Journal: Arthritis Research & Therapy

Article Title: Expansion of myeloid-derived suppressor cells contributes to metabolic osteoarthritis through subchondral bone remodeling

doi: 10.1186/s13075-021-02663-z

Figure Lengend Snippet: High-fat diet increases monocytic-MDSC expansion during PTOA. A Gating strategy used to define the M-MDSC subpopulation (CD11b + Ly6C + Ly6G − cells) in mouse bone marrow. Following the initial FS/SC discrimination, the gate was set on CD11b + cells. After exclusion of doublets (not shown), live CD11b + cells were gated and Ly6C + and Ly6G + populations. B Cytospin images of M-MDSC cells from the bone marrow from LFD and HFD mice (at 16 weeks). Scale bar denotes 10 microns. C HFD increases the BM M-MDSC population in mice ( n = 4 in each group) by total number and percentage with concomitant suppression of CD8 + and CD4 + T-cells compared to LFD mice ( n = 6 in each group, at 16 weeks). D HFD increases the BM M-MDSC population in DMM mice ( n = 3 in each group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-way ANOVA Tukey’s multiple comparison test

Article Snippet: The following anti-human antibodies were purchased from Miltenyi Biotec (Germany): CD11b-APC (clone M1/70, # 130-113-793), HLA-DR–PE (clone AC122, #130-113-402), CD14-fluorescein isothiocyanate (FITC) (clone Tuk4, #130-113-146), CD15-APCvio770 (clone VIMC6, #130-104-992), CD33-viobright515 (clone REA775, #130-111-027), CD66b-PEvio770 (cloneREA306, #130-119-808), and their corresponding isotype controls, along with corresponding isotype controls.

Techniques: Comparison

Journal: Arthritis Research & Therapy

Article Title: Expansion of myeloid-derived suppressor cells contributes to metabolic osteoarthritis through subchondral bone remodeling

doi: 10.1186/s13075-021-02663-z

Figure Lengend Snippet: MDSCs are expanded in obese patients with post-traumatic osteoarthritis. A Demographic of the patient population. B Gating strategy used to define the M-MDSC subpopulation (CD11b + HLADR − CD14 + CD15 − cells). The M-MDSC population is expanded in C peripheral blood and D synovial fluid of obese ( BMI > 30; N = 10) compared to non-obese ( BMI < 30; N = 5) patients diagnosed with post-traumatic osteoarthritis. Two-tailed unpaired Student t -test

Article Snippet: The following anti-human antibodies were purchased from Miltenyi Biotec (Germany): CD11b-APC (clone M1/70, # 130-113-793), HLA-DR–PE (clone AC122, #130-113-402), CD14-fluorescein isothiocyanate (FITC) (clone Tuk4, #130-113-146), CD15-APCvio770 (clone VIMC6, #130-104-992), CD33-viobright515 (clone REA775, #130-111-027), CD66b-PEvio770 (cloneREA306, #130-119-808), and their corresponding isotype controls, along with corresponding isotype controls.

Techniques: Two Tailed Test

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 1. Cell surface expression of CD11b on different cell populations. The expression was quantified by flow cytometry using ICRF44 (A) and CBRM1/5 (B) antibodies. The latter only recognises the headpiece of CD11b in its active state. Data are presented in mean fluorescence intensity (MFI), closed symbols 77R/R donors, open symbols 77R/H-77H/H donors. The two groups were not statistically different. Bars indicate means. doi:10.1371/journal.pone.0057082.g001

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 2. Phagocytosis of hiC3b-coated fluorescent beads. The uptake by macrophages (A), PMNs (B), monocytes (C) and DCs (D) was quantified by flow cytometry and the data are represented as percentage of phagocytosis. Data are expressed as mean+/2SEM. Filled columns 77R/R individuals, open columns 77R/H-77H/H individuals. Paired T test was applied and the p values are indicated. doi:10.1371/journal.pone.0057082.g002

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Flow Cytometry

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 3. Rosetting assay. Percentage of rosettes formed by CFSE- labelled RBC-hiC3b with freshly isolated PMNs from donors carrying the susceptible allele (77H/H, open symbol) or the common allele (77R/R, closed symbol). One representative assay out of 3 independent experiments. doi:10.1371/journal.pone.0057082.g003

