Journal: iScience

Article Title: Derived myeloid lineage induced pluripotent stem as a platform to study human C-C chemokine receptor type 5Δ32 homozygotes

doi: 10.1016/j.isci.2023.108331

Figure Lengend Snippet: Functionality of primary and iPSC-derived macrophages Phagocytic ability of M0, M1 and M2 primary macrophages (pMac, white bar, n = 4) and iPSC-derived macrophages (iMac) obtained from both controls (iMac-Con, light gray bar; n = 3) and individuals homozygous for CCR5Δ32 variant (iMac-CCR5Δ32, dark gray bar; n = 4) was evaluated by using pHrodo Green Zymosan A Bioparticles and was assessed by flow cytometry and confocal microscopy. (A) Macrophage phagocytic ability assessed by flow cytometry was shown as frequency of Zymosan+ cells. (B) Representative photographs showing phagocytized zymosan particles (in green) in primary macrophages (pMac, first row, n = 3) and iMac obtained from healthy controls (iMac-Con, second row, n = 3) and iMac obtained from homozygous CCR5Δ32 individuals (iMac-CCR5Δ32, third row, n = 4) polarized in M0, M1, and M2 macrophages (First, second and third column, respectively). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (C) The chemotactic ability of primary monocytes (pMono, white circle; n = 3) and iPSC-derived monocytes (iMono) obtained from healthy controls (iMono-Con, light gray square; n = 3) and from homozygous CCR5Δ32 individuals (iMono-CCR5Δ32, dark gray square; n = 3) to migrate in response to increasing the concentration of RANTES was assessed in vitro by a cell migration assay. The number of migrated cells was evaluated at different time points. ∗ iMono-Con vs. iMono-CCR5Δ32; # pMono-Con vs. iMono-CCR5Δ32. (D and E) Macrophage infectability with HIV was assessed in M1 and M2 polarized primary and iPSC-derived macrophages obtained from healthy controls (iMac-Con) and from homozygous CCR5Δ32 individuals (iMac-CCR5Δ32) by evaluating through qPCR the presence of (D) specific HIV-1 DNA Gag and (E) specific HIV-1 DNA LTR RU5. Amplification of genomic GAPDH as a reference gene was used to control the amount of DNA in each sample. Data shown as mean ± SEM. Statistical significance was calculated using ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Article Snippet: In the lower part of the transwell, DMEM medium (Lonza) was added with recombinant–soluble human RANTES (rh-RANTES; R&D) that was used as an attractant chemokine at different concentrations (1, 3, and 10 ng/ml).

Techniques: Derivative Assay, Variant Assay, Flow Cytometry, Confocal Microscopy, Staining, Concentration Assay, In Vitro, Cell Migration Assay, Amplification, Control

Journal: iScience

Article Title: Derived myeloid lineage induced pluripotent stem as a platform to study human C-C chemokine receptor type 5Δ32 homozygotes

doi: 10.1016/j.isci.2023.108331

Figure Lengend Snippet:

Article Snippet: In the lower part of the transwell, DMEM medium (Lonza) was added with recombinant–soluble human RANTES (rh-RANTES; R&D) that was used as an attractant chemokine at different concentrations (1, 3, and 10 ng/ml).

Techniques: Virus, Recombinant, Modification, Purification, Blocking Assay, Control, Library Quantification, Luminex, Pore Size, Amplification, Software

Journal: Frontiers in Cardiovascular Medicine

Article Title: Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells

doi: 10.3389/fcvm.2022.979745

Figure Lengend Snippet: Monocyte adhesion induced by siSOX13 is prevented by treating HAECs with MetCCL5. (A) HAECs overexpressing SOX13 or RFP control were exposed to OSS or ULS for another 24 h. Following shear, THP1 monocytes were added to the conditioned media (CM), and adhered monocytes were counted. (B) HAECs treated with siSOX13 or siCtrl were exposed to OSS or ULS for another 24 h. Following shear, THP1 monocytes were added to the CM, and adhered monocytes were counted. (C,D) HAECs treated with siSOX13 or siCtrl were exposed to OSS or ULS in the presence or absence of MetCCL5 for another 24 h. Following shear, THP1 monocytes were added to the CM, and adhered monocytes were counted (C) . In panel (D) , following shear, CM was removed, HAECs were washed with fresh medium, THP1 monocytes were added to the CM, and adhered monocytes were counted. Mean±SEM are shown, n = 3 to 7, p -values determined by one-way ANOVA.

Article Snippet: MetCCL5 (R&D 335-RM-025) was added to the fresh media at a concentration of 50ng/ml at the beginning of the 24 h shear exposure.

