Journal: Journal of microbiology and biotechnology

Article Title: The Gut Microbiota of Pregnant Rats Alleviates Fetal Growth Restriction by Inhibiting the TLR9/MyD88 Pathway.

doi: 10.4014/jmb.2304.04020

Figure Lengend Snippet: Fig. 3. TLR9 could exacerbate inflammation by mediating the expression of inflammatory cytokines. (A) The litter size, pregnancy weight and fetal weight were assessed. (B) ELISA was performed to detect the contents of IL-1β, TNF-α, IL-10, LPS and LBP in maternal rats' serum; (C) The level of relative inflammatory markers was measured using ELISA in cord blood; (D) HE staining was employed to assess morphological changes in placental tissue; (E) The abundance of relative proteins via Western blot; (F) The effects of ODN1668 and HCQ on TRAF3, TBK1, IRF3, and NF-κBp65 expression in placental tissue were determined by Western blot; (G) The abundance of PCNA, Ki67, and MMP9 via Western blot; (H) The effects of ODN1668 and HCQ on neutrophils number were assessed by flow cytometry; (I) The correlation between the expression of TLR9 and relative cytokines were analyzed by Pearson's correlation; (J) Relative mRNA levels of TLR9, ZO-1, Occludin, and Claudin-1 were detected by qRT-PCR; (K) Heatmap showing the correlation of TLR9, ZO-1, Occludin and Claudin-1 with LPS. *p < 0.05 compared with the Control group. &p < 0.05 compared with the FGR group. In the heatmaps, *p < 0.05 indicates a significant correlation between variables.

Article Snippet: The protein samples were then separated and transferred onto the nitrocellulose membrane, followed by incubation with their corresponding primary antibodies, including TLR2 (1:1000 dilution, Abcam, ab209217), TLR4 (1:500 dilution, Proteintech, 19811-1-AP), TLR9 (1-5 μg/ml, Abcam, ab134368), MyD88 (1:1000 dilution, Abcam, ab219413), premature IL-1β (1:1000 dilution, Abcam, ab254360), TNF-α (1:5000 dilution, Proteintech, 60291-1-Ig), IL-10 (1:1000 dilution, Abcam, ab33471), PCNA (1:5000 dilution, Proteintech, 10205-2-AP), Ki67 (1:1000 dilution, Abcam, ab16667), MMP9 (1:1000 dilution, Abcam, ab228402), TRAF3 (2 μg/ml, Abcam, ab36988), TBK1 (1: 5000, Abcam, ab40676), IRF3 (1: 1000, Abcam, ab238521), p-IRF3 (1: 5000, Abcam, ab76493), NF-κBp65 (1:1000, Proteintech, 10745-1-AP, USA), p-NF-κBp65 (1:1000, Abcam, ab76302), and β-actin (1:5000 dilution, Proteintech, 66009-1-Ig).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Flow Cytometry, Quantitative RT-PCR, Control

Journal: Journal of microbiology and biotechnology

Article Title: The Gut Microbiota of Pregnant Rats Alleviates Fetal Growth Restriction by Inhibiting the TLR9/MyD88 Pathway.

doi: 10.4014/jmb.2304.04020

Figure Lengend Snippet: Fig. 7. TLR9 regulates the pathogenesis of FGR through the gut microbiota. (A) The litter size, pregnancy weight, and fetal weight were determined. (B) ELISA was performed to examine the contents of IL-1β, TNF-α, IL-10, LPS and LBP in maternal rats' serum; (C) ELISA was applied to measure relative inflammatory marker levels in cord blood; (D) IHC staining was performed to assess the expression of TLR9 in placental tissue; (E) The abundance of relative proteins was shown by Western blot; (F) The effects of ODN1668 and HCQ on TRAF3, TBK1, IRF3, and NF-κBp65 expression in placental tissue were determined by Western blot; (G) The number of CD11b-positive cells was assessed by flow cytometry; (H) The abundance of PCNA, Ki67, and MMP9 was assessed by Western blot. *p <0.05 compared with the FGR group. &p < 0.05 compared with the HCQ group. # p < 0.05 compared with the FMT group.

