modulates SPP1-mediated Th17 cell differentiation to ameliorate MASLD. (A–C) Mice were derived from the experiments of SPP1 KD, wherein (A) presented that the protein levels of ITGB1 and CD44 expressed in the pull-down products were conspicuously surged by HFD administration, whereas a precipitous decline in their expression was observed in the liver tissue lysates procured from the SPP1 KD mice, while (B) demonstrated that such alterations were concomitant with trends in the phosphorylation level of ERK protein; and concurrently, (C) displayed a similar trend in hepatic Th17 cell populations. (D, E) Mice were derived from the first part of experiments, those were fed with HFD and different concentrations of ursolic acid, wherein (D) suggested that ursolic acid dose-dependently ameliorated the escalated Th17 cell populations induced by the high-fat dietary regimen, and (E) revealed consistent protein levels of SPP1, ITGB1, CD44, as well as the phosphorylation level of ERK. Data are represented as mean±SD. n=3-6. * P <0.05, ** P <0.01, *** P <0.001. SPP1, secreted phosphoprotein 1; MASLD, metabolic dysfunction-associated steatotic liver disease; KD, knockdown; ITGB1, integrin β1; CD44A, CD44 antagonists; HFD, highfat diets; ERK, extracellular signal-regulated kinase; SD, standard deviation.

Article Snippet: To assess the influence of ITGB1 and CD44 modulation on Th17 differentiation, cells were additionally co-incubated with ITGB1 inhibitor GLPG0187 (1 nM, HY-100506; MedChemExpress, Monmouth, NJ, USA) or CD44 antagonist (1 μg/ mL, 553131; BD Biosciences, San Jose, CA, USA) at specific concentrations recommended by preliminary dose-response studies.

Techniques: Cell Differentiation, Derivative Assay, Expressing, Phospho-proteomics, Knockdown, Standard Deviation

Journal: International journal of stem cells

Article Title: Integrin α 4 Positive Subpopulation in Adipose Derived Stem Cells Effectively Reduces Infarct Size through Enhanced Engraftment into Myocardial Infarction.

doi: 10.15283/ijsc22209

Figure Lengend Snippet: Fig. 2. Integrin α4 ＋ adipose-derived stem cells (ASCs) subpopulation were isolated and sorted from ASCs by fluo- rescence-activated cell sorting. (A) The population of integrin α4 ＋ ASCs were about 5.11% and 70.7% in the pre- and postsorted ASCs, respectively. (B) Immunofluorescent staining revealed integrin α4 were robustly expressed in integrin α4 ＋ ASCs subpopulation. (C) Western blotting showed that the ex- pression of integrin α4 with 135 kDa was obviously more in integrin α4 ＋

Article Snippet: A mouse anti-integrin α4 antibody was used to detect the grafted integrin α4+ ASCs. von Willebrand Factor (vWF), an endothelial cell marker, was identified by an mouse anti-vWF antibody (Proteintech).

Techniques: Derivative Assay, Isolation, FACS, Staining, Western Blot

Journal: International journal of stem cells

Article Title: Integrin α 4 Positive Subpopulation in Adipose Derived Stem Cells Effectively Reduces Infarct Size through Enhanced Engraftment into Myocardial Infarction.

doi: 10.15283/ijsc22209

Figure Lengend Snippet: Fig. 4. Identification of Integrin α4 ＋/green fluorescent protein＋ (GFP＋) adipose-derived stem cells (ASCs) and incorporation of injected ASCs into the vasculature by immunofluorescent staining in infarcted myocardium 4 weeks posttransplantation. (A) Immunofluorescent staining was performed for GFP (green) and integrin α4 (red), and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, blue), co-localized expression of integrin α4 and GFP were detected in the hearts receiving integrin α4 ＋ ASCs subpopulation, and integrin α4 ＋ ASCs sub- population showed much higher engraftment than unfractionated ASCs. (B) Immunofluorescent staining was conducted for GFP (green) and von Willebrand Factor (vWF, red), and nuclei were stained with DAPI (blue), GFP-positive ASCs directly incorporated into vasculature.

Article Snippet: A mouse anti-integrin α4 antibody was used to detect the grafted integrin α4+ ASCs. von Willebrand Factor (vWF), an endothelial cell marker, was identified by an mouse anti-vWF antibody (Proteintech).

