Journal: Biology

Article Title: Chemical Composition, Antioxidant Capacity, and Anticancerous Effects against Human Lung Cancer Cells of a Terpenoid-Rich Fraction of Inula viscosa

doi: 10.3390/biology13090687

Figure Lengend Snippet: IVL DCM decreased the viability of A549 cells. ( A ) MTT assay was used to measure the viability of A549 and HDFn cells treated with the indicated concentrations of IVL DCM for 24, 48, or 72 h. Data represents the mean ± SEM of the percent viability of treated cells compared to vehicle-treated cells. Data are the mean of three independent experiments ( n = 3). *** p < 0.001, **** p < 0.0001. ( B ) A549 cells treated with the indicated concentrations of IVL DCM for 24 h were stained with DAPI nuclear stain. DAPI fluorescence was imaged at 20X magnification using a BioTek Cytation 5 reader. ( C ) A549 cells were treated with the indicated concentrations of IVL DCM for 24 h and then stained with crystal violet. The micrographs represent stained cells imaged at 20X magnification by light microscopy using the Invitrogen EVOS ® FL Cell Imaging System.

Article Snippet: A549 human lung adenocarcinoma cells, the most common subtype of NSCLC, which accounts for approximately 85% of lung cancer cases [ ], human normal neonatal fibroblasts (HDFn), SK-OV-3 (human ovarian cancer), MCF-7 (human breast cancer), MDA-MB-231 (human breast cancer), HepG2 (human liver cancer), and HCT116 (human colorectal cancer) cells were obtained from ATCC and cultured at 37 °C and 5% CO 2 in a humidified incubator.

Techniques: MTT Assay, Staining, Fluorescence, Light Microscopy, Imaging

Journal: bioRxiv

Article Title: Intrarenal myeloid subsets associated with kidney injury are comparable in mice and patients with lupus nephritis

doi: 10.1101/2023.06.24.546409

Figure Lengend Snippet: (A ) Schematic of experimental workflow. Single cell suspensions were prepared from kidneys harvested from 4 lupus mouse strains with preclinical or clinical nephritis for analysis by single cell transcriptomic profiling or flow cytometry. Clinical nephritis was defined by fixed proteinuria of >300 mg/dl for >2 weeks and was confirmed by light microscopy. (B) Dot plot showing the expression of enriched cell-type canonical markers used to identify 10 intrarenal myeloid subsets. Genes used for RNA FISH staining ( , , , 23) are marked. (C ) Individual UMAP plots of intrarenal myeloid cells from the integrated analysis of preclinical and clinical nephritis for each strain (n=2,993 cells in each UMAP). ( D ) tSNE plots of flow cytometry depicting intrarenal myeloid subsets from preclinical or clinical nephritis in B6.Sle1.Yaa. Each plot contains 24,000 cells from the CD45+ CD11b+ XCR1- Ly6G- gate (6,000 cells from each mouse).

Article Snippet: This single cell suspension was finally enriched for CD45+ cells using Mouse CD45 MicroBeads (Miltenyi) and LS magnetic Columns (Miltenyi) for FACS sorting.

Techniques: Flow Cytometry, Light Microscopy, Expressing, Staining

Journal: bioRxiv

Article Title: Intrarenal myeloid subsets associated with kidney injury are comparable in mice and patients with lupus nephritis

doi: 10.1101/2023.06.24.546409

Figure Lengend Snippet: A . Sorting strategy for intrarenal myeloid populations. Using suspensions of kidney cells collected from mice with preclinical (top) or clinical (bottom) nephritis we positively selected cells using anti-CD45 beads. After gating for live single cells in the lymphocyte/myeloid gate, cDC1 and neutrophils were excluded using anti-XCR1 and anti-LyG respectively. The remaining cells were analyzed in the CD45+/CD11b+ gate (R5). cDC2 were identified as CD11chi/CD26+. Ly6Chi and Ly6Cint cells (R7) were subsetted using antibodies to Dectin2 and anti-CCR2. Classical 1 monocytes were defined as Ly6Chi/CCR2hi/Dectinlo and Classical 2 monocytes were defined as Ly6Cint/Dectin+/CCR2lo. Populations that were Dectin2lo/CCR2lo (DN) and Dectinhi/CCR2hi (DP) were also distinguished in the R7 gate. Ly6Clo cells (R8) were then used to gate resident macrophages (F4/80hi/CD81hi). Non-classical 2 cells were defined as Itgalhi/Fcgr4hi/Fcrl5hi and non-classical 1 cells were defined as Itgalhi/Fcgr4hi/Fcrl5lo. A small population of unidentified cells remained that comprised <5% of all myeloid cells. B . Expression histograms of antibody surface markers (x-axis) from intrarenal myeloid subsets shown from A (above). C . Sorting peripheral myeloid populations collected from mice with preclinical (top) or clinical (bottom) nephritis. We positively selected cells using anti-CD45 beads, gated on live single cells, and used a similar gating scheme as in A.

