FICZ reduces microglial activation in vitro by inhibiting NF-κB/MAPK signaling. (A) Scatter dot plot showing fluorescence-activated cell sorting (FACS)–based quantification of CD68 expression in the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using unpaired t test. (B) Representative histogram for the data shown in panel A. (C) Scatter dot plot showing FACS-based quantification of P2RY12 expression in the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using unpaired t test. (D) Representative histogram for the data shown in panel C. (E-H) Representative western blots showing the expression of phospho–NF-κB p65 (E) and total NF-κB p65 (G) isolated from the primary microglia that were treated with DMSO or 500 nM FICZ for 48 hours as indicated. Scatter dot plots showing quantification (FC, normalized to vinculin) of phospho–NF-κB p65 (F) and total NF-κB p65 (H) protein isolated from the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (I) Representative IF images depicting phospho–NF-κB p65 (green), CD11b (red), and DAPI (blue) derived from the primary microglia treated with either 1 μg/mL LPS or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. Primary antibodies and goat anti-rabbit IgG (H + L) Alexa Fluor 488 and anti-CD11b Alexa Fluor 647 antibodies were incubated for 1 hour at room temperature. Nuclei were stained using DAPI and high-content screening/image cytometry was performed using Olympus ScanR microscope (UPLSAPO 20×/0.75). Dotted circle, nucleus; scale bar, 100 μm. (J) Scatter dot plot showing quantification of the translocation of phospho–NF-κB p65 in the primary microglia treated with DMSO, 1 μg/mL LPS, or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. Quantification was done using Olympus ScanR analysis software 3.4.1. Cells were segmented using DAPI and CD11b, nuclei were defined by DAPI, and cytoplasm was defined by CD11b. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (K) Representative histograms showing the quantification of nuclear translocation for phospho–NF-κB p65 in the primary microglia treated with DMSO, 1 μg/mL LPS, or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. (L-N) Scatter dot plots showing concentration (picograms per milliliter) of IL-6 (L), MCP-1 (M), and IL-10 (N) from the supernatant (MCM) of the primary microglia that were treated with DMSO, 1 μg/mL LPS, or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. Concentrations were determined by cytometric bead array. Dots represent independent biological replicates. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (O) Representative images indicating pHrodo intake (red) of the primary microglia treated with DMSO (left) or 500 nM FICZ for 48 hours (right) after 0 minute (upper) and 195 minutes (lower) as indicated. Nuclei stained with Hoechst 33342 (blue). High-content screening/image cytometry with live cell imaging was performed using Olympus ScanR microscope (UPLSAPO 20×/0.75). Scale bar, 100 μm. (P) Line diagram showing quantification (FC total intensity) of pHrodo intake of the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Analysis was done using the Olympus ScanR system and software 3.4.1. Cells were detected by an artificial intelligence–based approach.25 Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using nonlinear fit. (Q-R) Scatter dot plots showing quantification of migration distance (Q) and mean migration speed (R) of the primary microglia during pHrodo intake. The cells were treated with DMSO or 500 nM FICZ for 48 hours as indicated. Analysis was done using the Olympus ScanR system and software 3.4.1. Cells were detected by an artificial intelligence–based approach.25 Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using unpaired t test. MCM, microglia-conditioned medium.

FICZ reduces microglial activation in vitro by inhibiting NF-κB/MAPK signaling. (A) Scatter dot plot showing fluorescence-activated cell sorting (FACS)–based quantification of CD68 expression in the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using unpaired t test. (B) Representative histogram for the data shown in panel A. (C) Scatter dot plot showing FACS-based quantification of P2RY12 expression in the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using unpaired t test. (D) Representative histogram for the data shown in panel C. (E-H) Representative western blots showing the expression of phospho–NF-κB p65 (E) and total NF-κB p65 (G) isolated from the primary microglia that were treated with DMSO or 500 nM FICZ for 48 hours as indicated. Scatter dot plots showing quantification (FC, normalized to vinculin) of phospho–NF-κB p65 (F) and total NF-κB p65 (H) protein isolated from the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (I) Representative IF images depicting phospho–NF-κB p65 (green), CD11b (red), and DAPI (blue) derived from the primary microglia treated with either 1 μg/mL LPS or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. Primary antibodies and goat anti-rabbit IgG (H + L) Alexa Fluor 488 and anti-CD11b Alexa Fluor 647 antibodies were incubated for 1 hour at room temperature. Nuclei were stained using DAPI and high-content screening/image cytometry was performed using Olympus ScanR microscope (UPLSAPO 20×/0.75). Dotted circle, nucleus; scale bar, 100 μm. (J) Scatter dot plot showing quantification of the translocation of phospho–NF-κB p65 in the primary microglia treated with DMSO, 1 μg/mL LPS, or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. Quantification was done using Olympus ScanR analysis software 3.4.1. Cells were segmented using DAPI and CD11b, nuclei were defined by DAPI, and cytoplasm was defined by CD11b. Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (K) Representative histograms showing the quantification of nuclear translocation for phospho–NF-κB p65 in the primary microglia treated with DMSO, 1 μg/mL LPS, or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. (L-N) Scatter dot plots showing concentration (picograms per milliliter) of IL-6 (L), MCP-1 (M), and IL-10 (N) from the supernatant (MCM) of the primary microglia that were treated with DMSO, 1 μg/mL LPS, or 500 nM FICZ for 48 hours + 1 μg/mL LPS as indicated. Concentrations were determined by cytometric bead array. Dots represent independent biological replicates. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (O) Representative images indicating pHrodo intake (red) of the primary microglia treated with DMSO (left) or 500 nM FICZ for 48 hours (right) after 0 minute (upper) and 195 minutes (lower) as indicated. Nuclei stained with Hoechst 33342 (blue). High-content screening/image cytometry with live cell imaging was performed using Olympus ScanR microscope (UPLSAPO 20×/0.75). Scale bar, 100 μm. (P) Line diagram showing quantification (FC total intensity) of pHrodo intake of the primary microglia treated with DMSO or 500 nM FICZ for 48 hours as indicated. Analysis was done using the Olympus ScanR system and software 3.4.1. Cells were detected by an artificial intelligence–based approach.25 Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using nonlinear fit. (Q-R) Scatter dot plots showing quantification of migration distance (Q) and mean migration speed (R) of the primary microglia during pHrodo intake. The cells were treated with DMSO or 500 nM FICZ for 48 hours as indicated. Analysis was done using the Olympus ScanR system and software 3.4.1. Cells were detected by an artificial intelligence–based approach.25 Experiment was performed twice and results were pooled. Dots represent independent biological replicates. Error bars showing mean ± SEM. P value was calculated using unpaired t test. MCM, microglia-conditioned medium.

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