Journal: Frontiers in Immunology

Article Title: miR-210 promotes the anti-inflammatory phenotype and M2 polarization in murine macrophages

doi: 10.3389/fimmu.2025.1633163

Figure Lengend Snippet: Effects of miR-210-KO in M0 macrophages. (A) Enrichr dot-plot representation for pathways enriched among differentially expressed genes in miR-210-KO versus WT M0 macrophages. The signed odds ratio (x-axis) indicates over-representation in up-regulated (positive) or down-regulated (negative) genes. (B) Western blot analysis of pro-IL1β levels in M0 macrophages, with α-tubulin used as a loading control. Densitometry analysis performed using TotalLab. Data are presented as the mean ± SEM, n = 3 mice per condition. Statistical significance was determined using Student’s t test, *p< 0.05. (C) ELISA quantification of IL-6, TNF-α and IL-1β in the supernatant of M0 macrophages. Data are presented as mean ± SEM, n = 3 for IL-6 and IL-1β and n=4 for TNF-α.

Article Snippet: The membranes were incubated overnight at 4°C with antibodies against p53 (Abcam, #ab90363, 1:1000), TGF-β (Abcam, #ab215715, 1:1000), GAPDH (Abcam, #ab37168, 1:1000), IL-1β (Abcam, ab9722, 1:1000), αTubulin (R&D Systems, MAB9344 1:1000), β-actin (Sigma-Aldrich, A2228), and MMP-9 antibody (Novus Biologicals, NBP1-57840), prepared in 5% BSA in TBST.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

doi:

Figure Lengend Snippet: vv early mRNAs and intracellular cores are aligned on microtubules by immunofluorescence. (A) Cells were infected and transfected with BrUTP for 2 h. Cells were extracted with TX-100 before fixation and double-labeling with either anti-tubulin and anti-BrU (left panel) to reveal the viral mRNA structures or anti-p39 (right panel) to label intracellular cores. The high-magnification view (indicated with 1 and 2) of the indicated areas 1 and 2 in the merge of anti-tubulin and p39 shows that intracellular cores are aligned on MT tracks (arrowheads). In B in the panel indicated with + nocodazole, cells were treated with 20 μM nocodazole 1 h before infection and BrUTP-transfection and fixed at 2 h postinfection in the continued presence of the drug. In the + latrunculin panel, cells were infected and transfected and treated 20 min before fixation with 1 μM Lat A and 2.5 μM taxol. Fixed cells were then double-labeled with anti-BrU and with either anti-tubulin or with rhodamine-phalloidin. Cells were fixed without prior TX-100 extraction. MTs (α-tubulin) as well as the viral mRNAs (α-BrU) show a diffuse pattern in the cytosol after nocodazole treatment. In the presence of Lat A, the phalloidin labeling is diffuse too, but the BrU pattern seems unaffected. Scale bars, 10 μm. In the table at the bottom of B, cells were infected and BrUTP-transfected for 2 h 20 min before fixation cells were mock treated or treated with either 40 μM of nocodazole or with 1 μM Lat A and 2.5 μM taxol. The cells were then extracted with TX-100, fixed, and labeled with anti-BrU. The amount of BrU-positive structure was counted in 30 infected/transfected cells. Because nocodazole-treated cells mostly lacked any BrU-positive structures, making it difficult to estimate which cells were transfected, cells that showed some residual BrU labeling were considered for counting. The values represent the average amount of BrU structures per cell and the SEM

Article Snippet: The membranes were blocked in PBS, 0.2% Tween 20, and 5% milk powder for 2 h before incubation with anti–β-tubulin or anti–β-actin antibodies followed by horseradish peroxidase-tagged goat anti-mouse antibody ( Bio-Rad ).

Techniques: Immunofluorescence, Infection, Transfection, Labeling

Journal:

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

doi:

Figure Lengend Snippet: Biochemical evidence for MT association of viral mRNAs. (A–C) Cells were infected for 2 h. They were either left untreated or were treated with either 20 μM of nocodazole from 1 h before infection onward or with 1 μM Lat A together with 2.5 μM taxol 20 min before cell lysis. (A and B) Total RNA was extracted from infected cells from either the detergent-soluble (S) or -insoluble cytoskeletal fraction (CK), and the fractions were analyzed by an RNase protection assay with the use of probes to H5R and β-actin mRNA. The position of the H5R (A) and β-actin (B) protected mRNA is indicated on the right. The percentage of protected mRNAs in the cytoskeleton (CK) versus soluble (S) fraction of eachsample as assessed by phosphoimager analysis is indicated at the bottom of each panel. (C) Western blot analysis of actin and tubulin (the position of the respective protein is indicated) after nocodazole or Lat A treatment in the soluble (S) and cytoskeleton (CK) fraction. The proteins were detected with the use of antibodies to β-actin or β-tubulin followed by enhanced chemiluminescence.