Article Snippet: This single cell suspension was finally enriched for CD45+ cells using Mouse CD45 MicroBeads (Miltenyi) and LS magnetic Columns (Miltenyi) for FACS sorting.

Techniques: Expressing

Journal: bioRxiv

Article Title: Intrarenal myeloid subsets associated with kidney injury are comparable in mice and patients with lupus nephritis

doi: 10.1101/2023.06.24.546409

Figure Lengend Snippet: (A) & (C) Percentage of Cd11b+ cells identified in flow cytometry as Classical 1, Classical 2, Non-classical 1, Non-classical 2 in preclinical or clinical nephritis. Cells were collected from CD45 enriched kidney cell suspensions (light blue) and matched blood (red) from B6.Sle1.Yaa (A) and NZB/W (C) mice. 5 - 8 mice per condition. (ns: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). ( B) & (D) . Top panel: Co-clustering of single myeloid cells collected from CD45 enriched kidney cell suspensions (light blue) and matched blood (red) from NZW/BXSB (B) and NZB/W (D) mice during clinical nephritis. Bottom panels in ( B ) & ( D ) feature plots of canonical markers for identifying Classical 1, Classical 2, Non-classical 1, Non-classical 2 monocytes. ( E) Normalized bar plots comparing myeloid subset frequencies in preclinical or clinical nephritis for each lupus mouse strain determined by scRNA-seq droplet proportions. ( F ) Pairwise comparison of PAGA transcriptional relatedness of myeloid subclusters subclusters. ( G ) KEGG gene sets enriched in C2 over C1 from mice. ( H) Volcano plot depicting differentially expressed genes between C1 (left) and C2 (right). Labeling reflects differentially expressed genes identified in injury associated macrophages from other studies (see text).

Article Snippet: This single cell suspension was finally enriched for CD45+ cells using Mouse CD45 MicroBeads (Miltenyi) and LS magnetic Columns (Miltenyi) for FACS sorting.

Techniques: Flow Cytometry, Comparison, Labeling

Journal: bioRxiv

Article Title: Intrarenal myeloid subsets associated with kidney injury are comparable in mice and patients with lupus nephritis

doi: 10.1101/2023.06.24.546409

Figure Lengend Snippet: A . tSNE plots of intrarenal myeloid subsets in preclinical or clinical nephritis in NZB/W using flow cytometry. Each tSNE contains 16,500 cells from the CD45+ CD11b+ XCR1- Ly6G- gate (5,500 cells from each mouse). B . Average frequency of each myeloid subset measured via flow cytometry in B6.Sle1Yaa or NZB/W during preclinical or clinical nephritis. 5-8 mice were used for each condition.

Article Snippet: This single cell suspension was finally enriched for CD45+ cells using Mouse CD45 MicroBeads (Miltenyi) and LS magnetic Columns (Miltenyi) for FACS sorting.

Techniques: Flow Cytometry

Journal: Scientific reports

Article Title: Human platelet lysate combined with mesenchymal stem cells pretreated with platelet lysate improved cardiac function in rats with myocardial infarction.

doi: 10.1038/s41598-024-79050-6

Figure Lengend Snippet: Fig. 2. The effect of HPL on MSC morphology and gene expression. (A): The MSCs exposed to HPL (8%) at passage 4 appear elongated, longer, and spindle-shaped whereas the MSCs not treated with HPL exhibit a broader morphology with more prominent and larger nuclei (light microscopy, Optika, 10X magnification). (B): Among the four cardiomiRs, miR-499, and miR-208a showed increased expression in the HPL + MSCs group. In addition, two stem cell marker genes (Nanog and Oct-4) and two cardiac-related genes (GATA-4 and Nkx2.5) exhibited a significant increase in the HPL + MSCs group compared to the MSCs group. HPL: human platelet lysate, MSCs: mesenchymal stem cells, PMSCs: bone marrow mesenchymal stem cells pretreated with HPL. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. MSCs.

Article Snippet: Rabbit polyclonal primary antibody for NKX2 (No: orb540657), rabbit polyclonal antibody for cTnI (No: orb304638), and goat anti-rabbit IgG (H + L) antibody (FITC) (No orb688925) were purchased from Biorbyt, United Kingdom.