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Isolation

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 4. In vitro and in vivo PMN migration. (A) Migration of neutrophil cell lines through transwells seeded with mouse endothelial cells. PMNs migrated into the bottom chamber in response to MIP-2 were counted at different time points as indicated. Pooled results from at least 4 independent experiments are presented as mean 6 SEM. Itgam2/2 and wild type C57BL/6 neutrophil cell lines were used as controls. CD11b- deficient PMNs, known to have weaker endothelial interactions, migrated faster than the C57BL/6 and the hCD11b expressing cell lines (p,0.001 at 60 mns and p,0.05 at 90 mns). Statistical analysis by Bonferroni’s multiple comparison test. (B) Time course of the migration of freshly isolated human 77R/R or 77R/H neutrophils through a HUVEC layer in response to MIP-2. Pooled results from at least 4 independent experiments are presented as mean 6 SEM. (C, D) In vivo peritoneal migration of hCD11b-77R and hCD11b-77H PMN cells lines following i.p. injection of MIP-2 (C) and thioglycollate (D). The two hCD11b expressing PMN lines were labelled with DDAO or CFSE and adoptive transferred at a 1:1 ratio into C57BL/6 mice. Absolute numbers of labelled PMNs recovered from the peritoneum are shown. Data of one out of at least 3 independent experiments are presented. Bars indicate means. doi:10.1371/journal.pone.0057082.g004

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: In Vitro, In Vivo, Migration, Expressing, Comparison, Isolation, Injection

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 5. Cytokine response. Monocytes (A), DCs (B) were stimulated with 2 mg/ml and 10 mg/ml of TLR7/8 ligand (R848) respectively for 24 h. Cytokines quantified using a bead multiplex assay. Closed symbols: 77R/R cells, open symbols: 77R/H-77H/H cells. Each dot represents a single individual, bars denote means. No significant differences between the two CD11b genotypes. Statistical analysis by paired t test. (C, D) Modulation of TLR7/8-induced cytokine release by hiC3b-coated beads. Monocytes (C), DCs (D) were fed with hiC3b-coated beads one hour prior to R848 stimulation. The cytokine changes between the samples with and without CR3 pre-engagement with iC3b are shown with the p values indicated. Data are expressed as mean+/2SEM. The cytokine responses of 77R/R cells (black column) and 77R/H-77H/H cells (white columns) were not statistically different in paired assays. IL, interleukin; TNF-a, tumour necrosis factor alpha; IP-10, Interferon gamma-induced protein 10. doi:10.1371/journal.pone.0057082.g005

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Multiplex Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 2. CD11b is involved in the regulation of α-syn–mediated microglial migration. (A) Effect of fMLP receptor antagonist cyclosporin H and Ab against either α-syn or CD11b on migration of rat microglia toward purified rH α-syn aggregates or fMLP. Here 1.0 × 105 microglia were loaded onto each insert. (B) Effect of anti–α-syn or anti-CD11b Ab on migration of WT mouse microglia toward purified rH α-syn aggregates or fMLP. (C) Migration of CD11b−/−mouse microglia toward purified rH α-syn aggregates or fMLP. In A, B, and C, the transmigrated microglia were measured in the 96-well Boyden chambers using a CytoQuant assay kit. (D) Migration of rat primary microglia toward rat neuron-enriched cultures in which α-syn expression was intact, enhanced or knocked down. “Input” control reflects addition of 1.0 × 105 microglia directly to the well. (E) Migration of mouse WT or CD11b−/−microglia toward mouse or rat neuron-enriched cultures with or without an anti-CD11b Ab. Microglial migration in D and E was measured in the 24-well Boyden chambers. In A–E, n = 4. The Student t test was performed in E, whereas ANOVA followed by the Newman–Keuls multiple-comparisons test were performed in A–D. (F) Representative images showing rat primary microglial migration toward rat neurons with or without an anti-CD11b blocking Ab. Here 3.0 × 105 microglia were loaded onto each insert. (G) Quantitative analysis of microglia that overlapped neurons, counted in images as represented in F. n = 5, Student’s t test. In A–E and G, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding BSA or IgG controls; ##P < 0.01 and ###P < 0.001 compared as indicated. (Scale bar: 100 μm.)

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: Migration, Purification, Expressing, Control, Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 3. α-Syn aggregates directly bind to CD11b, which activates Nox2 to induce a migratory conformation of microglia. (A) In vitro binding assays. Myc-DDK– fused human CD11b was incubated with rH α-syn aggregates, and binding was detected via immunoblot for DDK and α-syn. Incubation of CD11b or α-syn alone served as controls. n = 3. (B) In vivo binding assays. Purified rH α-syn aggregates were mixed with WT or CD11b−/−microglial lysates to allow α-syn to react with CD11b, and the mixtures were further incubated in the IgG- or Ab-conjugated magnetic beads. Binding was detected via immunoblot for CD11b and α-syn. n = 3. (C) O2

