Journal: Regenerative Therapy

Article Title: In vitro and in vivo study of concentrated growth factor (CGF) mediating macrophage polarization in bone defect repair

doi: 10.1016/j.reth.2025.04.013

Figure Lengend Snippet: Surface characteristics of C GF and its effect on macrophages. A: CGF and its three-dimensional structure under electron microscopy; The proliferation of BMDM treated with different concentrations of CGF; B: ELISA method is used to detect the secretion of IL-1 β 、TNF- α 、VEGF-A、IL-10 after CGF acts on M0 and M1 macrophages; C: Western blot detection of protein expression of Arg-1 and iNOS after CGF acts on M0 and M1 macrophages; D: Western blot detection of JAK/STAT pathway related protein expression after CGF acts on M0 and M1 macrophages, respectively (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. #, P < 0.05; ##, P < 0.01; ###, P < 0.001; ####, P < 0.0001. # compared with the control group, ns, P > 0.05,one-way ANOVA).

Article Snippet: BMDM macrophage cell lines (Procell Life Science & Technology Co., Ltd.) were used for in vitro studies.

Techniques: Electron Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Journal: Nature Communications

Article Title: Coordination of cohabiting phage elements supports bacteria–phage cooperation

doi: 10.1038/s41467-019-13296-x

Figure Lengend Snippet: The Lm 10403S strain harbors two functional lytic phage elements. a Growth analysis of WT Lm and mutants harboring deletions of the elements lysis modules; LMRG_01552–4 of ϕ10403S ( Δ(hol-lys) ϕ ), and LMRG_02377-8 of monocin cluster ( Δ(hol-lys) mon ) or a mutant deleted of both lysis modules ( Δhol-lys) ϕ /Δ(hol-lys) mon ), in the presence of mitomycin C (MC). Growth analysis of the mutants without MC is presented in Supplementary Fig. . Error bars represent the standard deviation of three independent biological repeats, and are hidden by the symbols. b Plaque forming assay of WT Lm and a mutant deleted for the ϕ10403S integrase gene, Δint ( LMRG_01511 ) with MC treatment (+MC). Virions obtained from MC treated bacterial cultures (4 h post MC treatment) or from bacteria grown to exponential phase (3 h, OD 600 0.5) without MC treatment ( WT Lm -MC), tested on an indicator strain for plaque forming units (PFU). Error bars represent the standard deviation of three independent experiments. c A monocin killing assay performed on monocins obtained from MC treated bacterial cultures of the Lm ϕ10403S-phage cured strain (Δϕ) and a mutant lacking both the monocin cluster ( LMRG_02362-02378 ) and ϕ10403S-phage (Δ mon/ Δϕ), as a control. Five-fold serial dilutions of filtered supernatants (containing monocins) were applied on a lawn of different Listeria strains (target cells), and incubated for 1–2 days. The dark zones of growth inhibition indicate monocin killing activity. The experiment was performed three times. Monocins from Δϕ bacteria that were not treated with MC (Δϕ –MC) are shown at the bottom, as a reference. d Intracellular growth analysis of WT Lm and a deletion mutant lacking both lysis modules ( Δ(hol-lys) ϕ /Δ(hol-lys) mon ) in bone marrow derived macrophage (BMDM) cells. Growth curves represent one biological replicate, more independent experiments are shown in the source data file. Error bars represent standard deviation of a technical triplicate, sometimes hidden by the symbols. e Transcription analysis of the two phage elements holin and endolysin genes under SOS and intracellular growth conditions using NanoString technology (6 h post BMDM infection). Transcription levels are presented as fold change of relative counts for the indicated gene mRNA, relative to the levels observed during lysogeny (i.e., exponentially grown bacteria in BHI medium). Data represent 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: BMDM cells used for infection experiments were isolated from 6 to 8 week-old female C57BL/6 mice (Envigo, Israel) and cultured in Dulbecco’s Modified Eagle Medium (DMEM)-based media supplemented with 20% fetal bovine serum, sodium pyruvate (1 mM), L-glutamine (2 mM), β-Mercaptoethanol (0.05 mM) and monocyte-colony stimulating factor (M-CSF, L929-conditioned medium); BMDM medium .

Techniques: Functional Assay, Lysis, Mutagenesis, Standard Deviation, Bacteria, Control, Incubation, Inhibition, Activity Assay, Derivative Assay, Infection

Journal: Nature Communications

Article Title: Coordination of cohabiting phage elements supports bacteria–phage cooperation