Techniques: Gene Expression, Light Microscopy, Expressing, Marker

Journal: Scientific reports

Article Title: Human platelet lysate combined with mesenchymal stem cells pretreated with platelet lysate improved cardiac function in rats with myocardial infarction.

doi: 10.1038/s41598-024-79050-6

Figure Lengend Snippet: Fig. 9. Immunofluorescent analysis indicates the expression of NKX2.5 and cTnI (green) in the hearts of rats with MI two and four weeks after PMSCs and HPL + PMSCs injection: (A, B): Sample micrographs of NKX2.5 and cTnI in an animal of each group. Quantification of the levels of NKX2.5 + and cTnI+ (C and E) and the number of Dil+/NKX2.5 + and Dil+/ cTnI+ (D and F) in the study groups. Stem cells stained with Dil dye (red) and nuclei with DAPI (blue). *** P < 0.001 vs. PMSCs 2 weeks; # < P < 0.05, ### P < 0.001 vs. HPL + PMSCs 2 weeks; &&& P < 0.01 vs. PMSCs; $$ P < 0.001, $$$ P < 0.001 vs. HPL + PMSCs. n = 3 in each group.

Article Snippet: Rabbit polyclonal primary antibody for NKX2 (No: orb540657), rabbit polyclonal antibody for cTnI (No: orb304638), and goat anti-rabbit IgG (H + L) antibody (FITC) (No orb688925) were purchased from Biorbyt, United Kingdom.

Techniques: Expressing, Injection, Staining

Journal: Molecular Cancer

Article Title: A MCP1 fusokine with CCR2-specific tumoricidal activity

doi: 10.1186/1476-4598-10-121

Figure Lengend Snippet: Phenotype analysis . A . The amino acid sequence of GMME1. B . GMME1 Mechanism of Action. GMME1 is capable of blocking CCR2 homodimerization and recruitment of β-arrestin. As a result, various biochemical responses take place such as an increase in p38 phosphorylation while p44/42, AKT, STAT3 are inhibited. In addition, a strong Ca 2+ influx s triggered leading to the activation of caspase 3 and apoptosis. C . The phenotype of expanded C57BL/6 MSCs was analyzed by flow cytometry for various cell surface markers. D . An RT-PCR analysis demonstrates that MSC do not express CCR2. E . MSC culture under adipogenic or osteogenic conditions leads to their differentiation. Photographs were taken under light microscopy using a Contax167MT camera (Kyocera) with a 400 ISO film attached to an Axiovert25 Zeiss microscope (Carl Zeiss) F . Following the retroviral transduction of MSC, the GFP expression levels was monitored by flow cytometry with GMME1 secretion level at 33 ng/10 6 cell/24 hrs as detected by ELISA (P < 0.05; n = 3/group).

Article Snippet: Mouse recombinant CCL2 protein (CCL2 1-76), ELISA kits for mouse CCL2 and human IL6, anti-human CCR2 antibody, CCR2 primers, and Annexin-V/PI detection kits were purchased from R&D systems (Minneapolis, USA).

Techniques: Sequencing, Blocking Assay, Phospho-proteomics, Activation Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Light Microscopy, Microscopy, Retroviral, Transduction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer

Article Title: A MCP1 fusokine with CCR2-specific tumoricidal activity

doi: 10.1186/1476-4598-10-121

Figure Lengend Snippet: Pharmacological properties of GMME1 on EG7 tumor cells . A . Following the confirmation that EG7 cells express CCR2 by RT-PCR, 10 5 EG7 cells/well were cultured for 48 hrs in presence of increasing amounts of CCL2 5-76, CCL2 1-76 or GMME1 and the proliferative response measured by MTT. CCL2 5-76, and to a lesser extent, CCL2 1-76 were capable of inducing the proliferation of EG7 cells as opposed to GMME1 (P < 0.05; n = 6/group). B . Following the addition of 1.5 pmol of GMME1 on EG7 cells for 48 hrs (lower panel), a PI/Annexin-V co-staining demonstrates that GMME1 leads to apoptosis induction (32% dead cells). None of the B16 cells, which are CCR2 null, were affected by the addition of GMME1. C . EG7 cells cultured with GMME1 for 48 hrs induce de novo expression of the pro-apoptotic BAX protein. D . Following the stimulation of EG7 cells for different time points, cell lysate was analysis by a pSTAT3 ELISA (P < 0.05; n = 6/group). The experiments were repeated using the 5 min time point, then lysate was probed by WB. Total STAT3 was used as loading control. E . WT or CCR2 -/- monocytes were purified and cultured with 1.5 pmol of GMME1 for 48 hrs before a PI/Annexin-V co-staining. Even though 58% of WT monocytes died, no major cell death was detected with CCR2 -/- monocytes.