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: In Vitro, Binding Assay, Incubation, Western Blot, In Vivo, Purification, Magnetic Beads

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 5. H2O2, a product of α-syn–activated Nox2, serves as a direct signal to regulate microglial directional migration on the interaction between α-syn and CD11b. (A) Microglial chemotaxis toward H2O2 with or without catalase (Cat) based on an under-agarose gel migration assay. n = 4. ANOVA, followed by the Newman–Keuls multiple-comparisons test. (B) Polarized microglial morphology and F-actin distribution after direct exposure of cells to 10 μM H2O2 for 30 min. (Scale bar: 10 μm.) (C) Effect of α-syn stimulation on extracellular H2O2 in WT mouse microglia with or without catalase, as measured by an Abcam kit. BSA served as a control (Ctr). (D) Extracellular H2O2 in stimulated CD11b−/−mouse microglial cultures. (E) Extracellular H2O2 in stimulated gp91phox−/−mouse microglial cultures. (F) Quantitative analysis of the changes in intracellular H2O2 concentration in WT mouse microglia. (G) Quantitative analysis of the changes in intracellular H2O2 concentration in WT mouse microglia pretreated with catalase overnight. In C, D, E, F, and G, ANOVA was performed comparing with the BSA-treated control. n = 4. (H) BSA- or catalase-preincubated microglia migrated toward BSA, fMLP, or α-syn. Assays were performed in the 96-well Boyden chambers using a CytoQuant kit, with 1.0 × 105 microglia loaded onto each insert. n = 4. ANOVA followed by the Newman–Keuls multiple-com- parisons test were performed. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding BSA-treated control. ###P < 0.001 as indicated.

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: Migration, Chemotaxis Assay, Agarose Gel Electrophoresis, Control, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 6. Lyn acts as an H2O2 sensor to regulate the phosphorylation of cortactin. (A) Exposure to either α-syn or H2O2 induced the phosphorylation of SFKs (pSFK) in WT primary mouse microglia. Tubulin (Tub) blots served as protein loading controls. (B) Lyn, an SFK family member, is phosphorylated in WT mouse microglia stimulated using either α-syn or H2O2. (C) Effect of DMSO (DM) or catalase (Cat) on α-syn– or H2O2-induced Lyn phosphorylation in WT mouse microglia. (D) Effect of DMSO (DM) or PP2 on α-syn– or H2O2-induced Lyn phosphorylation in WT mouse microglia. (E) Lyn phosphorylation (pLyn) in CD11b−/−

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: Phospho-proteomics

Journal: Biomedicines

Article Title: Targeting the Glutamine Transporter SLC1A5 Enhances Sensitivity of Acute Myeloid Leukemia to MLN4924

doi: 10.3390/biomedicines14030667

Figure Lengend Snippet: Effect of MLN4924 in combination with SLC1A5 inhibitor (V9302) on AML cell proliferation and cell differentiation. ( A – C ) Evaluate the interference effect of shRNA on SLC1A5 in AML cells through RT-qPCR and Western blot. ( D , E ) The effect of SLC1A5 knockdown on the sensitivity to MLN4924 of AML cells was detected through CCK8 assay (24 h after treatment). ( F , G ) Kasumi-1 and MOLM-13 cells were treated with V9302 for 24 h and IC50 was detected through CCK8 assay. ( H – M ) CCK8 assay was used to verify the effect of MLN4924 combined with V9302 on AML cell viability. ( N – V ) CD11b, CD14, and CD16 expression on AML cell surface treated with MLN4924 and V9302 for 48 h was detected through flow cytometry. ( W ) Wright–Giemsa staining to detect morphological changes of AML cells treated with MLN4924 and V9302 for 24 h captured using an oil immersion lens (1000×). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Antibody against CD11b (Anti-Human CD11b Mouse IgG2a Recombinant Antibody, FITC-65582) was purchased from Proteintech (China).

Techniques: Cell Differentiation, shRNA, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Expressing, Flow Cytometry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Bupleuri Radix Polysaccharides Alleviate MASLD by Regulating Muribaculaceae -Derived SCFAs in the Gut–Liver Axis