Article Snippet: The membranes were blocked in PBS, 0.2% Tween 20, and 5% milk powder for 2 h before incubation with anti–β-tubulin or anti–β-actin antibodies followed by horseradish peroxidase-tagged goat anti-mouse antibody ( Bio-Rad ).

Techniques: Infection, Lysis, Rnase Protection Assay, Western Blot

Journal:

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

doi:

Figure Lengend Snippet: P25 (4RL) colocalizes with the viral mRNAs early in infection and may associate with MTs. Top panels in A: Infected and BrUTP-transfected HeLa cell were fixed at the indicated times postinfection and double-labeled with anti-BrU and anti-p25 antibodies. At 30 min postinfection the viral mRNAs partially colocalize with the diffuse p25 labeling, whereas at 45 min postinfection colocalization of the two markers is detected in the perinuclear region where the mRNAs appear still unorganized. At 60 min postinfection, when all of the mRNAs are organized into the typical punctate structures, no colocalization is detected. In the merge anti-BrU is shown in green and anti-p25 in red. Note that over time the p25 labeling decreases. Bottom panels in B: Cells were extracted with TX-100 before fixation at 30 min postinfection and double-labeling with antitubulin and anti-p25. Anti-p25 labeling appears to follow MT tracks (arrowheads). In the merge p25 is in the green and antitubulin in the red channel. Scale bars, top, 10 μm; bottom, 2 μm. (B) Infected HeLa cells were mock treated or treated with either nocodazole or Lat A (together with taxol) and Triton-extracted before fixation at 1 h postinfection. The cells were then labeled with antibodies to β-tubulin, p25, or rhodamine-phalloidin. Ten random pictures of both nocodazole- and mock-treated cells were taken with the use of the same parameters (the same amplitude, gain, and offset). The total fluorescence for p25 or actin was measured with the use of NIH image. The graph represents the average and SD of the total fluorescence of 10 cells for both treated and untreated cells.

Article Snippet: The membranes were blocked in PBS, 0.2% Tween 20, and 5% milk powder for 2 h before incubation with anti–β-tubulin or anti–β-actin antibodies followed by horseradish peroxidase-tagged goat anti-mouse antibody ( Bio-Rad ).

Techniques: Infection, Transfection, Labeling, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Ptbp2 re-expression rescues axon growth defects in Smn-deficient motoneurons

doi: 10.3389/fnmol.2024.1393779

Figure Lengend Snippet: Ptbp2 interacts with Smn in motoneurons. (A) Representative images of Smn-Ptbp2 PLA signal in cultured motoneurons at DIV 6 using anti-Smn and anti-Ptbp2 antibodies. Motoneuron morphology was visualized with anti-Tubb3 antibody. Scale bars, 10 and 5 μm (magnified areas). (B) Co-immunoprecipitation of Smn by anti-Ptbp2 from motoneuron lysate pre-treated with RNase A as indicated. (C) Representative images showing Smn immunofluorescence and Hnrnpr FISH in cultured motoneurons at DIV 6. Arrowheads indicate colocalization of Smn and Hnrnpr in granules. Scale bars, 10 and 2 μm (magnified areas). (D) Fluorescence intensity profiles of Smn and Hnrnpr at the location indicated by arrow 4 in (C) . (E) Representative images showing Ptbp2 and Smn immunofluorescence and Hnrnpr FISH in cultured motoneurons at DIV 6. Scale bars, 10 and 2 μm (magnified areas). (F) Fluorescence intensity profiles of Ptbp2, Smn and Hnrnpr at the location indicated by a line in (E) .

Article Snippet: Cells were fixed again for 10 min at room temperature in PLP, washed with DPBS, and stained with FITC-conjugated anti-Tubb3 antibody (130-131-158, Miltenyi Biotec).

Techniques: Cell Culture, Immunoprecipitation, Immunofluorescence, Fluorescence