Techniques: Control, Shear

Journal: Oncology Letters

Article Title: Cancer-associated fibroblast-derived CCL5 contributes to cisplatin resistance in A549 NSCLC cells partially through upregulation of lncRNA HOTAIR expression

doi: 10.3892/ol.2021.12957

Figure Lengend Snippet: CAFs secrete elevated CCL5 to promote cell viability in NSCLC cancer cells via paracrine activation. (A) mRNA expression levels of CCL5 in CAFs, NFs and two NSCLC cell lines by RT-qPCR using GAPDH gene as the internal control. (B) CCL5 in conditioned medium secreted by NFs and CAFs were quantified by ELISA. *P<0.05, ***P<0.001 and ****P<0.0001 vs. CAFs. (C) A549 and H1299 cancer cells were incubated with CAF-CM or NF-CM in combination with anti-CCL5 antibody (0.1 µg/ml) for 6 h followed by treatment with DDP for 48 h. Cell viability was determined by the MTT assay. (D) mRNA expression levels of CCL5 and CCR5 in NSCLC cell lines cultured with CAF-CM were assessed by RT-qPCR using GAPDH gene as the normalization control. (E) A549 and H1299 cancer cells were incubated with CAF-CM or NF-CM in combination with CCR5 inhibitor (Met-RANTES; 0.1 µg/ml) for 6 h followed by treatment with DDP for 48 h. Cell viability was determined by the MTT assay. *P<0.05, **P<0.01 and ***P<0.001 vs. control; # P<0.05 and ## P<0.01 vs. DDP; && P<0.01 vs. CAF-CM and DDP. Results were expressed as the mean ± SD, and the means were calculated from ≥3 independent experiments. RT-qPCR, reverse transcription-quantitative PCR; CAF, cancer-associated fibroblast; NF, normal fibroblast; CM, conditioned medium; DDP, cisplatin; CCL5, C-C motif chemokine ligand 5; CCR5, C-C motif chemokine receptor 5; NSCLC, non-small cell lung cancer.

Article Snippet: For cell treatment, cancer cells were incubated with CAF-CM or NF-CM in combination with either anti-CCL5 antibody (0.1 μg/ml; cat. no. MAB678-SP; R&D Systems, Inc.), CCR5 antagonist (Met-RANTES; 0.1 μg/ml; cat. no. 335-RM-025; R&D Systems, Inc.) or recombinant human CCL5 (3 ng/ml; cat. no. 300-06; PeproTech, Inc.) for 6 h, followed by treatment with 50 μM DDP (Sigma-Aldrich; Merck KGaA) in the presence of CM for another 48 h.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Cancer-associated fibroblast-derived CCL5 contributes to cisplatin resistance in A549 NSCLC cells partially through upregulation of lncRNA HOTAIR expression

doi: 10.3892/ol.2021.12957

Figure Lengend Snippet: CAF-CM increases HOTAIR expression in tumor cells in vitro . (A) HOTAIR expression in human NSCLC A549 and H1299 cell lines treated with CAF-CM or NF-CM was determined by qPCR. HOTAIR expression in human NSCLC A549 and H1299 cell lines treated with CAF-CM and (B) CCL5 neutralizing antibody or (C) CCR5 inhibitor (Met-RANTES) was determined by qPCR. ***P<0.001 and ****P<0.0001 vs. control; ## P<0.01 and ### P<0.001 vs. CAF-CM. Results were expressed as the mean ± SD, and the means were calculated from ≥3 independent experiments. qPCR, quantitative PCR; CAF, cancer-associated fibroblast; NF, normal fibroblast; CM, conditioned medium; CCL5, C-C motif chemokine ligand 5; CCR5, C-C motif chemokine receptor 5; NSCLC, non-small cell lung cancer; HOTAIR, HOX transcript antisense RNA.

Article Snippet: For cell treatment, cancer cells were incubated with CAF-CM or NF-CM in combination with either anti-CCL5 antibody (0.1 μg/ml; cat. no. MAB678-SP; R&D Systems, Inc.), CCR5 antagonist (Met-RANTES; 0.1 μg/ml; cat. no. 335-RM-025; R&D Systems, Inc.) or recombinant human CCL5 (3 ng/ml; cat. no. 300-06; PeproTech, Inc.) for 6 h, followed by treatment with 50 μM DDP (Sigma-Aldrich; Merck KGaA) in the presence of CM for another 48 h.

Techniques: Expressing, In Vitro, Control, Real-time Polymerase Chain Reaction