Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

doi: 10.1186/s12964-024-01797-2

Figure Lengend Snippet: Schematic summary of HDAC2-mediated kcnq2/kcnq3 genes transcription repression in bone cancer pain. The HDAC2-mediated transcriptional repression of kcnq2 and kcnq3 genes, induced by the activation of EREG/EGFR-ERK-Runx1 signaling, contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Note that HDAC2 needs to form corepressor complex with MeCP2 and Sin3A, and EREG is an upstream signal molecule for HDAC2-mediated gene transcription repression. EREG/EGFR-ERK-Runx1 signaling underlies the HDAC2-mediated gene transcription repression

Article Snippet: Then, tissues or cultured cells were incubated with the corresponding primary antibody (see Supplementary Table S1) in PBS at 4 °C overnight, which includes rabbit anti-KCNQ2 (1:200, Abcam), rabbit anti-KCNQ3 (1:200, Abcam), rabbit anti-mouse NeuN (1:200, Sigma-Aldrich), mouse anti-pig GFAP (1:200, Cell Signaling Technology), mouse anti-NF200 (1:200, Sigma-Aldrich), mouse anti-CGRP (1:200, Abcam), rabbit anti-HDAC2 (1:200, Abcam), rabbit anti-MeCP2 (1:200, Abcam), goat anti-EGFR antibody (1:200, GeneTex), rabbit anti-Runx1(1:200, Abcam), goat anti-Sin3A (1:200, R&D systems).

Techniques: Activation Assay

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Developmental expression profile of Sin3a/SIN3A. A) Sin3a mRNA levels were measured by qRT-PCR at the indicated stages. Data were normalized against the detected levels of exogenously added GFP and expressed relative to the value obtained for mid-1-cell embryos. The experiment was conducted three times, and the data are expressed as mean ± SEM. GV, full-grown GV-intact oocytes; MII, metaphase II-arrested egg; 1C, 2C, and 8C refer to 1-cell, 2-cell, and 8-cell stages, respectively; BL, blastocyst. B) The relative amount of SIN3A was measured by immunoblot analysis. MII in vitro, oocytes were matured in vitro to MII; MII in vivo, MII eggs were collected following maturation in vivo. The TUBA signal was used to normalize total protein loading. The experiment was performed three times, and similar results were obtained in each case; shown is a representative example. C) Immunocytochemical analysis of SIN3A expression during preimplantation development. The experiment was conducted twice, and at least 15 oocytes/embryos were analyzed for each experiment. Bar = 50 μm.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, In Vivo

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Sin3a 3′-UTR contains elements that drive translational recruitment during oocyte maturation and following activation. Full-grown oocytes (A) or MII eggs (B) were microinjected with the luciferase reporter cRNAs. Firefly luciferase reporter activities were normalized to the coinjected Renilla luciferase. The data are expressed as mean ± SEM, and at least 10 oocytes/embryos were analyzed for each group.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Activation Assay, Luciferase

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Time course for SIN3A degradation. A) Immunoblot analysis for SIN3A was conducted at the indicated developmental stages. The TUBA signal was used to normalize total protein loading. The experiment was performed three times, and similar results were obtained in each case; shown is a representative example. B) Quantification of the relative amount of SIN3A shown in A. The data are expressed as mean ± SEM.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Western Blot

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: SIN3A degradation is proteasome-dependent. A) Mid-1-cell embryos were isolated and cultured in vitro in the presence of the proteasome inhibitor MG132 or DMSO, the vehicle. SIN3A abundance was measured by immunoblot analysis at the indicated times. The TUBA signal was used to normalize total protein loading. The experiment was performed three times, and a representative example is shown. B) Quantification of the data shown in A. The data are expressed as the mean ± SEM, and the SIN3A signal is relative to the mid-1-cell embryo. The times refer to the number of hours post-hCG injection. *P < 0.05.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Isolation, Cell Culture, In Vitro, Western Blot, Injection