Article Snippet: Mouse recombinant CCL2 protein (CCL2 1-76), ELISA kits for mouse CCL2 and human IL6, anti-human CCR2 antibody, CCR2 primers, and Annexin-V/PI detection kits were purchased from R&D systems (Minneapolis, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Control, Purification

Journal: Molecular Cancer

Article Title: A MCP1 fusokine with CCR2-specific tumoricidal activity

doi: 10.1186/1476-4598-10-121

Figure Lengend Snippet: Pharmacological properties of GMME1 on human U266 tumor cells . A . U266 cells were analyzed by flow cytometry and were negative for the expression of CD19 while CD138 and CCR2 were detected. B . 10 5 U266 cells were cultured with increasing amounts of CCL2 5-76, CCL2 1-76 or GMME1 and the proliferative response measured by MTT. CCL2 5-76 was capable of inducing U266 proliferation whereas GMME1 completely suppressed the proliferative response (P < 0.05; n = 6/group). C . Using 1.5 pmol of GMME1 on U266 cells for 48 hrs, a PI/Annexin-V co-staining demonstrates that GMME1 leads to apoptosis (42% cell death). D . A similar set-up was used for the assessment of STAT3 activation on U266 cells. Following the stimulation of U266 cells using different time points, cell lysate was analysis by a pSTAT3 ELISA. Since STAT3 is inhibited as of 10 min following GMME1 addition on U266 cells, the experiment was repeated at this time point then the lysate was probed by WB. Total STAT3 was used as loading control. To further confirm the inhibitory effect of GMME1 on these cells, the U266 conditioned-media was collected following 48 hrs post-treatment with the different test conditions and analyzed using a human IL-6 ELISA kit. No detectable levels of human IL6 could be observed in the GMME1 group as opposed to the remaining test conditions (*P < 0.05; n = 6/group).

Article Snippet: Mouse recombinant CCL2 protein (CCL2 1-76), ELISA kits for mouse CCL2 and human IL6, anti-human CCR2 antibody, CCR2 primers, and Annexin-V/PI detection kits were purchased from R&D systems (Minneapolis, USA).

Techniques: Flow Cytometry, Expressing, Cell Culture, Staining, Activation Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular Cancer

Article Title: A MCP1 fusokine with CCR2-specific tumoricidal activity

doi: 10.1186/1476-4598-10-121

Figure Lengend Snippet: Pharmacological properties of GMME1 on CCR2 + medulloblastoma cells . A . Confirmation of CCR2 expression on mouse medulloblastoma cell line PS125. B . PS125 cells were seeded in 6-well plates (10 4 cells/well) and cultured with CCL2 -/- or GMME1 CM. After 48 hrs, apoptosis was measured by Annexin-V/PI staining. C . To further see a dose-response effect, the same experiment was repeated using 3 different concentrations and cell death was analyzed (P < 0.05; n = 3/group). D . Human medulloblastoma cells (Daoy) were analyzed by WB to confirm the presence of CCR2 on cell surface. E . Human medulloblastoma cells were treated with GMME1 as described in B . Cell apoptosis was measured by Annexin-V/PI staining after 48 hrs culture. F . Human medulloblastoma cells were cultured in presence of GMME1 in condition medium or affinity-purified GMME1 protein. Cell growth was measured by MTT assay. (*P < 0.05; **P < 0.01; n = 3/group).

Article Snippet: Mouse recombinant CCL2 protein (CCL2 1-76), ELISA kits for mouse CCL2 and human IL6, anti-human CCR2 antibody, CCR2 primers, and Annexin-V/PI detection kits were purchased from R&D systems (Minneapolis, USA).

Techniques: Expressing, Cell Culture, Staining, Affinity Purification, MTT Assay

Journal: Molecular Cancer

Article Title: A MCP1 fusokine with CCR2-specific tumoricidal activity

doi: 10.1186/1476-4598-10-121

Figure Lengend Snippet: GMME1 induced apoptosis of primary myeloma cells from patients . A . White blood cells were isolated from bone marrow aspirates from patients with myeloma, and stained with anti-human CD38, CD45 and CD138 antibodies. CD38 + CD45 - CD138 + cells were considered as myeloma cells. B-C . CCR2 highly expressed on myeloma cells (black field is antibody isotype control). D-E . Isolated lymphocytes were cultured in absence ( D ) or presence ( E ) of GMME1, and the apoptotic myeloma cells were determined by annexin staining ( D and E ). F . Percentage of apoptotic myeloma cells from patients was presented as the mean+/-SD (*P < 0.05; n = 5).

Article Snippet: Mouse recombinant CCL2 protein (CCL2 1-76), ELISA kits for mouse CCL2 and human IL6, anti-human CCR2 antibody, CCR2 primers, and Annexin-V/PI detection kits were purchased from R&D systems (Minneapolis, USA).

Techniques: Isolation, Staining, Control, Cell Culture