Article Snippet: The protein samples were then separated and transferred onto the nitrocellulose membrane, followed by incubation with their corresponding primary antibodies, including TLR2 (1:1000 dilution, Abcam, ab209217), TLR4 (1:500 dilution, Proteintech, 19811-1-AP), TLR9 (1-5 μg/ml, Abcam, ab134368), MyD88 (1:1000 dilution, Abcam, ab219413), premature IL-1β (1:1000 dilution, Abcam, ab254360), TNF-α (1:5000 dilution, Proteintech, 60291-1-Ig), IL-10 (1:1000 dilution, Abcam, ab33471), PCNA (1:5000 dilution, Proteintech, 10205-2-AP), Ki67 (1:1000 dilution, Abcam, ab16667), MMP9 (1:1000 dilution, Abcam, ab228402), TRAF3 (2 μg/ml, Abcam, ab36988), TBK1 (1: 5000, Abcam, ab40676), IRF3 (1: 1000, Abcam, ab238521), p-IRF3 (1: 5000, Abcam, ab76493), NF-κBp65 (1:1000, Proteintech, 10745-1-AP, USA), p-NF-κBp65 (1:1000, Abcam, ab76302), and β-actin (1:5000 dilution, Proteintech, 66009-1-Ig).

Techniques: Enzyme-linked Immunosorbent Assay, Marker, Immunohistochemistry, Expressing, Western Blot, Flow Cytometry

Journal: JOR spine

Article Title: Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix.

doi: 10.1002/jsp2.1286

Figure Lengend Snippet: FIGURE 1 Effect of bafilomycin-mediated autophagy flux inhibition in NP cells in vitro. (A) Experimental design for the bafilomycin treatment in rat NP cell culture. Rat NP cells were treated with Bafilomycin A1 in high nutrients (HN) and low nutrients (LN) media for indicated time and analyzed for different measures. (B) Expression of two common autophagy markers LC3-II and SQSTM1/p62 as determined by western blot following baf A1 treatment for 8–48 h in HN and LN media. (C) Cellular morphology of NP cells in LN media with baf (10 nM, 8–48 h) treatment was visualized using light microscopy. Cellular metabolism of NP cells in LN media with 10 nM baf treatment (8–48 h) was quantified using CCK-8 assay. (D) Representative images of live NP cells (stained green) and dead NP cells (stained red) following incubation in LN media +/ baf (8–48 h) treatment. The ratio of live (green)/ dead (red) cells in LN media +/ 10 nM baf treatment was quantified using image J. Data shown are mean ± SEM of 3 technical replicates. Two-tailed Student's t test was used to quantify significance. *p < 0.05 baf A1, bafilomycin A1.

Article Snippet: Primary antibodies to LC3 (12741, cell signaling technology), ATG7 (8558, cell signaling technology), ATG12-ATG5 (4180, cell signaling technology), SQSTM1/p62 (5114, Cell Signaling Technology), TNF-α (3707, cell signaling technology), IL-1β (12 242, cell signaling technology), p53 (2524, cell signaling technology), GAPDH (2118, cell signaling technology) and β-actin (A2066, Millipore Sigma), and secondary anti-rabbit HRP antibody (31460, Thermo Fisher) were used.

Techniques: Inhibition, In Vitro, Cell Culture, Expressing, Western Blot, Light Microscopy, CCK-8 Assay, Staining, Incubation, Two Tailed Test

Journal: JOR spine

Article Title: Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix.

doi: 10.1002/jsp2.1286

Figure Lengend Snippet: FIGURE 5 Successful inhibition of autophagy in NP tissue. Autophagy was inhibited primarily in NP tissue of Col2-Cre; Atg7fl/fl mice. (A) Expression of ATG7, p62, LC3-II, and ATG12-ATG5 protein in AF and NP tissue from Atg7fl/fl control and Col2-Cre; Atg7fl/fl experimental mice at 3, 6, and 12 months (mo) of age was determined by Western blotting. β-actin was used as an internal loading control. (B) Densitometric analysis of the proteins in AF and NP tissue were quantified by dividing the amount of each protein by β-actin and normalizing to control protein amount for each age group. Data shown are mean ± SD of 3 independent mice per tissue per age group. Two-tailed unpaired t test was used to quantify significance and p-values are shown in the graphs. fl/fl, Atg7fl/fl, KO or Atg7 KO, Col2-Cre; Atg7fl/fl.

Article Snippet: Primary antibodies to LC3 (12741, cell signaling technology), ATG7 (8558, cell signaling technology), ATG12-ATG5 (4180, cell signaling technology), SQSTM1/p62 (5114, Cell Signaling Technology), TNF-α (3707, cell signaling technology), IL-1β (12 242, cell signaling technology), p53 (2524, cell signaling technology), GAPDH (2118, cell signaling technology) and β-actin (A2066, Millipore Sigma), and secondary anti-rabbit HRP antibody (31460, Thermo Fisher) were used.

