Journal: Molecular Medicine Reports

Article Title: Expression and function of Toll-like receptors in peripheral blood mononuclear cells in patients with ankylosing spondylitis

doi: 10.3892/mmr.2019.10631

Figure Lengend Snippet: TNF-α mediates TLR expression and NF-κB p65 signaling in PBMCs. (A) Western blotting was performed to detect the protein levels of TLR3, TLR4, TLR5, p65 and p-p65. Protein expression of (B) TLR3, (C) TLR4, (D) TLR5 and (E) p-p65/p65 presented as bar diagrams. β-actin was used as an internal control. *P<0.05, **P<0.01 vs. control. NF-κB, nuclear factor-κB; p-, phosphorylated; PBMC, peripheral blood mononuclear cell; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α.

Article Snippet: The membranes were blocked in 5% dried skimmed milk in TBS buffer at 37°C for 1 h and incubated overnight at 4°C with antibodies against TLR3 (1:700; ab62566, Abcam), TLR4 (1:600; ab13556, Abcam), TLR5 (1:800; ab62460, Abcam), p65 (1:1,000; ab32536, Abcam), phosphorylated (p)-p65 (1:600; ab86299, Abcam) and β-actin (1:1,000; MAB8969, R&D Systems, Inc.).

Techniques: Expressing, Western Blot, Control

Journal: Virology

Article Title: SNAP23 is essential for germination of EV-D68 replication organelles.

doi: 10.1016/j.virol.2022.11.011

Figure Lengend Snippet: Fig. 2. SNAP23 expression is not altered by EV-D68 infection but SNAP23 knockdown inhibits VP3 expression. (A) Cells were infected with EV-D68 over a course of 5 h (MOI = 25). Samples were collected at each indicated time point for Western blot analysis. (B) Cells were transfected with non-targeting or SNAP23 siRNAs for 48 h and then infected with EV-D68 (MOI = 10) for 4 h. Cell lysates were collected at the end of the infection and used for Western blot analysis. Subsets of non-targeting and siSNAP23 transfected cells in B were also treated with 2 mM guanidine hydrochloride (GuHCl) as a control to inhibit viral replication. (C) SNAP23 bands from three indepen dent experiments, completed for (A), were semi- quantified and normalized to beta-actin. (D) Bands from full length SQSTM1, in (B), were semi- quantified from three independent experiments and normalized to beta-actin. (E) VP3 bands were semi- quantified from three independent experiments and independent from the blots, in (B), probed against SQSTM1 due to the cleavage product from SQSTM1 is close in size to VP3. n = 3 independent experiments. Unless noted by an asterisk, results were otherwise not statistically significant. * = p-value <0.05 and error bars represent the mean 土SEM.

Article Snippet: Primary antibodies used include: SNAP23 ([EPR8538], ab131242, Abcam), LC3 (NB600-1384, Novus), SQSTM1 (H00008878-M01, Abnova), Beta Actin (NB600-532, Novus), and Enterovirus pan Monoclonal Antibody to detect VP3 (MA5-18206, Invitrogen).

Techniques: Expressing, Infection, Knockdown, Western Blot, Transfection, Control

Journal: Scientific Reports

Article Title: Histological analysis of sleep and circadian brain circuitry in cranial radiation-induced hypersomnolence (C-RIH) mouse model

doi: 10.1038/s41598-022-15074-0

Figure Lengend Snippet: In vitro radiosensitivity of astrocytic cell lines based on region of isolation and treatment timing. ( A ) Photomicrographs of SCN2.2 and CTXTNA2 cells, two astrocytic cell lines with similar morphology. B. Quantification of trypan blue postitive cells to determine cell viability 1, 6 and 24 h after 8 Gy radiation, there were significant main effects for time (F(1,18) = 96.834, p < 0.001), cell type (F(1,18) = 235.937, p < 0.001), and an interaction between time and cell type (F(2,17) = 151.927, < 0.001). Posthoc tests (tukey) were significant if p < 0.05. SCN2.2 cells (green bars) survived at significantly higher level 1 ( p < 0.001) and 6 h ( p = 0.024) after radiation when compared to the cortical astrocytes (purple bars). ( C ) Clonogenic assay to quantify survival fractions from 0 to 8 Gy in SCN and cortical cells. Again SCN2.2 cells (green line) had better overall survival after radiation when compared to CTXTNA2 cells (purple line). There were significant main effects of both cell line (F(1,24) = 33.228, p < 0.001) and radiation dose (F(5,24) = 402.558, p < 0.001), and an interaction between cell line and dose (F(5,24) = 6.172, p = 0.001). The dotted black line across the graph shows the dose modifying fractors (DMF 10 ) levels for both cells lines which was also significantly different between the two cell lines. ( D ) The chronotherapeutic experimental timeline, 1.0 × 10 6 cells were plated on day 0. Cells were then serum shocked with a 50% horse serum solution for 2 h at circadian time(CT) 8 (01:00, Day 2) or 20 (13:00, Day 1) on the subsequent days. All cells were irradiated on Day 2 at 11:00 and then colonies stained and counted after Day 5. ( E ) Clonogenic assay of SCN2.2 cells irradiated at CT8 (dotted line) and CT20 (solid line). Astrocytes irradiated at CT8 were significantly more sensitive to radiotherapy. There were significant main effects of time (F(1,24) = 7.041, p = 0.014) and dose (F(5,24) = 26.995, p < 0.001) but no interaction (F(5,24) = 1.081, p = 0.396). ( F ) Clonogenic assayof CTXTNA2 cells irradiated at CT8 (dotted line) and CT20 (solid line). Again, astrocytes irradiated at CT8 were significantly more sensitive to radiotherapy. There were significant main effect of time (F(1,24) = 14.099, p = 0.001) and dose (F(5,24) = 135.997, p < 0.001) but no interaction (F(5,24) = 1.349, p = 0.278). ( G ) ATM western analysis of both cell lines when samples were collected at two timepoint post-synchronization. Western blot of ATM (blue bands) and control β-actin (teal bands) in both cell lines show higher presence of ATM after serum shock entrainment (Left panel). A graph of the ATM/β-actin relative ratio (Right panel), shows that CTXTNA cells express more of the protein than SCN2.2 cells. Four replicates of each cell line at the 3 timepoints were compared, there were significant main effects of time (F(2,21) = 83.154, p < 0.001) and cell type (F(1,21) = 8.312, p = 0.009) and an interaction (F(2,21) = 5.230, p = 0.014). Posthoc tests (tukey) were significant if p < 0.05. Both cell lines had more protien during CT08 when compared to CT20 but only CTXTNA cells had significant differences between the two ( p = 0.007). Significance was defined as p < 0.05 and indicated by * or a, b, and c with different letters indicating significant differences.

Article Snippet: Membranes were stained using primary antibodies for ATM (1:1000; ab81292, abcam, MA) and β-actin (1:5000; ab8226, abcam, MA) for 24 h at 4°C, followed by 1 h at room temperature in secondary antibodies (1:5000) for IRDye 800CW Goat anti-Mouse (926-32210; Li-Cor Biotechnology, NE) and IRDye 680CW Goat anti-Rabbit (926-68071; Li-Cor Biotechnology, NE).

Techniques: In Vitro, Isolation, IF-P, Clonogenic Assay, Irradiation, Staining, Western Blot, Control