Journal: Molecular Vision

Article Title: Osmotic induction of cyclooxygenase-2 in RPE cells: Stimulation of inflammasome activation

doi:

Figure Lengend Snippet: Transcription factor activity involved in mediating NaCl-induced expression of cyclooxygenase-2 ( COX2 ) in retinal pigment epithelial (RPE) cells. The mRNA level was determined with real-time reverse transcription (RT)–PCR in cells cultured 6 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and is expressed as folds of the unstimulated control. A: The following compounds were tested: a hypoxia-inducible transcription factor (HIF)-1 inhibitor (HIF-Inh; 5 µM), the signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic (1 µM), the nuclear factor (NF)-κB inhibitor CAPE (5 µM), the activator protein-1 (AP-1) inhibitor SR11302 (5 µM), and the cAMP response element-binding protein (CREB) inhibitor 666–15 (250 nM). B: Knocking down nuclear factor of activated T cell 5 (NFAT5) with small interfering RNA (siRNA) reduced the level of COX2 mRNA under hyperosmotic conditions. Nontargeted siRNA (siNon) had no effects. Each bar represents data obtained in three to nine independent experiments with cell lines from different donors. Each experiment was performed with cell lines from three to six donors; in total, cell lines from 21 different donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ●p<0.05. Statistically significant difference versus nontargeted siRNA: ○p<0.05.

Article Snippet: The compounds 666–15, amiloride, caffeic acid phenethyl ester (CAPE), GSK650394, NS-398, SB203580, and SR11302 were purchased from Tocris (Ellisville, MO).

Techniques: Activity Assay, Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Control, Binding Assay, Small Interfering RNA

Journal: Nature Communications

Article Title: Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia

doi: 10.1038/s41467-021-27455-6

Figure Lengend Snippet: a Decorin (800 pg/ml) attenuated activated TCM (act-Tm)-mediated microglia Ccl5 production ( n = 3). Two-tailed Student’s t test. Exact P values are indicated within each panel (ns, not significant). b Decorin reduces microglia Ccl5 production in response to Ccl4 exposure (6 ng/ml) over a physiologic dose range in vitro ( n = 4). One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. c TCM (activated T cell-conditioned media) induced microglial Ccl5 production was significantly attenuated by decorin. The addition of Dcn with CCR5 has the greatest inhibition similar to the combination of CCR5 and CCR8 inhibitors ( n = 6). One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. d Schematic representation of decorin binding to the CCR8 receptor on microglia, culminating is reduced microglial Ccl5 production by inhibiting NFκB activation. e Schematic representation of decorin treatment of Nf1 OPG mice. Nf1 OPG mice were treated between 4 and 6 weeks of age with decorin, while control Nf1 OPG mice received PBS only. Isolated optic nerves were analyzed at 12 weeks of age. f Decorin treatment has no change on optic nerve volume, but ( g ) decreased proliferation (%Ki67 + cells) of Nf1 OPG mice relative to the vehicle-treated Nf1 OPG controls (PBS, n = 8, Decorin, n = 8). No difference in microglia (%Iba1 + cells) or T cell (CD3 + cells) content was observed between decorin-treated mice and their respective controls. Two-tailed Student’s t test (ns, not significant). g Scale bars, 40 µm. From left to right in each panel: a P = 0.0134, b P < 0.0001, P = 0.0035; c P < 0.0001, ns, P = 0.0086, ns; f ns; g P = 0.0010, ns, ns.

Article Snippet: Four-week-old Nf1 OPG mice were treated with 10 mg/kg NFκB inhibitor (NFκB-IN; Caffeic acid phenethyl ester, Fisher Scientific, 274310) or 1 mg/ml Decorin (R&D System 1060-DE-100) intraperitoneally every other day for 2 weeks between 4 and 6 weeks of age.

Techniques: Two Tailed Test, In Vitro, Inhibition, Binding Assay, Activation Assay, Control, Isolation

Journal: Nature Communications

Article Title: Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia

doi: 10.1038/s41467-021-27455-6

Figure Lengend Snippet: a Nf1 OPG mouse optic nerves ( n = 3) have increased Iκbα phosphorylation relative to WT mice ( n = 3). b The increased Iκbα phosphorylation in PBS-treated Nf1 OPG mouse optic nerves ( n = 3) was reduced by OVA treatment ( n = 3). c Activated TCM (act-Tm) increased Iκbα phosphorylation in microglia, which was reduced following the addition of decorin (800 pg/ml). d Decorin (800 pg/ml) blocks the increased p65-NFκB Ser 536 phosphorylation induced by activated TCM (act-Tm) treatment ( n = 3 ). e Decorin (800 pg/ml) blocks the increased total p65-NFκB expression, ( f ) as well as the nuclear localization of p65-NFκB, induced by activated TCM (act-Tm) treatment ( n = 3). β-actin and HDAC are used as controls for total protein expression and the nuclear fractions, respectively. g Schematic representation of the NFκB inhibitor treatment used. Nf1 OPG mice were treated between 4 and 6 weeks of age with the CAPE NFκB inhibitor ( n = 8), whereas control Nf1 OPG mice received PBS only ( n = 8). Isolated optic nerves were analyzed at 12 weeks of age. h NFκB inhibitor treatment reduced optic glioma volume and ( i ) proliferation (%Ki67 + cells), as well as microglia (%Iba1 + cells) and T-cell (CD3 + ) content within the optic nerves of Nf1 OPG mice relative to vehicle-treated controls. Two-tailed Student’s t test. j Reduced Ccl5 RNA expression was observed in the optic nerves from Nf1 OPG mice treated with the CAPE NFκB inhibitor (NFκB-IN) ( n = 5). Two-tailed Student’s t test. k Proposed model of asthma-induced decorin suppression of the Nf1 OPG neuron-immune-cancer cell axis. Asthma induces T cell production of decorin, which reduces T cell Ccl4-mediated microglia Ccl5 expression through inhibition of NFκB signaling. Data are presented as the means ± SEM. Exact P values are indicated within each panel. i Scale bars 40 µm. From left to right in each panel: h P = 0.0288; i P = 0.0003, P = 0.0196, P = 0.0025; j P = 0.0018.

Article Snippet: Four-week-old Nf1 OPG mice were treated with 10 mg/kg NFκB inhibitor (NFκB-IN; Caffeic acid phenethyl ester, Fisher Scientific, 274310) or 1 mg/ml Decorin (R&D System 1060-DE-100) intraperitoneally every other day for 2 weeks between 4 and 6 weeks of age.

Techniques: Phospho-proteomics, Expressing, Control, Isolation, Two Tailed Test, RNA Expression, Inhibition