Techniques: Inhibition, Expressing, Control, Western Blot, Two Tailed Test

Journal:

Article Title: Critical roles of PPAR?/? in keratinocyte response to inflammation

doi: 10.1101/gad.207501

Figure Lengend Snippet: External signals stimulate PPARβ expression and cell differentiation in primary mouse keratinocyte cultures. (A) Apoptosis-derived CM and necrosis-derived CM induce keratinocyte differentiation. Primary keratinocytes from wild-type (first, third, and fifth panels) or from PPARβ−/− mice (second and fourth panels) were exposed at time 0 (usually after two or three passages) to conditioned medium (CM) from mixed leukocyte reactions (MLR), prepared either with apoptotic fibroblasts (first and second panels) or with necrotic cells (third, fourth, and fifth panels). Keratinocytes were lysed after different times of exposure to CM as indicated, and the expression of PPARβ was evaluated by RPA. The expression of involucrin (INV), transglutaminase I (TgaseI), was used as markers of keratinocyte differentiation. The expression of keratin (K) K6, K10, and K17 reflects different pathways of keratinocyte differentiation. Cyclin A (Cyc A) expression indicated the status of the cell with respect to cell cycle. In the fifth panel, the necrosis-derived CM was precleared of TNF-α. (B) The major pro-inflammatory mediators TNF-α, IFN-γ, and TPA up-regulate PPARβ expression. Primary keratinocytes from wild-type (first, third, and fifth panels) or from PPARβ−/− mice (second, fourth, and sixth panels) were cultured in KSFM and exposed at time 0 to TNF-α 5 ng/ml (first and second panels) IFN-γ 5 ng/ml (third and fourth panels). or TPA 20 ng/ml (fifth and sixth panels). In all three treatments, the expression of all differentiation markers is strongly reduced and delayed in PPARβ−/− cells.

Article Snippet: Anti-mouse-TNF-α antibodies were from R & D Systems.

Techniques: Expressing, Cell Differentiation, Derivative Assay, Cell Culture

Journal:

Article Title: Critical roles of PPAR?/? in keratinocyte response to inflammation

doi: 10.1101/gad.207501

Figure Lengend Snippet: Identification of the region in the PPARβ promoter that mediates the TNF-α, TPA, and ceramide responses. (A) PPARβ promoter 5′-deletion mutants. Plasmids are named according to the length of the promoter region they contain upstream from the transcription start site. Keratinocytes in culture were cotransfected with a CAT reporter driven by the various PPARβ promoter constructs and pCMV-β-galactosidase as a control of transfection efficiency. Keratinocyte cultures were treated for 24 h with the inducers indicated. Results were normalized to β-galactosidase activity and are presented as relative CAT activity compared with that of the promoterless vector pBLCAT alone. (B) TNFα-, TPA-, and Cer-signaling target an AP-1(−414) of the PPARβ promoter. The hatched bar depicts site-directed mutations of the transcription factor binding sites. The mutations introduced into the site are indicated as underlined nucleotides above the respective hatched bar. The numbers associated with the sites indicate their position relative to the transcription start site. Data are means of six independent experiments. Vehicles for TPA, TNF-α, and Cer were ethanol, PBS, and DMSO, respectively.

Article Snippet: Anti-mouse-TNF-α antibodies were from R & D Systems.

Techniques: Construct, Transfection, Activity Assay, Plasmid Preparation, Binding Assay

Journal:

Article Title: Critical roles of PPAR?/? in keratinocyte response to inflammation

doi: 10.1101/gad.207501

Figure Lengend Snippet: Signaling cascades that mediate the effects of TNF-α, TPA, and Cer on the PPARβ gene in keratinocytes. (A) (Overall scheme) The solid arrows represent the major TNFα-activated, FAN-mediated, pathway regulating PPARβ expression. The cascade invoked by TPA is depicted by the dashed arrows. (B) TNF-α induces PPARβ gene expression through FAN and Cer. Keratinocytes were cotransfected with expression vectors for wild-type FAN (wtFAN, second panel) or a dominant-negative form of FAN (dnFAN, third and fourth panels) and pCMV-β-galactosidase as internal control. Transfected keratinocytes were treated with TNF-α and RPA were performed at the indicated times. (fourth panel) Suppression of PPARβ expression by dnFAN was rescued by addition of exogenous ceramide at 12 h post-transfection. (first panel) Control experiment with TNF-α alone; (fifth panel) effect of ceramide in absence of TNF-α treatment. (C) Regulation of PPARβ via the stress-associated kinase pathway. Keratinocytes were transfected with a CAT reporter driven by the pPPARβ(−445) promoter region together with expression vectors encoding dominant negative (dn) or constitutively active (ca) kinases. The empty vector pCDNA 3.1 was used as a control (vector). Transfected keratinocytes were treated with an inducer (TNF-α, ceramide, or TPA) and CAT activity was measured 24 h post-treatment. SB203580 (10 μM) is a specific inhibitor of p38 and was added 1 h prior to treatment with the inducers. Values are means of six independent experiments.

