Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Reprogramming Macrophage Polarization, Depleting ROS by Astaxanthin and Thioketal-Containing Polymers Delivering Rapamycin for Osteoarthritis Treatment.

doi: 10.1002/advs.202305363

Figure Lengend Snippet: Fig. 3 Altruistic cancer cells regenerate via epigenetic mechanism. A Mathematical model explains the persistence of altruistic subclone in breast cancer cell population (Upper), and a spatial model simulates changes in the percentage of altruistic cells over time for hypothesized genetic- and epigenetics-mediated altruism (Lower). Dotted lines link events in upper panel to corresponding points in lower panel. See Supplementary Note 2 & 3. B Indicated cancer cell lines were sorted according to Mi/EGFP levels and the fluorescence monitored over indicated time. C Mi/EGFP fluorescence of non-sorted MDA-MB-231miR-125bprom-EGFP cells, treated as indicated, as determined by FACS. D Mi/EGFPLow cells were transfected with indicated siRNAs and the Mi/EGFP fluorescence determined after four days. E Schema showing putative consensus sites for KLF2 binding along the hsa-miR-125b-1 promoter and gRNA targeting sequence of CRISPRi. F Mi/EGFPLow cells were transduced with lentivirus for expression of CRISPRi targeting KBS-1 or non-specific sequence, and Mi/EGFP fluorescence determined after four days. G,H Changes in Mi/ EGFP fluorescence as treated in (F) were monitored in indicated cell lines (G). Percentage effect of KBS-1 CRISPRi expression on cell viability relative to control CRISPRi was also measured. Cancer cells were exposed to indicated concentration of docetaxel (H). I MDA-MB-231miR-125bprom-EGFP cells, as treated in (F), were grown as xenografts in NSG mice. Upper: excised tumors at end point of monitoring period. Lower: Percentage change in tumor size with and without docetaxel treatment. J Immunoblotting to detect for IGFBP2 and CCL28 in conditioned media from MDA-MB-231miR-125bprom-EGFP cells as treated in (F) and used to establish xenograft model for (I). Ponceau S was used to visualize protein load. Quantification of band intensities (relative to Control CRISPRi) is shown. Experiments repeated two times (B, C, D, F, G, H, J). Representative data are shown for (J). Mean percentage ± s.d. cells for technical triplicates of representative set are shown in same colours as corresponding histograms (C, D, F, G) or in black only (B). Data are mean ± s.d. from two independent biological sets of triplicates (H). n = 4 independent animals per group (I). Statistical analysis was performed using two-tailed one sample t-test against 0 (H) and two-tailed unpaired t-test (I lower). NT: no treatment; DTX: docetaxel treatment. Exact P values are shown

Article Snippet: CD45 (MCA87A647; Biorad), EpCAM (324206; BioLegend), digoxigenin (DIG) (11093274910; Roche), GFP (2555; Cell Signaling Technology), polyclonal IGFBP2 (3922; Cell Signaling Technology), monoclonal IGFBP2 (ab109284; Abcam), monoclonal IGFBP2 (MAB6741-SP; R&D Systems), monoclonal CCL28 (ab192600; Abcam), polyclonal CCL28 (ab196567; Abcam), monoclonal CCL28 (MAB717-SP; R&D Systems), monoclonal CCL28 (sc-376654, Santa Cruz Biotechnology), Bak1 (12105; Cell Signaling Technology), beta actin (sc-47778; Santa Cruz), H3ac (61637; Active Motif ), GAPDH (sc-137179; Santa Cruz), histone H4ac (39243; Active Motif ), PCAF (3378; Cell Signaling Technology), KLF2 (sc-28675X; Santa Cruz), Cas9 (61757; Active Motif ), ChIP negative control IgG (53026; Active Motif ), luciferase (NB600307; Novus Biologicals), IGF-1R (AF-305-NA; R&D Systems), integrin α5 (AF1864; R&D Systems), integrin β1 (AF-1778-SP; R&D Systems), integrin αV (ab94704; Abcam), integrin β3 (AF-2266-SP; R&D Systems), integrin α2b (ab63983; abcam), CCR10 (MAB3478; R&D Systems), CCR3 (PAB13065; Abnova), CD24 (311118; BioLegend), CCNA2 (4656, Cell Signaling Technology).

Techniques: Fluorescence, Transfection, Binding Assay, Sequencing, Transduction, Expressing, Control, Concentration Assay, Western Blot, Two Tailed Test