GCB Research Data for Jiang et al

Original data for the figures</p

Tetracycline Inhibits Local Inflammation Induced by Cerebral Ischemia via Modulating Autophagy

<div><h3>Background</h3><p>Tetracycline exerts neuroprotection via suppressing the local inflammation induced by cerebral ischemia. However, the underlying mechanism is not completely clear.</p> <h3>Methodology/Principal Findings</h3><p>The mRNA and protein expressions of tumor necrosis factor α and interleukin 6 and the number of activated microglia were measured to detect the inflammatory process in the ischemic hemisphere. The key proteins of nuclear factor kappa B pathway and the binding activity of nuclear factor kappa B were also measured. Two key components of autophagy, Beclin 1 and LC3, were detected by western blotting. Pretreatment with tetracycline inhibited the mRNA and protein expressions of tumor necrosis factor α and interleukin 6 and decreased the numbers of activated and phagocytotic microglia. Tetracycline down regulated the total and phosphorylated expressions of IKK, IκB and p65 (<em>P</em><0.05). The autophagy inhibitor, 3-methyladenine, inhibited inflammation and activation of nuclear factor kappa B pathway. The levels of Beclin 1 and LC3 were decreased by 3-methyladenine and tetracycline.</p> <h3>Conclusions/Significance</h3><p>Our data suggested that pretreatment of tetracycline may inhibit autophagy in the ischemic stroke brain and then suppress the inflammatory process via inhibiting the activation of nuclear factor kappa B pathway.</p> </div

Table_1_Detection of differentially expressed genes in spatial transcriptomics data by spatial analysis of spatial transcriptomics: A novel method based on spatial statistics.XLSX

BackgroundSpatial transcriptomics (STs) simultaneously obtains the location and amount of gene expression within a tissue section. However, current methods like FindMarkers calculated the differentially expressed genes (DEGs) based on the classical statistics, which should abolish the spatial information.Materials and methodsA new method named spatial analysis of spatial transcriptomics (saSpatial) was developed for both the location and the amount of gene expression. Then saSpatial was applied to detect DEGs in both inter- and intra-cross sections. DEGs detected by saSpatial were compared with those detected by FindMarkers.ResultsSpatial analysis of spatial transcriptomics was founded on the basis of spatial statistics. It was able to detect DEGs in different regions in the normal brain section. As for the brain with ischemic stroke, saSpatial revealed the DEGs for the ischemic core and penumbra. In addition, saSpatial characterized the genetic heterogeneity in the normal and ischemic cortex. Compared to FindMarkers, a larger number of valuable DEGs were found by saSpatial.ConclusionSpatial analysis of spatial transcriptomics was able to effectively detect DEGs in STs data. It was a simple and valuable tool that could help potential researchers to find more valuable genes in the future research.</p

Data_Sheet_1_Detection of differentially expressed genes in spatial transcriptomics data by spatial analysis of spatial transcriptomics: A novel method based on spatial statistics.ZIP

BackgroundSpatial transcriptomics (STs) simultaneously obtains the location and amount of gene expression within a tissue section. However, current methods like FindMarkers calculated the differentially expressed genes (DEGs) based on the classical statistics, which should abolish the spatial information.Materials and methodsA new method named spatial analysis of spatial transcriptomics (saSpatial) was developed for both the location and the amount of gene expression. Then saSpatial was applied to detect DEGs in both inter- and intra-cross sections. DEGs detected by saSpatial were compared with those detected by FindMarkers.ResultsSpatial analysis of spatial transcriptomics was founded on the basis of spatial statistics. It was able to detect DEGs in different regions in the normal brain section. As for the brain with ischemic stroke, saSpatial revealed the DEGs for the ischemic core and penumbra. In addition, saSpatial characterized the genetic heterogeneity in the normal and ischemic cortex. Compared to FindMarkers, a larger number of valuable DEGs were found by saSpatial.ConclusionSpatial analysis of spatial transcriptomics was able to effectively detect DEGs in STs data. It was a simple and valuable tool that could help potential researchers to find more valuable genes in the future research.</p

Tetracycline down-regulating the expressions of mRNA and protein of cytokines.

<p>The mRNA and protein were detected by ELISA and real time PCR respectively. (A) Both tetracycline and 3-MA significantly decreased the levels of TNF-α. (B) IL6 was significantly decreased by tetracycline and 3-MA. (C) Tetracycline down-regulated mRNA expressions of TNF-α. (B) The mRNA level of IL6 in the brain was inhibited by tetracycline and 3-MA. Bars represent mean±SD (n = 6); *<i>P</i><0.05 vs. control group.</p

Suppressed activation of microglia by tetracycline.

<p>Activated microglia marked by OX-42 (A–D) and ED1 (E–H) were detected by immunofluorescence. Tetracycline decreased the number of activated microglia (OX-42 positive) in the brain (<i>P</i><0.05). The number of ED1 positive microglia was also decreased (288±20 vs. 692±50/mm²). Scale bars: 25 µm. *<i>P</i><0.05 versus control group.</p

Tetracycline inhibiting the protein levels of LC3 and Beclin 1.

<p>(A) LC3 and Beclin 1 in brain tissues detected by western blotting using β-actin as loading control. (B) Relative density of LC3 II/LC3 I and Beclin 1. (C) LC3 detected by immunofluorescence. Scale bar = 5 µm. Bars represent mean±SD (n = 6); *<i>P</i><0.05 vs. control group, #<i>P</i><0.05 vs. 3-MA group.</p

Tetracycline inhibiting the DNA binding activity of NF-κB.

<p>The binding activity of of NF-κB was assessed by Electrophoretic mobility shift assay (EMSA). (A) EMSA autoradiography of NF-κB DNA binding activity. (B) Levels of NF-κB DNA binding activity expressed as arbitrary densitometric units (A.U.) using Image J. Compared to the control group, NF-κB binding activity was significantly decreased in the tetracycline group. Bars represent mean±SD; *<i>P</i><0.05 versus control group.</p

Tetracycline inhibiting the autophagy to suppress the activation of NF-κB pathway.

<p>Ischemia could induce the autophagy which in turns activated the IKK via phosphorylation. The activated IKK phosphorylated the IκBα to liberate NF-κB and allow it to translocate to the nucleus. The phosphorylated NF-κB binds to its binding sites in the target genes to initiate their transcriptions. These proteins played essential roles in inflammatory process.</p

Relationship between NSFC disease-specific funding in 2012 and disability-adjusted life-years (DALYs) in 2012.

<p>Both X and Y axes are submitted to logarithmic scale. The line represents funding predicted on the basis of a linear regression with DALYs.</p