MSS1 gene knockdown reduced the expression of MSS1 at protein levels.

<p>In order to assess the efficacy of gene silencing, western blot analysis was performed after transfection with siRNAs targeting MSS1. The specificity of MSS1 gene silencing was determined by comparing with cells transfected with the scrambled RNA duplex. The BV2 cells were transfected with either MSS1-specfic or control siRNAs. At 24 h post-incubation, cell lysates were analyzed for the protein expression of MSS1 using western blot. Compared with the negative control group, the expression of MSS1 was significantly reduced by incubation with MSS1-targeted siRNAs. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p

No significant activation of the IREα-XBP1 or ATF6 pathway by rifampicin.

<p>PC12 cells were incubated with 150 μM rifampicin for indicated lengths of time, ranging from 3 to 24 h. Cells treated with 1 μM Tg served as positive controls. (A) Cell lysates were subjected to western blotting using p-IREα and β-actin antibodies. Rifampicin did not stimulate IREα phosphorylation. (B) Total RNA was subjected to RT-PCR to measure XBP1u/XBP1s expression. Rifampicin did not induce splicing of XBP1 mRNA significantly. (C) Cell lysates were analyzed by western blotting using the ATF6 antibody. Rifampicin did not activate ATF6 cleavage in PC12 cells. Data present mean ± SEM of three independent experiments, with four to six replicates each.</p

Rifampicin-induced neuroprotection was GRP78-dependent.

<p>PC12 cells were transfected with GRP78-specific siRNAs or control siRNAs for 24 h. After that, cells were treated with or without 150 μM rifampicin for 2 h, followed by 1 μM rotenone for 24 h. (A) Western blot analysis verified the efficient gene silencing of GRP78. (B) After the above treatment, cell viability was measured and presented as the relative viability (% control). (C) Morphological evaluation of PC12 cells under the above-mentioned treatment by light microscopic observation and DAPI staining. The apoptotic cells were marked with arrows. Scale bar = 25 μm. (B–C) GRP78 gene silencing significantly exacerbated rotenone-triggered neuron injury, with or without the presence of rifampicin. Neither GRP78-specific nor control siRNAs decreased cell viability. Data present mean ± SEM of three independent experiments. *p<0.05.</p

2D-DIGE gel images of proteins isolated from rifampicin-treated PC12 cells.

<p>(A) Arrows indicate proteins that were differentially expressed in PC12 cells treated with or without rifampicin. (B) Representative peptide mass fingerprint spectra generated by MALDI-TOF-MS. The x-axis indicates the mass-to-charge ratio, m/z. The y-axis indicates the relative abundance. Peptide masses are labeled and the corresponding m/z is annotated.</p

Rifampicin significantly suppressed the expression of MSS1 in LPS-stimulated BV2 microglia.

<p>Cells were treated with the indicated doses of rifampicin for 2 h prior to the addition of LPS (1000 ng/ml). At 24 h post-LPS incubation, cell lysates were analyzed for the protein production of MSS1 using western blot. Rifampicin significantly inhibited the LPS-induced MSS1 expression at protein levels. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with untreated cells and cells treated with LPS in the absence of rifampicin.</p

The PERK-eIF2α-ATF4 pathway regulated rifampicin-induced GRP78 activation.

<p>(A) PC12 cells were treated with 150 μM rifampicin for indicated lengths of time, ranging from 3 to 24 h. Cell lysates were analyzed by western blotting using antibodies against phosphorylated PERK (p-PERK), PERK, phosphorylated eIF2a (p- eIF2a), eIF2a, ATF4 and β-actin. Rifampicin induced a transient PERK phosphorylation at 3 h post-incubation. eIF2a was activated at 3 h post-treatment, which persisted for 9 h and started to decline thereafter. ATF4 was activated at 6 h post-treatment, which persisted up to 24 h post-treatment. (B) PC12 cells were transfected with ATF4-specific siRNAs or control siRNAs for 24 h, followed by rifampicin incubation at 150 μM for 24 h. Western blot analysis showed that ATF4 gene silencing reduced GRP78 protein expression. Data present mean ± SEM of three independent experiments. *p<0.05.</p

Rifampicin induced a time- and dose-dependent GRP78 activation.

<p>(A) Cells were treated with rifampicin at 150 μM for indicated periods of time. Western blot analysis showed that rifampicin significantly upregulated GRP78 protein expression at 6 h post-treatment. Prolonged rifampicin incubation further enhanced GRP78 induction up to 24 h after treatment. (B) Cells were treated with rifampicin at indicated concentrations, followed by western blot analysis to measure GRP78 activation. Rifampicin induced a dose-dependent upregulation of GRP78 expression. Protein expression was relative to control cells, in which GRP78 expression was deemed to be 1. Data present mean ± SEM of three independent experiments. *p<0.05 compared with control groups.</p

Decreased iNOS expression and NO production by MSS1 gene silencing in LPS-activated microglia.

<p>The BV2 cells were transfected with either MSS1-specfic or control siRNA for 24 h, then cells were stimulated for 24 h with LPS (1000 ng/mL). At 24 h post-LPS incubation, cell lysates were analyzed for the protein production of iNOS using western blot. The Griess assay was performed to measure the production of the NO metabolite, nitrite. Transfection with MSS1-specific siRNA suppressed the LPS-induced iNOS expression at protein levels, along with the production of nitrites Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p

Differential MSS1 protein expression identified by MALDI-TOF-MS.

<p>Differential MSS1 protein expression identified by MALDI-TOF-MS.</p

MSS1 gene silencing inhibited microglial NF-kB activation in response to LPS stimulation.

<p>The BV2 cells were transfected with either MSS1-specfic or control siRNA for 24 h, then cells were incubated with LPS at 1000 ng/mL for 8 h. After that, cells were transfected with NF-kB-luciferase reporter plasmid and pCMV-gal control vectors using Lipofectamine reagents. NF-kB activation was detected and expressed as relative luciferase activity. Compared with the negative control group, treatment with MSS1-targetd siRNA significantly suppressed the enhancement of NF-kB activity by LPS. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p