Journal: Journal of Personalized Medicine

Article Title: Exosomes from Microvascular Endothelial Cells under Mechanical Unloading Inhibit Osteogenic Differentiation via miR-92b-3p/ELK4 Axis

doi: 10.3390/jpm12122030

Figure Lengend Snippet: ELK4 is a direct target of miR-92b-3p and is responsible for miR-92b-3p-mediated suppression of osteogenic differentiation in MC3T3-E1 cells. ( A ) qRT–PCR analysis of ELK4 mRNA expression in MC3T3-E1 cells after transfection of mimic-92b-3p, inhibitor-92b-3p, or the corresponding control ( n = 3). ( B ) Western blotting analysis of the protein expression of ELK4 in MC3T3-E1 cells ( n = 3). ( C ) The relative luciferase activities of the ELK4 WT and MUT reporters were assessed after 293T cells were treated for 48 h with mimic-92b-3p and the equivalent controls ( n = 3). ( D ) Schematic representation of the luciferase reporters containing ELK4 3′-UTR WT or MUT sequences. ( E ) mRNA levels of ELK4 analyzed by qRT–PCR in MC3T3-E1 cells treated with Con Exos/Clino Exos (200 μg/mL) ( n = 3). ( F ) Protein levels of ELK4 analyzed by Western blotting ( n = 3). ( G ) qRT–PCR analysis of ALP, Osx, Runx2, and Ocn in MC3T3-E1 cells after the co-transfection of inhibitor-92b-3p, si-ELK4 and their negative controls in MC3T3-E1 cells ( n = 3). ( H ) Western blotting analysis of Osx, Runx2, and Ocn expression in MC3T3-E1 cells ( n = 3). ( I ) ALP activity analysis in MC3T3-E1 cells ( n = 3). ( J ) Representative images of ALP staining in MC3T3-E1 cells ( n = 3). * p < 0.05, ** p < 0.01 vs. control.

Article Snippet: After incubation with 5% skim milk (5% w / v ) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Luciferase, Cotransfection, Activity Assay, Staining

Journal: Journal of Personalized Medicine

Article Title: Exosomes from Microvascular Endothelial Cells under Mechanical Unloading Inhibit Osteogenic Differentiation via miR-92b-3p/ELK4 Axis

doi: 10.3390/jpm12122030

Figure Lengend Snippet: A schematic diagram illustrating the molecular mechanisms of which exosomes derived from MVECs cultured under mechanical unloading regulate osteogenic differentiation. miR-92b-3p expression was increased in MVEC-secreted exosomes after mechanical unloading, resulting in the upregulation of miR-92b-3p expression in MC3T3-E1 cells cocultured with Clino Exos. ELK4, the direct target of miR-92b-3p, is decreased in MC3T3-E1 cells treated with Clino Exos, thus inhibiting osteogenic differentiation. The blue arrow represents inhibition, and the red arrow represents promotion.

Article Snippet: After incubation with 5% skim milk (5% w / v ) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA).

Techniques: Derivative Assay, Cell Culture, Expressing, Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LSD1 modulates stress-evoked transcription of immediate early genes and emotional behavior

doi: 10.1073/pnas.1511974113

Figure Lengend Snippet: LSD1, CoREST, and HDAC2 are previously unidentified SRF corepressors. (A and B) Hippocampus protein extracts immunoprecipitated with anti-SRF antibody (A) or with anti-LSD1 (B) were separated by SDS/PAGE and detected with the indicated antibodies. In addition to the 67-KDa band in A and B, the SRF antibody detected a 57-KDa band described as a neurospecific splicing isoform (28) (also see Fig. S3). (C) HeLa cells overexpressing HA-LSD1 or HA-neuroLSD1 immunoprecipitated with anti-SRF antibody were separated by SDS/PAGE and were immunodecorated with anti-HA. (D–F) qChIP experiments performed on hippocampal chromatin using anti-SRF, anti-panLSD1, and anti-HDAC2 antibodies together with IgG as mock treatment (dashed lines represent the highest mock treatment of the two genotypes) on egr1 (D) and c-fos (E) promoters and an unrelated control (the egr1 distal genomic region) (F). Data are shown as mean ± SEM; *P = 0.035, Student t test. (G and H) In neurons, LSD1/neuroLSD1 regulation of IEGs’ activity-dependent transcription requires SRF binding to DNA. (G) Reporter assay in primary wild-type and neuroLSD1HET hippocampal neurons transfected with the pGL3-egr1(−370) construct (treatment: F1,28 = 34.74, P < 0.0001; genotype: F1,28 = 2.459, P = 0.1281; treatment × genotype: F1,28 = 5.384, P = 0.0278) and the mutated version lacking the five SREs, pGL3-egr1(−370m) (treatment × genotype: F1,20 = 0.2209, P = 0.6435). (H) Reporter assay in primary wild-type and neuroLSD1HET hippocampal neurons transfected with the pGL3-egr1(−370) construct and the pCGN-HA vector (treatment: F1,11 = 207.2, P < 0.0001; genotype: F1,11 = 5.319, P = 0.0416; treatment × genotype: F1,11 = 7.116, P = 0.0219) or pCGN-HA-neuroLSD1 (treatment × genotype: F1,11 = 0.6388, P = 0.4411). Reporter activity was assayed in basal conditions and after treatment with BDNF. (n = 3–8 per condition). Results are shown as mean ± SEM; *P < 0.01, two-way ANOVA, Bonferroni post hoc test.