Article Snippet: Anti-mouse-TNF-α antibodies were from R & D Systems.

Techniques: Expressing, Dominant Negative Mutation, Transfection, Plasmid Preparation, Activity Assay

Journal:

Article Title: Critical roles of PPAR?/? in keratinocyte response to inflammation

doi: 10.1101/gad.207501

Figure Lengend Snippet: PPARβ accelerates keratinocyte differentiation. (A) Overexpression and activation of PPARβ stimulates keratinocyte differentiation. Keratinocytes were transfected with a wtPPARβ expression vector and lysed for RPA at various time points post-transfection, as indicated. Overexpression of PPARβ alone is not sufficient to trigger keratinocyte differentiation (first panel), which requires a 6-h exposure to a PPARβ-specific ligand L165041 (LD) (second panel). Better results were obtained with a repeated exposure to LD [6 h, followed by an additional 18 h with fresh medium containing the second dose (third panel)]. This latter dose regime was also able to slightly induce the expression of differentiation markers in absence of ectopical expression of PPARβ (fourth panel). (B) Activation of the PPARβ LBD by cytokines. Keratinocytes were cotransfected with an expression vector for a GAL4 DBD–PPARβ LBD, the Gal4 responsive reporter vector pG5CAT (Clontech), and the pCMV-β-galactosidase vector. CAT and β-galactosidase activities were measured after 24-h exposure to two different concentrations (open and solid bars) of PPARβ selective ligand L165041 (LD; 1, 5 μM), IFN-γ (10, 25 ng/ml), TNF-α (10, 25 ng/ml), TGF-β (10, 25 ng/ml), TPA (10, 20 ng/ml), Ceramide (Cer; 10, 20 μM), or arachidonic acid (AA; 10, 20 μM). Exposure to IFN-γ, TNF-α, TPA, and Cer resulted in a dose-dependent increase of the CAT reporter activity. (C) Total lipid extract of cytokine-activated keratinocytes activates PPARβ. Total lipid extract were prepared from keratinocytes untreated (control), or exposed to vehicle or to various pro-inflammatory mediators, as indicated. (Left) Transfections were performed as in A. Cells were then exposed to 1 or 2 μL of a total lipid extract for 24 h and CAT activity was measured. (Right) Keratinocytes were transfected with the wtPPARβ expression vector, pGL-3xPPRE-tk-luc reporter gene, and pCMV-β-galactosidase. Normalized reporter activity is shown as fold increase as compared with control.

Article Snippet: Anti-mouse-TNF-α antibodies were from R & D Systems.

Techniques: Over Expression, Activation Assay, Transfection, Expressing, Plasmid Preparation, Activity Assay

Journal:

Article Title: Critical roles of PPAR?/? in keratinocyte response to inflammation

doi: 10.1101/gad.207501

Figure Lengend Snippet: Inflammation-induced PPARβ expression protects keratinocytes from apoptosis. (A) Elevated PPARβ expression inhibits TNFα-induced apoptosis. Keratinocytes prepared from PPARβ+/+,PPARβ+/−, and PPARβ−/− mice were either transfected with expression vectors harboring the wtPPARβ and/or cultured in the absence or presence of TNF-α and L165041 (LD, 5 μM) as indicated. The level of TNFα-induced apoptosis was monitored by caspase 8 activity. Keratinocytes expressing wtPPARβ were more resistant to apoptosis signals, whereas PPARβ+/− cells showed an increase susceptibility. PPARβ−/− keratinocytes exhibited higher basal caspase 8 activity and were more sensitive to TNFα-induced apoptosis, but could be rescued by transfection with the vector expressing wtPPARβ. Values are means of three independent experiments. (B) PPARβ−/− keratinocytes are more susceptible to TNFα-induced apoptosis. Culture of primary keratinocytes from wild-type and from PPARβ−/− mice were exposed to TNF-α. Apoptosis was measured by radioactive DNA fragmentation assay over indicated periods of time. Values are means of three independent experiments.

Article Snippet: Anti-mouse-TNF-α antibodies were from R & D Systems.

Techniques: Expressing, Transfection, Cell Culture, Activity Assay, Plasmid Preparation, DNA Fragmentation Assay