doi: 10.3390/ijms27020637

Figure Lengend Snippet: Bupleuri radix polysaccharides (BRP) attenuates liver injury and metabolic dysfunction in methionine- and choline-deficient (MCD) diet-induced metabolic dysfunction-associated steatotic liver disease (MASLD) mice. ( A ) Schematic illustration of the experimental design. BRP (200 or 400 mg/kg) was administered to MCD diet-induced mice. ( B ) The body weight of mice in different groups. ( C ) Liver coefficient of mice in each group. ( D ) Hematoxylin and eosin (H&E) staining of liver tissues and ( E ) quantification of the extent of liver damage using the MASLD activity score (MAS) method. Scale bar = 100 μm. ( F ) The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglyceride (TG), and non-esterified fatty acids (NEFA) in the serum. ( G ) The levels of TC, TG, and NEFA in the livers. Relative mRNA expression levels of ( H ) fibrosis-related genes Acta2 and Col1a1 and ( I ) inflammation-related markers Cd11b , Cd68 , Ccl2 , Tgfβ1 , and Tnfα in livers. Statistical significance: Data are presented as mean ± standard deviation (SD) (n = 6 mice per group). Statistical analysis was performed using one-way ANOVA followed by appropriate post hoc tests, as described in . # p < 0.05, ## p < 0.01, ### p < 0.001 compared between the control (CT) group and the model group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared between the model group and the BRP treatment groups.

Article Snippet: Sections were then blocked with 0.2% Triton X-100 + 2.5% BSA + 10% goat serum blocking buffer at RT for 30 min. After blocking, primary antibody cocktails were applied: Villin (Proteintech Group Inc., Rosemont, IL, USA; 66096-1-Ig) + Ki67 (Proteintech Group Inc., Rosemont, IL, USA; 27309-1-AP) or CD11b (Proteintech Group Inc., Rosemont, IL, USA; 66519-1-Ig) + E-cadherin (Cell Signaling Technology, Inc., Danvers, MA, USA; 3195S).

Techniques: Staining, Activity Assay, Expressing, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Bupleuri Radix Polysaccharides Alleviate MASLD by Regulating Muribaculaceae -Derived SCFAs in the Gut–Liver Axis

doi: 10.3390/ijms27020637

Figure Lengend Snippet: BRP alleviates colonic mucosal injury and inflammation during MASLD. ( A – D ) Representative ( A ) H&E staining and ( C ) AB-PAS staining of colon tissues, and ( B , D ) the quantification of colonic injury area was performed using Image J software, version 1.53. In H&E staining, nuclei are stained blue-purple and cytoplasm appears pink; in AB-PAS staining, acidic mucins are stained blue and neutral mucins magenta. Scale bar = 100 μm. Representative immunofluorescence co-staining images of ( E ) Villin (green) and Ki67 (red), as well as ( F ) Cd11b (green) and E-cadherin (red), in colon tissue. DAPI was used for nuclear labeling (blue), and yellow signals in merged images indicate colocalization. Scale bar = 100 μm. ( G ) Protein levels of E-cadherin and Occludin in colonic tissues. ( H , I ) Relative mRNA levels of Ecad , Ocln , Muc2 , Zo1 , Cd11b , Il1b , and Ccl2 in colon tissues. Statistical significance: Data are presented as mean ± SD (n = 6 mice per group). Statistical analysis was performed using one-way ANOVA followed by appropriate post hoc tests, as described in the . # p < 0.05, ## p < 0.01, ### p < 0.001 compared between the CT group and the model group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared between the model group and the BRP treatment groups.

Article Snippet: Sections were then blocked with 0.2% Triton X-100 + 2.5% BSA + 10% goat serum blocking buffer at RT for 30 min. After blocking, primary antibody cocktails were applied: Villin (Proteintech Group Inc., Rosemont, IL, USA; 66096-1-Ig) + Ki67 (Proteintech Group Inc., Rosemont, IL, USA; 27309-1-AP) or CD11b (Proteintech Group Inc., Rosemont, IL, USA; 66519-1-Ig) + E-cadherin (Cell Signaling Technology, Inc., Danvers, MA, USA; 3195S).

Techniques: Staining, Software, Immunofluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Bupleuri Radix Polysaccharides Alleviate MASLD by Regulating Muribaculaceae -Derived SCFAs in the Gut–Liver Axis

doi: 10.3390/ijms27020637

Figure Lengend Snippet: The hepatoprotective effect of BRP is suppressed by removing gut microbiota in MASLD mice. ( A ) Animal experimental flowchart. ( B ) The body weight of mice in different groups. ( C ) Liver coefficient of mice in each group. ( D ) H&E staining of liver tissues, and ( E ) quantification of the extent of liver damage using the MAS method. Scale bar = 100 μm. ( F ) The levels of AST, ALT, TC, and TG in the serum. ( G ) The levels of TG and TC in the livers. ( H , I ) Relative mRNA expression levels of Col1a1 , Acta2 , Cd11b , Cd68 , Tgfβ1 , Tnfα , and Ccl2 in livers. Statistical significance: Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by appropriate post hoc tests, as described in the . n = 6 mice per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared between the CT group and either the CT + ABX group or the model + ABX group.