Article Snippet: The following antibodies were used for Western blot or immunoprecipitation experiments: anti-LSD1 (C69G12; Cell Signaling Technology); anti-SRF (D71A9; Cell Signaling Technology); anti-CoREST (07-455; Merck Millipore); anti-HDAC2 (ab7029; Abcam); anti-GAPDH (NB300-320; Novus Biological); anti-α/β tubulin (2148; Cell Signaling Technology); and anti-HA (sc-7392; Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, SDS Page, Activity Assay, Binding Assay, Reporter Assay, Transfection, Construct, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LSD1 modulates stress-evoked transcription of immediate early genes and emotional behavior

doi: 10.1073/pnas.1511974113

Figure Lengend Snippet: Graphical model of SRF-LSD1/neuroLSD1–mediated transcriptional modulation of the IEGs. Given the dimeric nature of the corepressor complex, it is conceivable that, depending on the relative amounts of LSD1 and neuroLSD1, both LSD1/LSD1 homodimers and LSD1/neuroLSD1 heterodimers could be present in vivo (11). (A) In wild-type mice, in resting conditions, IEG transcription is repressed but permits activity-induced transcription. (B) Upon stressful stimuli, IEG transcription is fully activated. (C and D) In neuroLSD1-mutant mice, in resting conditions (C), a more condensed chromatin structure at the IEG proximal promoters does not permit stress-induced transcription (D). (E and F) Chronic treatment with the HDAC inhibitor SAHA leads to normalization of anxiety in neuroLSD1 mice.

Article Snippet: The following antibodies were used for Western blot or immunoprecipitation experiments: anti-LSD1 (C69G12; Cell Signaling Technology); anti-SRF (D71A9; Cell Signaling Technology); anti-CoREST (07-455; Merck Millipore); anti-HDAC2 (ab7029; Abcam); anti-GAPDH (NB300-320; Novus Biological); anti-α/β tubulin (2148; Cell Signaling Technology); and anti-HA (sc-7392; Santa Cruz Biotechnology).

Techniques: In Vivo, Activity Assay, Mutagenesis

Journal:

Article Title: Srf –/– ES cells display non-cell-autonomous impairment in mesodermal differentiation

doi: 10.1093/emboj/19.21.5835

Figure Lengend Snippet: Fig. 1. Genotypes and SRF protein activities of ES cells mutated at the Srf locus. (A) Genotyping by Southern blotting of ES cell lines used in this study. Genomic DNAs from the ES cell lines E14.1 Srf+/+ (lane 1), 226 Srf–/+ (lane 7) and 226-100 Srf–/– (lane 2), 226-100 Srf–/– plus human Srf cDNA expression construct pSGSRF1 (rescue) (lanes 3–5) or 226-100 Srf–/– plus an empty vector (vector) (lane 6) were digested with BglII, electrophoresed and blotted. Filters were successively hybridized with either a murine Srf probe discriminating between wild-type and targeted alleles (top panel) or a human Srf probe detecting the integrated human Srf cDNA (bottom panel). The plasmid pSGSRF1 was used as a hybridization control (Srf cDNA) (lane 8). Positions of the BglII fragments representing the wild-type allele (4.6 kb) or the targeted allele (5.4 kb) are indicated. Lanes 3, 4 and 5 represent the ‘rescue’ lines 226-100-2, 226-100-37 and 226-100-42, respectively. (B) Western analysis of SRF protein in extracts of ES cell lines used in this study. The blot (left) and quantification of the density of the bands on it (right) are shown. No specific SRF western signal was observed in the homozygous Srf–/– ES cell lines. (C) SRF-mediated SRE binding activities in extracts of ES cell lines used in this study. A radiolabeled c-fos SRE oligonucleotide probe was incubated with recombinant His-tagged SRF (lanes 1 and 2) or protein extracts from ES cells of the different genotypes, as indicated. Even-numbered lanes represent extracts with added polyclonal anti-SRF serum. SRF-containing DNA–protein complexes were supershifted by this antiserum. No SRF-containing complexes were detected in extracts from Srf–/– ES cells (lanes 7–10). Lanes 11 and 12 contained 4 µg of protein extract only, whereas 20 µg of protein was used for other cell extracts.

Article Snippet: To detect specific binding of SRF to the probe, anti-SRF antiserum (rabbit IgG, Santa Cruz) was included for super-shift analyses.

Techniques: Southern Blot, Expressing, Construct, Plasmid Preparation, Hybridization, Western Blot, Binding Assay, Incubation, Recombinant