Techniques: Derivative Assay, Injection, Staining, Expressing

Journal: International journal of stem cells

Article Title: Integrin α 4 Positive Subpopulation in Adipose Derived Stem Cells Effectively Reduces Infarct Size through Enhanced Engraftment into Myocardial Infarction.

doi: 10.15283/ijsc22209

Figure Lengend Snippet: Fig. 5. Integrin α4 ＋ adipose-derived stem cells (ASCs) subpopu- lation more robustly migrated and engrafted into the infartced my- ocardium after cell transplantation. (A) Four weeks posttranspla- ntation, green fluorescent protein-positive ASCs were more fre- quently detected in the hearts receiving integrin α4 ＋ ASCs sub- population than in the hearts undergoing unfractionated ASCs treatment. (B) The high reproducibility of the standard curve of re- al-time PCR. Serial dilution (10−2∼10−8) of DNA was made 7 times to construct the standard curve. Each pink circle corresponded to one dilution in one experiment. The blue solid line represented the regression analysis. (C) Four weeks after cell transplantation, in- tegrin α4 ＋ ASCs subpopulation treated rats showed the signifi- cantly greater ASC numbers per heart as compared to unfractio- nated ASCs treated rats. *p＜0.05 vs. unfractionated ASCs.

Article Snippet: A mouse anti-integrin α4 antibody was used to detect the grafted integrin α4+ ASCs. von Willebrand Factor (vWF), an endothelial cell marker, was identified by an mouse anti-vWF antibody (Proteintech).

Techniques: Derivative Assay, Transplantation Assay, Serial Dilution, Construct

Journal: International journal of stem cells

Article Title: Integrin α 4 Positive Subpopulation in Adipose Derived Stem Cells Effectively Reduces Infarct Size through Enhanced Engraftment into Myocardial Infarction.

doi: 10.15283/ijsc22209

Figure Lengend Snippet: Fig. 6. Integrin α4 + adipose-derived stem cells (ASCs) subpopulation more effectively reduced infarct size post-implantation in vivo. (A) Representative tomographic histograms of the 18F-fluorodeoxyglucose (FDG) positron emission tomography images at 4 weeks posttrans- plantation. Yellow colors were indicative of higher tracer uptake, while blue colors stood for lower tracer uptake. (B) Four weeks after transplantation, unfractionated ASCs transplantation markedly elevated the 18F-FDG uptake in apical-septal, apical-anterior, and apex seg- ments as compared with phosphate-buffered saline (PBS) control. Of importance, integrin α4 + ASCs subpopulation implantation further increased the 18F-FDG uptake in apical-septal, apical-inferior, and apex segments compared with unfractionated ASCs injection. (C) Representative transverse, coronal, and sagittal myocardial 18F-FDG images at 4 weeks after cell transplantation. The infarcted area can be visually detected as an area of low glucose metabolism. (D) Four weeks after transplantation, integrin α4 + ASCs subpopulation markedly reduced infarct size as compared to unfractionated ASCs and PBS control. (E) Integrin α4 + ASCs subpopulation significantly increased global 18F-FDG uptake compared with unfractionated ASCs and PBS control. *p＜0.05 vs. unfractionated ASCs. #p＜0.05 vs. PBS.

Article Snippet: A mouse anti-integrin α4 antibody was used to detect the grafted integrin α4+ ASCs. von Willebrand Factor (vWF), an endothelial cell marker, was identified by an mouse anti-vWF antibody (Proteintech).

Techniques: Derivative Assay, In Vivo, Positron Emission Tomography, Transplantation Assay, Saline, Control, Injection

Journal: International journal of stem cells

Article Title: Integrin α 4 Positive Subpopulation in Adipose Derived Stem Cells Effectively Reduces Infarct Size through Enhanced Engraftment into Myocardial Infarction.

doi: 10.15283/ijsc22209

Figure Lengend Snippet: Fig. 7. Integrin α4 ＋ adipose-derived stem cells (ASCs) subpopulation more effectively improve cardiac fucntion post-implantation in vivo. (A, B) Four weeks after transplantation, integrin α4 ＋ ASCs subpopulation reduced left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) compared with unfractionated ASCs and phosphate-buffered saline (PBS) control. (C) Integrin α4 ＋

Article Snippet: A mouse anti-integrin α4 antibody was used to detect the grafted integrin α4+ ASCs. von Willebrand Factor (vWF), an endothelial cell marker, was identified by an mouse anti-vWF antibody (Proteintech).