doi: 10.1038/s41467-019-13296-x

Figure Lengend Snippet: The monocin element affects ϕ10403S excision within macrophage cells. a Intracellular growth analysis of WT Lm and mutants lacking each of the phage elements, Δ mon and Δ ϕ (monocin cluster and ϕ10403S, respectively), and a double mutant lacking both phage elements Δ mon/ Δ ϕ , in BMDM cells. Growth curves represent one biological replicate, more independent experiments are shown in the source file. Error bars represent standard deviation of triplicate samples, and are hidden by the symbols. b A bacterial phagosomal escape assay. Percentage of bacteria that escaped the macrophage phagosomes at 2.5 h post infection, as determined by a microscope fluorescence assay. Macrophages were infected with WT Lm , Δ mon and Δ mon/ Δϕ bacteria, as well as with a Δ mon mutant that was complemented with an intact comK gene on the pPL2 plasmid (Δ mon+ pPL2 -comK ). The data is a mean of three independent experiments. The error bar represent standard deviation. Asterisk (*) indicates statistical significance of p = 0.01 calculated using Student's t -test. c qRT-PCR analysis of intact comK gene (representing ϕ10403S attB site) in WT Lm and indicated mutants grown intracellularly in BMDM cells (6 h post infection). Presented as relative quantity (RQ), relative to the levels in WT bacteria. The data represent three independent experiments. Error bars indicate a 95% confidence interval. d Intracellular growth analysis of WT Lm , Δmon and the Δmon mutant complemented with an intact comK gene with its native promoter (Δ mon+ pPL2 -comK ) in BMDM cells. Growth curves represent one biological replicate, more independent experiments are shown in Supplementary Fig. and in the source data file. Error bars represent standard deviation of triplicate samples, and are hidden by the symbols. Source data are provided as a Source Data file.

Article Snippet: BMDM cells used for infection experiments were isolated from 6 to 8 week-old female C57BL/6 mice (Envigo, Israel) and cultured in Dulbecco’s Modified Eagle Medium (DMEM)-based media supplemented with 20% fetal bovine serum, sodium pyruvate (1 mM), L-glutamine (2 mM), β-Mercaptoethanol (0.05 mM) and monocyte-colony stimulating factor (M-CSF, L929-conditioned medium); BMDM medium .

Techniques: Mutagenesis, Standard Deviation, Bacteria, Infection, Microscopy, Fluorescence, Plasmid Preparation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Coordination of cohabiting phage elements supports bacteria–phage cooperation

doi: 10.1038/s41467-019-13296-x

Figure Lengend Snippet: MpaR is required for ϕ10403S excision. a Upper panel: schematic representation of the monocin locus. The mon consists of 17 genes comprising: a regulatory module, a bacteriocin module encoding tail-like structures, and a lysis module. Lower panel: transcription analysis of the monocin cluster genes under SOS and intracellular growth (in BMDM cells) conditions, using NanoString technology. Transcription levels are presented as relative counts, relative to the levels observed at the lysogenic state (i.e., exponentially grown bacteria in BHI medium at 37˚C). Data represent three independent experiments. b qRT-PCR analysis of intact comK gene (representing ϕ10403S attB site) in WT Lm and indicated mutants grown intracellularly in BMDM cells (6 h post infection). Presented as relative quantity (RQ), relative to the levels in WT bacteria. The data is a mean of three independent experiments. Error bars indicate a standard deviation. c Intracellular growth analysis of WT Lm , Δ mpaR and Δ mpaR mutant complemented with an intact comK gene under its native promoter (Δ mpaR+ pPL2- comK ). Growth curves represent one biological replicate, more independent experiments are shown in Supplementary Fig. and in the source file. Error bars represent standard deviation of triplicate samples, sometimes are hidden by the symbols. d A bacterial phagosomal escape assay. Percentage of bacteria that escaped the macrophage phagosomes at 2.5 h post infection, as determined by a microscope fluorescence assay. Macrophages were infected with WT Lm , Δ mpaR and Δ mpaR mutant complemented with an intact comK gene on the pPL2 plasmid (Δ mpaR+ pPL2 -comK ). The data is a mean of three independent experiments. The error bar represent standard deviation. Asterisk (*) indicates statistical significance of p < 0.01 calculated using Student's t -test. Source data are provided as a Source Data file.

Article Snippet: BMDM cells used for infection experiments were isolated from 6 to 8 week-old female C57BL/6 mice (Envigo, Israel) and cultured in Dulbecco’s Modified Eagle Medium (DMEM)-based media supplemented with 20% fetal bovine serum, sodium pyruvate (1 mM), L-glutamine (2 mM), β-Mercaptoethanol (0.05 mM) and monocyte-colony stimulating factor (M-CSF, L929-conditioned medium); BMDM medium .

Techniques: Lysis, Bacteria, Quantitative RT-PCR, Infection, Standard Deviation, Mutagenesis, Microscopy, Fluorescence, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: A Novel Mechanism of Macrophage Activation by the Natural Yolkin Polypeptide Complex from Egg Yolk

doi: 10.3390/ijms23063125

Figure Lengend Snippet: Effect of yolkin on viability and proliferation of bone marrow-derived macrophages (BMDM). The cells (1 × 10 5 /mL) were seeded in 96-well plates in DMEM + 10% FBS and incubated overnight at 37 °C. Next, the cells were exposed to yolkin (10, 100 and 150 µg/mL) for 24 and 48 h. The cell proliferation activity of yolkin was assessed with an MTT assay. Non-stimulated cells were used as a negative control. The results represent three to five independent experiments and data are presented as median ± min–max. A one-sample t -test was used to examine the mean differences between samples and control (100). * p ≤ 0.05, *** p ≤ 0.0001.

Article Snippet: The murine bone marrow-derived macrophages of the BMDM cell line (Bei Resources) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, antibiotics (penicillin, streptomycin and gentamycin) and 3% L-glutamine.

Techniques: Derivative Assay, Incubation, Activity Assay, MTT Assay, Negative Control, Control