Article Snippet: Sections were then blocked with 0.2% Triton X-100 + 2.5% BSA + 10% goat serum blocking buffer at RT for 30 min. After blocking, primary antibody cocktails were applied: Villin (Proteintech Group Inc., Rosemont, IL, USA; 66096-1-Ig) + Ki67 (Proteintech Group Inc., Rosemont, IL, USA; 27309-1-AP) or CD11b (Proteintech Group Inc., Rosemont, IL, USA; 66519-1-Ig) + E-cadherin (Cell Signaling Technology, Inc., Danvers, MA, USA; 3195S).

Techniques: Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Bupleuri Radix Polysaccharides Alleviate MASLD by Regulating Muribaculaceae -Derived SCFAs in the Gut–Liver Axis

doi: 10.3390/ijms27020637

Figure Lengend Snippet: P. intestinale synergistically enhances the hepatoprotective effect of BRP on MALFD mice. ( A ) Animal experimental flowchart. ( B ) The body weight of mice in different groups. ( C ) Liver coefficient of mice in each group. ( D ) H&E staining of liver tissues and ( E ) quantification of the extent of liver damage using the MAS method. Scale bar = 100 μm. ( F ) The levels of AST, ALT, TC, TG, and NEFA in the serum. ( G ) The levels of TC, TG, and NEFA in the livers. ( H , I ) Relative mRNA expression levels of Acta2 , Col1a1 , Cd11b , Cd68 , Ccl2 , Tgfβ1 , and Tnfα in livers. ( J ) The levels of SCFAs (acetic, propionic, butyric, pentanoic, and isovaleric acids) in cecum. Statistical significance: Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by appropriate post hoc tests, as described in the . n = 6 mice per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared between the CT group and the model group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared between the model group and the treatment groups.

Article Snippet: Sections were then blocked with 0.2% Triton X-100 + 2.5% BSA + 10% goat serum blocking buffer at RT for 30 min. After blocking, primary antibody cocktails were applied: Villin (Proteintech Group Inc., Rosemont, IL, USA; 66096-1-Ig) + Ki67 (Proteintech Group Inc., Rosemont, IL, USA; 27309-1-AP) or CD11b (Proteintech Group Inc., Rosemont, IL, USA; 66519-1-Ig) + E-cadherin (Cell Signaling Technology, Inc., Danvers, MA, USA; 3195S).

Techniques: Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Bupleuri Radix Polysaccharides Alleviate MASLD by Regulating Muribaculaceae -Derived SCFAs in the Gut–Liver Axis

doi: 10.3390/ijms27020637

Figure Lengend Snippet: P. intestinale synergistically enhances BRP in improving colonic mucosal damage in MALFD mice. ( A – D ) Representative ( A ) H&E staining and ( C ) AB-PAS staining of colon tissues, and ( B , D ) the quantification of colonic injury area was performed using Image J software. In H&E staining, nuclei are stained blue–purple and cytoplasm appears pink; in AB-PAS staining, acidic mucins are stained blue and neutral mucins magenta. Scale bar = 100 μm. ( E , F ) Representative immunofluorescence co-staining images of Villin (green) and Ki67 (red), as well as Cd11b (green) and E-cadherin (red), in colon tissue. DAPI was used for nuclear labeling (blue). Scale bar = 100 μm. ( G ) Relative mRNA levels of barrier genes Muc2 , Zo1 , Ecad , and Ocln , stem/proliferation markers Lgr5 and Ki67 , and inflammatory genes Ccl2 , Il1b , Tnfα , and Il6 in colon tissues. Statistical significance: Data are presented as mean ± SD. The p values were calculated by one-way ANOVA followed by appropriate post hoc tests, as described in the . n = 6 mice per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared between the CT group and the model group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared between the model group and the treatment groups.

Article Snippet: Sections were then blocked with 0.2% Triton X-100 + 2.5% BSA + 10% goat serum blocking buffer at RT for 30 min. After blocking, primary antibody cocktails were applied: Villin (Proteintech Group Inc., Rosemont, IL, USA; 66096-1-Ig) + Ki67 (Proteintech Group Inc., Rosemont, IL, USA; 27309-1-AP) or CD11b (Proteintech Group Inc., Rosemont, IL, USA; 66519-1-Ig) + E-cadherin (Cell Signaling Technology, Inc., Danvers, MA, USA; 3195S).

Techniques: Staining, Software, Immunofluorescence, Labeling