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Combined Sin3a siRNA and morpholino inhibit maturation-associated increase in the amount of SIN3A. A) Full-grown oocytes microinjected with control or Sin3a morpholino (MO) and siRNA were cultured for 1 h in medium containing 2.5 μM milrinone and then cultured in inhibitor-free medium for maturation. MII eggs were collected 16 h after maturation and used for immunoblot analysis to detect SIN3A protein levels. TUBA was used to normalize total protein loading. The experiment was performed three times, and a representative example is shown. B) Quantification of the relative amount of SIN3A shown in A. The data are expressed as mean ± SEM.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Control, Cell Culture, Western Blot

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Effect of inhibiting the maturation-associated increase in SIN3A on histone acetylation in 1-cell embryos. A) The indicated histone were assayed by immunocytochemistry for their acetylation status using maternal SIN3A-depleted, control 1-cell embryos (Ctrl), and 1-cell embryos that were incubated with TSA (an HDAC inhibitor) to generate the maximum increase in histone acetylation. The experiment was performed twice, and at least a total of 10 embryos were analyzed for each sample. Bar = 50 μm. B) Quantification of the data shown in A. The data are expressed relative to the control 1-cell embryos and are expressed as mean ± SEM. *P < 0.05.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Immunocytochemistry, Control, Incubation

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Inhibiting the maturation-associated increase in SIN3A leads to arrest at the 2-cell stage. After inhibiting the maturation-associated increase in SIN3A, the maternal SIN3A-depleted and control MII eggs were in vitro fertilized (IVF) and embryo development was assessed at 24, 48, and 72 h after fertilization; hpf, hours postfertilization. The experiment was performed three times and the data pooled. A total of 56 experimental and 43 control embryos were analyzed.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Control, In Vitro

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Transcription is reduced in 2-cell embryo when maternal SIN3A is depleted. A) The experiment was performed four times and shown are representative images. At least 10 embryos were analyzed for each treatment group. Bar = 25 μm. B) Quantification of the images shown in A. The data are expressed as mean ± SEM. *P < 0.05.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques:

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Heat map of all samples from different treatment groups constructed using hierarchical clustering. Replicate sample numbers are indicated at the bottom of the figure. GV, full-grown GV-intact oocytes; a-am, 1-cell embryos incubated in α-amanitin to the 2-cell stage; 2C, 2-cell embryos derived from oocytes injected with control siRNA and morpholino matured and fertilized in vitro; KD, 2-cell embryos derived from oocytes injected with Sin3a siRNA and morpholino matured and fertilized in vitro. Colors correspond to relative RNA abundance (on the log2 scale) for the detected genes each of which is represented by one horizontal bar. The numbers correspond to each replicate within each group.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Construct, Incubation, Derivative Assay, Injection, Control, In Vitro

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Karyogram of genes up-regulated in 2-cell embryos depleted of maternal SIN3A (left panel) and density of genes (right panel). The comparison showed that most of the SIN3A sensitive genes emanated from gene dense regions.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Comparison

Journal: Biology of Reproduction

Article Title: Maternal SIN3A Regulates Reprogramming of Gene Expression During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.115.133504

Figure Lengend Snippet: Overexpressing SIN3A does not affect pre- and postimplantation development. A) Immunoblot analysis of SIN3A demonstrating that SIN3A is expressed at a similar level in 2-cell embryos (SIN3A o/e 2C) when compared to 1-cell embryos (1C). As shown in Figure 1, SIN3A is essentially absent in control 2-cell embryos (2C). B) Effect of overexpressing SIN3A on development to the blastocyst stage. Control embryos were injected with buffer. The experiment was conducted seven times, and at least 261 embryos were analyzed in each group. The data are expressed as mean ± SEM. C) Effect of overexpressing SIN3A on cell numbers in blastocysts. The experiment was conducted four times, and at least 32 embryos were analyzed in each group. The data are expressed as mean ± SEM. D) Immunocytochemical detection of lineage markers in blastocysts derived from embryos overexpressing SIN3A. Bar = 50 μm. E) Incidence of postimplantation development following blastocyst transfer of control and SIN3A-overexpressing embryos. The experiment was conducted six times.

Article Snippet: The SIN3A primary antibody (BMP004; MBL International) was diluted 1:2000 in blocking solution.

Techniques: Western Blot, Control, Injection, Derivative Assay