Techniques: Derivative Assay, In Vivo, Transplantation Assay, Saline, Control

Journal: Neural Regeneration Research

Article Title: Pre-clinical study of human umbilical cord mesenchymal stem cell transplantation for the treatment of traumatic brain injury: safety evaluation from immunogenic and oncogenic perspectives

doi: 10.4103/1673-5374.317985

Figure Lengend Snippet: Identification, immunogenicity and immunomodulation of huMSCs . (A) The huMSCs cultured in our experiment. The huMSCs were spindle-shaped and grew in a whorl pattern. Original magnification 100×, scale bars: 200 μm. (B) Flow cytometry detection of CD29, CD44, CD73 and CD105. The rate of positive expression for all factors was greater than 95%. (C) Detection of huMSC HLAII expression in PBMCs and huMSCs by western blot. No HLAII expression band was detected in the huMSCs. (D) Relative expression of HLAII in PBMCs and huMSCs. The bar graph shows no HLAII expression in huMSCs. (E) PCR amplification curves of HLA-DPA1 , HLA-DQA1 and HLA-DRA1 genes in PBMCs and huMSCs. (F) Relative expression of HLA-DPA1 , HLA-DQA1 and HLA-DRA1 genes in PBMCs and huMSCs. PCR results showed no expression of HLA-DPA1 , HLA-DQA1 or HLA-DRA1 genes in huMSCs. (G) Expression curves of serum IL-6, IL-10, IL-12, TGF-β, and TNF-α detected by ELISA method ( n = 5 rats per group). Compared with the TBI group, the serum pro-inflammatory cytokines IL-6, IL-12 and TNF-α in the Tail Vein and In Situ group were lower (### P < 0.001), whereas the inflammation inhibiting factors IL-10 and TGF-β were much higher (### P < 0.001), indicating that huMSCs exert good immunoregulatory effects. Data are expressed as the mean ± SD. *** P < 0.001, vs . PBMCs (one-way analysis of variance followed by least significant difference test). ELISA: Enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HLAII: human leukocyte antigen II; huMSCs: human umbilical cord mesenchymal stem cells; IL: interleukin; PBMCs: peripheral blood mononuclear cells; PCR: polymerase chain reaction; TBI: traumatic brain injury; TGF-β: transforming growth factor beta; TNF-α: tumor necrosis factor alpha.

Article Snippet: The sections were thereafter incubated overnight at 4°C with primary antibodies, including rabbit anti-human CD29 polyclonal antibody (Cat# PB9086, 1:100, Boster), proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody (Cat# 2586S, 1:4000, CST, Danvers, MA, USA), Caspase 3 (CASP3) rabbit polyclonal antibody (Cat# 19677-1-AP, 1:200, Proteintech) and F4/80 rabbit polyclonal antibody (Cat# 28463-1-AP, 1:200, Proteintech), diluted in 1% bovine serum albumin containing PBS.

Techniques: Immunopeptidomics, Cell Culture, Flow Cytometry, Expressing, Western Blot, Amplification, Enzyme-linked Immunosorbent Assay, In Situ, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Pre-clinical study of human umbilical cord mesenchymal stem cell transplantation for the treatment of traumatic brain injury: safety evaluation from immunogenic and oncogenic perspectives

doi: 10.4103/1673-5374.317985

Figure Lengend Snippet: Sites of accumulation of huMSCs in vivo in TBI rats . (A) Live imaging of the primary organs of experimental rats. (B, C) The average fluorescence intensity in the brain, liver and lung in the In Situ (B) and Tail Vein (C) groups. Data are expressed as the mean ± SD ( n = 5 rats at each time point). # P < 0.05, ## P < 0.01, vs . TBI group (one-way analysis of variance followed by least significant difference test). (D, E) CD29 (red, stained by CoraLite594) immunoreactivity in CFSE-labeled huMSCs in brain tissue (immunofluorescence staining, original magnification 200×, scale bars: 100 μm). CD29 and CFSE co-labeled huMSCs were identified in the In Situ group on days 1, 3 and 7 (D) in the brain lesions. A few CFSE-labeled huMSCs in the Tail Vein group were also observed in the brain lesions on days 1 and 3 (E). CFSE: Carboxyfluorescein succinimidyl ester; CON: TBI group; DAPI: 4′,6-diamidine-2′-phenylindole dihydrochloride; huMSCs: human umbilical cord mesenchymal stem cells; TBI: traumatic brain injury.

