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<t>Cytokine</t> array analysis revealed Pd1-dependent cytokines and chemokines in LPS-treated microglia . A , qPCR showing the decreased Pd1 mRNA in the microglia after Pd1 siRNA (20 nM and 50 nM) treatment for 48 h. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined by one-way ANOVA. A : ANOVA, F (2, 12) = 15.32, p = 0.0005; NC siRNA versus Pd1 siRNA 20 nM, p = 0.0022; NC siRNA versus . Pd1 siRNA 50 nM, p = 0.0004; n = 5 wells per group). B , array membranes of protein expression among NC siRNA, NC siRNA + LPS (5 μg/ml), Pd1 siRNA (50 nM), and Pd1 siRNA (50 nM) + LPS (5 μg/ml) groups in culture medium for microglia. C , the expression of one colony-stimulating factor (G-CSF), one intercellular cell adhesion molecule (CD54), two inflammatory factors (IL-6 and TNF-α), and four chemokines (CXCL10, CCL12, CXCL9, and CCL5) significantly increased following LPS treatment, while the expression of one chemokine (CCL4) decreased. A significant increase in the expression of one inflammatory factor (TNF-α) and one chemokine (CXCL9) after treatment with Pd1 siRNA + LPS. All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by two-way ANOVA. (Figure 6C: two-way ANOVA; G-CSF, NC siRNA versus Pd1 siRNA + LPS, p = 0.0016, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0021; CD54, NC siRNA versus Pd1 siRNA, p = 0.0245, NC siRNA versus Pd1 siRNA + LPS, p = 0.0185; IL-6, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001; Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; CXCL10, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0481; CCL12, NC siRNA versus NC siRNA + LPS, p = 0.0012, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017, NC siRNA + LPS versus Pd1 siRNA, p = 0.0012; Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0017; CXCL9, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0156, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0145; CCL4, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p = 0.0062, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0143; CXCL2, NC siRNA versus NC siRNA + LPS, p = 0.0121, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017; CCL5, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; TNF-α, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0442, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; n = 3–4 separate experiments per group). G-CSF, granulocyte colony-stimulating factor; IL-6, interleukin-6; LPS, lipopolysaccharide; NC, negative control; Pd1, programmed cell death protein 1; qPCR, quantitative real-time PCR; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-alpha.
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Cytokine array analysis revealed Pd1-dependent cytokines and chemokines in LPS-treated microglia . A , qPCR showing the decreased Pd1 mRNA in the microglia after Pd1 siRNA (20 nM and 50 nM) treatment for 48 h. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined by one-way ANOVA. A : ANOVA, F (2, 12) = 15.32, p = 0.0005; NC siRNA versus Pd1 siRNA 20 nM, p = 0.0022; NC siRNA versus . Pd1 siRNA 50 nM, p = 0.0004; n = 5 wells per group). B , array membranes of protein expression among NC siRNA, NC siRNA + LPS (5 μg/ml), Pd1 siRNA (50 nM), and Pd1 siRNA (50 nM) + LPS (5 μg/ml) groups in culture medium for microglia. C , the expression of one colony-stimulating factor (G-CSF), one intercellular cell adhesion molecule (CD54), two inflammatory factors (IL-6 and TNF-α), and four chemokines (CXCL10, CCL12, CXCL9, and CCL5) significantly increased following LPS treatment, while the expression of one chemokine (CCL4) decreased. A significant increase in the expression of one inflammatory factor (TNF-α) and one chemokine (CXCL9) after treatment with Pd1 siRNA + LPS. All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by two-way ANOVA. (Figure 6C: two-way ANOVA; G-CSF, NC siRNA versus Pd1 siRNA + LPS, p = 0.0016, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0021; CD54, NC siRNA versus Pd1 siRNA, p = 0.0245, NC siRNA versus Pd1 siRNA + LPS, p = 0.0185; IL-6, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001; Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; CXCL10, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0481; CCL12, NC siRNA versus NC siRNA + LPS, p = 0.0012, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017, NC siRNA + LPS versus Pd1 siRNA, p = 0.0012; Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0017; CXCL9, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0156, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0145; CCL4, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p = 0.0062, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0143; CXCL2, NC siRNA versus NC siRNA + LPS, p = 0.0121, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017; CCL5, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; TNF-α, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0442, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; n = 3–4 separate experiments per group). G-CSF, granulocyte colony-stimulating factor; IL-6, interleukin-6; LPS, lipopolysaccharide; NC, negative control; Pd1, programmed cell death protein 1; qPCR, quantitative real-time PCR; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-alpha.

Journal: The Journal of Biological Chemistry

Article Title: Effect of microglial Pd1 on glial scar formation after spinal cord injury in mice

doi: 10.1016/j.jbc.2025.108489

Figure Lengend Snippet: Cytokine array analysis revealed Pd1-dependent cytokines and chemokines in LPS-treated microglia . A , qPCR showing the decreased Pd1 mRNA in the microglia after Pd1 siRNA (20 nM and 50 nM) treatment for 48 h. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined by one-way ANOVA. A : ANOVA, F (2, 12) = 15.32, p = 0.0005; NC siRNA versus Pd1 siRNA 20 nM, p = 0.0022; NC siRNA versus . Pd1 siRNA 50 nM, p = 0.0004; n = 5 wells per group). B , array membranes of protein expression among NC siRNA, NC siRNA + LPS (5 μg/ml), Pd1 siRNA (50 nM), and Pd1 siRNA (50 nM) + LPS (5 μg/ml) groups in culture medium for microglia. C , the expression of one colony-stimulating factor (G-CSF), one intercellular cell adhesion molecule (CD54), two inflammatory factors (IL-6 and TNF-α), and four chemokines (CXCL10, CCL12, CXCL9, and CCL5) significantly increased following LPS treatment, while the expression of one chemokine (CCL4) decreased. A significant increase in the expression of one inflammatory factor (TNF-α) and one chemokine (CXCL9) after treatment with Pd1 siRNA + LPS. All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by two-way ANOVA. (Figure 6C: two-way ANOVA; G-CSF, NC siRNA versus Pd1 siRNA + LPS, p = 0.0016, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0021; CD54, NC siRNA versus Pd1 siRNA, p = 0.0245, NC siRNA versus Pd1 siRNA + LPS, p = 0.0185; IL-6, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001; Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; CXCL10, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0481; CCL12, NC siRNA versus NC siRNA + LPS, p = 0.0012, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017, NC siRNA + LPS versus Pd1 siRNA, p = 0.0012; Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0017; CXCL9, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0156, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0145; CCL4, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p = 0.0062, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0143; CXCL2, NC siRNA versus NC siRNA + LPS, p = 0.0121, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017; CCL5, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; TNF-α, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0442, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; n = 3–4 separate experiments per group). G-CSF, granulocyte colony-stimulating factor; IL-6, interleukin-6; LPS, lipopolysaccharide; NC, negative control; Pd1, programmed cell death protein 1; qPCR, quantitative real-time PCR; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-alpha.

Article Snippet: The levels of specific cytokines secreted by microglia were quantified using a mouse cytokine microarray kit (R&D Systems, ARY006).

Techniques: Expressing, Negative Control, Real-time Polymerase Chain Reaction, Small Interfering RNA