Article Snippet: The sections were thereafter incubated overnight at 4°C with primary antibodies, including rabbit anti-human CD29 polyclonal antibody (Cat# PB9086, 1:100, Boster), proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody (Cat# 2586S, 1:4000, CST, Danvers, MA, USA), Caspase 3 (CASP3) rabbit polyclonal antibody (Cat# 19677-1-AP, 1:200, Proteintech) and F4/80 rabbit polyclonal antibody (Cat# 28463-1-AP, 1:200, Proteintech), diluted in 1% bovine serum albumin containing PBS.

Techniques: In Vivo, Imaging, Fluorescence, In Situ, Staining, Labeling, Immunofluorescence

Journal: Frontiers in Nutrition

Article Title: Prevention of high-fat/high-sugar diet-induced type 2 diabetes mellitus-associated non-alcoholic fatty liver disease in rats with fermented and raw Rosa roxburghii Tratt (Cili) juice

doi: 10.3389/fnut.2025.1584551

Figure Lengend Snippet: Expression analyses of transcription or protein levels of selected genes. (A) Expression analyses of selected genes by qRT–PCR. The data represent the means ± SDs from six biological replicates with three technical replicates. (B–D) FCJ/RCJ upregulated ABCC3, IDI1, and APOA2 expression in the liver. FCJ and RCJ significantly upregulated hepatic ABCC3 (B) and IDI1 (C) expression, and RCJ significantly upregulated hepatic APOA2 (D) expression in T2DM rats. The data represent the means ± SDs from three biological replicates with three technical replicates. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: The western blotting antibodies used included APOA2 (BM5624, BOSTER, China), IDI1 (A07892-1, BOSTER, China), ABCC3 (DF3874, Affinity, United States), β-tubulin (Solarbio, China), goat anti-rabbit (BS13278, Bioworld, United States), and goat anti-mouse (BS12478, Bioworld, United States) antibodies.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Effect of Mixed Transplantation of Autologous and Allogeneic Microskin Grafts on Wound Healing in a Rat Model of Acute Skin Defect

doi: 10.1371/journal.pone.0085672

Figure Lengend Snippet: Aa, Ab, and Ac show integrin β1 expression at 2 post-graft weeks (PGWs) in groups II,III, and IV, respectively, in Experiment A. Ad shows integrin β1 expression in the hair follicle in group IV in Experiment A. Ae and Af show integrin β1 expression at 2 PGWs in groups II and III, respectively, in Experiment B. Ag and Ah show expression of integrin β1 in the hair follicle and no expression of integrin β1 in the epidermis, respectively, of normal Sprague-Dawley (SD) rat skin. Ai, Aj, and Ak show negative control staining of 2 PGW sections from groups II, III, and IV, respectively, in Experiment A. Scale bar, 50 µm in Aa–Ah and 100 µm in Ai–Ak. In Experiment A, the mean integrated optical density (IOD) of integrin β1 expression at 2 PGWs was higher in groups III and IV than in group II (* p<0.05), and, more interestingly, was highest in group III among these 3 groups (* p<0.05, # p<0.05). At 3 and 4 PGWs, the integrin β1 expression remained higher in group III than in groups II and IV (* p<0.05, # p<0.05). In Experiment B, the integrin β1 expression at 2 PGWs was stronger in group III than in group I (* p<0.05), and no intergroup difference was found at 4 PGWs.

Article Snippet: The sections were then sequentially incubated with normal goat serum for 40 minutes to block nonspecific binding, with the primary rabbit anti-integrin β1 polyclonal antibody (1∶100, Wuhan Boster Biological Technology LTD, Hubei, China) for 15 hours at 4°C, and with the SABC kit (Wuhan Boster Biological Technology, LTD, Hubei, China) at 37°C to bind the primary antibody.

Techniques: Expressing, Negative Control, Staining

Journal: Nature Communications

Article Title: Phc2 controls hematopoietic stem and progenitor cell mobilization from bone marrow by repressing Vcam1 expression

doi: 10.1038/s41467-019-11386-4

Figure Lengend Snippet: Blocking the interaction between VCAM-1 and VLA-4 restores steady-state HSPC mobilization in KO mice. a Relative ratio of migrated WT LSK cells (LSK) through WT or KO BMSCs in the presence of anti-VLA-4 Ab and/or anti-VCAM-1 Ab. n = 5 per group. Statistical significance was assessed by two-tailed Student’s t -test. * P < 0.05; ** P < 0.01. b Relative ratio of adhered WT or KO LSK to KO BMSCs in the presence of anti-VLA-4 Ab and/or anti-VCAM-1 Ab. n = 5 per group. Statistical significance was assessed by two-tailed Student’s t- test. * P < 0.05; ** P < 0.01. c , d CFSE-labeled LSK cells from WT mice were intravenously injected into lethally irradiated WT or KO mice. Anti-VCAM-1 Ab or its isotype control Ab (2 mg kg −1 ) was intravenously injected into WT and KO recipient mice 1 h before adoptive cell transfer. n = 5 per group. c The frequencies of CFSE + donor cells were measured in the BM, PB, and, spleen (SP) of recipients at 16 h after adoptive cell transfer. d The frequencies of donor-derived clonogenic progenitors (CFU-C) homing to the BM (femur), PB and SP of recipients at 16 h after adoptive cell transfer. Statistical significance was assessed by one-way ANOVA with Tukey HSD analysis. Mean values not sharing the same superscript letter ( a, b, c ) differ significantly at P < 0.05. e–g Anti-VCAM-1 Ab or its isotype control Ab (2 mg kg −1 ) was intravenously injected into WT and KO mice for 3 days. n = 5 per group. e Comparison of WBC counts between WT and KO mice on day 3 after Ab treatment. f Absolute number of splenocytes for WT and KO mice on day 3 after Ab treatment. g CFU-C in the BM (femur), PB, and SP of WT and KO mice on day 3 after Ab treatment. Statistical significance was assessed by two-tailed Student’s t- test. ** P < 0.01. All data are presented as the mean ± SEM. WT, WT recipient mice; KO, KO recipient mice. “−“, treatment with an isotype control Ab; “ + “, treatment with anti-VCAM-1 Ab. Source data are provided as a Source Data File

Article Snippet: To block the VLA-4 and VCAM-1 interaction, LSK cells were preincubated with anti-VLA-4 (10 μg mL −1 ; Merck CBL1304) Ab or an isotype control (10 μg ml −1 ; BD Biosciences 559478) for 30 min, and BMSCs were preincubated with anti-VCAM-1 Ab (10 μg mL −1 ) or an isotype control for 30 min.

Techniques: Blocking Assay, Two Tailed Test, Labeling, Injection, Irradiation, Derivative Assay, Mouse Assay

Journal: Nature Communications

Article Title: Phc2 controls hematopoietic stem and progenitor cell mobilization from bone marrow by repressing Vcam1 expression

doi: 10.1038/s41467-019-11386-4

Figure Lengend Snippet: Neutralizing interaction between VCAM-1 and VLA-4 reverses the defect involving regimen-induced HSPC mobilization in KO mice. a – c G-CSF-induced HSPC mobilization assays with anti-VCAM-1 Ab. n = 5 per group. a WBC counts from experimental animals until day 5 after G-CSF and Ab treatment. b Representative image of spleens (left) and absolute number of splenocytes from experimental animals on day 5 after G-CSF and Ab treatment (right). c CFU-C in the BM (femur), PB, and spleen (SP) from experimental animals on day 5 after G-CSF and Ab treatment. d , e WBC counts ( d ) and CFU-C ( e ) in PB from experimental animals 3 h after AMD3100 and Ab treatment. n = 5 per group. f – h 5-FU-induced HSPC cell mobilization assays with anti-VCAM-1 Ab. n = 5 per group. f Comparison of WBC counts between WT and KO mice at indicated time points after 5-FU and Ab treatment. g Representative image of spleen from a WT and KO mouse (left) and absolute number of splenocytes (right) of WT and KO mice on day 16 after 5-FU and Ab treatment. h CFU-C in the BM (femur), PB, and spleen (SP) of WT and KO mice on day 16 after 5-FU and Ab treatment. “−“, treatment with an isotype control Ab; “ + “, treatment with anti-VCAM-1 Ab. Statistical significance was assessed by two-tailed Student’s t -test. ** P < 0.01. All data are presented as the mean ± SEM. Source data are provided as a Source Data File

Article Snippet: To block the VLA-4 and VCAM-1 interaction, LSK cells were preincubated with anti-VLA-4 (10 μg mL −1 ; Merck CBL1304) Ab or an isotype control (10 μg ml −1 ; BD Biosciences 559478) for 30 min, and BMSCs were preincubated with anti-VCAM-1 Ab (10 μg mL −1 ) or an isotype control for 30 min.

Techniques: Two Tailed Test