Journal: Cells

Article Title: HDAC1 -Mediated lncRNA Stimulatory Factor of Follicular Development to Inhibit the Apoptosis of Granulosa Cells and Regulate Sexual Maturity through miR-202-3p- COX1 Axis

doi: 10.3390/cells12232734

Figure Lengend Snippet: HDAC1 regulates the proliferation and apoptosis of GCs. ( A ) Effects TSA treatment at concentration of 0.01, 0.1, 0.25, 0.5, and 1 μM for 24 and 48 h on the viability of porcine GCs. ( B ) Micrographs of GCs treated with 0.1 and 0.25 μM TSA at 24 and 48 h. ( C ) Effects of 0.01, 0.1, 0.25, 0.5, and 1 μM TSA treatment on the expression of HDAC1 for 24 and 48 h. ( D – F ) The mRNA and protein levels of cell cycle and cell apoptosis-related genes in GCs treated with TSA. ( G , H ) The proliferation and apoptosis of GCs treated with TSA were assessed using EdU and flow cytometry. ( I ) Effect of TSA on the activity of Caspase 3/7 in porcine GCs was detected using Caspase 3/7 activity assay. ( J ) The interference efficiency of HDAC1 . ( K – M ) The mRNA and protein levels of cell cycle and cell apoptosis-related genes in GCs treated with HDAC1 -siRNA. ( N , O ) The proliferation and apoptosis of GCs treated with HDAC1 -siRNA were assessed using EdU and flow cytometry. ( P ) Effect of HDAC1 -siRNA on the activity of Caspase 3/7 in porcine GCs was detected using Caspase 3/7 activity assay. * indicates p < 0.05, ** indicates p < 0.01.

Article Snippet: The Caspase 3/7 Activity Assay was also performed to detect the apoptosis of GCs using the Caspase 3/7 Activity Assay Kit (Elabscience, Wuhan, China).

Techniques: Concentration Assay, Expressing, Flow Cytometry, Activity Assay

Journal: Cells

Article Title: HDAC1 -Mediated lncRNA Stimulatory Factor of Follicular Development to Inhibit the Apoptosis of Granulosa Cells and Regulate Sexual Maturity through miR-202-3p- COX1 Axis

doi: 10.3390/cells12232734

Figure Lengend Snippet: SFFD as ceRNA for miR-202-3p- COX1 regulates the proliferation and apoptosis of porcine GCs. ( A – D ) Effects of SFFD overexpression and miR-202-3p mimic on the mRNA and protein levels of genes related to cell cycle, E2 secretion, and apoptosis. ( E – H ) Effects of SFFD knockdown and miR-202-3p inhibitor on the mRNA and protein levels of genes related to cell cycle, E2 secretion, and apoptosis. ( I ) Effects of SFFD overexpression and miR-202-3p mimic on the proliferation of GCs. ( J ) Effects of SFFD knockdown and miR-202-3p inhibitor on the proliferation of GCs. ( K ) Effects of SFFD overexpression with miR-202-3p mimic and SFFD knockdown with miR-202-3p inhibitor on the E2 secretion of GCs. ( L , M ) Effects of SFFD overexpression and miR-202-3p mimic on the apoptosis and Caspase 3/7 activity of GCs. ( N , O ) Effects of SFFD knockdown and miR-202-3p inhibitor on the apoptosis and Caspase 3/7 activity of GCs. * indicates p < 0.05, ** indicates p < 0.01.

Article Snippet: The Caspase 3/7 Activity Assay was also performed to detect the apoptosis of GCs using the Caspase 3/7 Activity Assay Kit (Elabscience, Wuhan, China).

Techniques: Over Expression, Knockdown, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: MicroRNA-9 regulates survival of chondroblasts and cartilage integrity by targeting protogenin

doi: 10.1186/1478-811X-11-66

Figure Lengend Snippet: MiR-9 affects cell proliferation and survival during chondrogenesis of chick limb mesenchymal cells. (A) Changes in the phosphorylation levels of Akt, GSK, and JNK were analyzed by Western blotting. (B) Total RNA was purified from chondroprogenitors cultured with or without 5 μM JNK inhibitor and the expression of mir-9 was measured with real-time PCR. (C) Chondroprogenitor cells were treated with 100 nM of anti-mir-9 oligonucleotides (mir-9inhibitor). The expression of mir-9 was measured with real-time PCR (upper panel) and Precartilage condensation was analyzed by PA staining at day 3 and Alcian blue staining at day 5 of culture (lower panel). The data shown are representative of at least four independent experiments. The diameter of typical standard culture is 5 mm. (D) Total cell numbers were counted at 2 day of culture. (E) Apoptotic cells were analyzed by FACS analysis. (F) Changes in the cleaved form of caspase-3 were analyzed by Western blotting. Results of cell adhesion experiments were pooled from 5 replicate samples derived from 4 independent experiments. The mean is plotted and the error bars represent 95% CI (lower/upper limit). ***, statistically different from control cells (p < 0.001).

Article Snippet: Activities of caspase-3 and caspase-7 were determined using a caspase colorimetric assay kit (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Western Blot, Purification, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Staining, Derivative Assay, Control

Journal: Cell Communication and Signaling : CCS

Article Title: MicroRNA-9 regulates survival of chondroblasts and cartilage integrity by targeting protogenin

doi: 10.1186/1478-811X-11-66

Figure Lengend Snippet: PRTG induced apoptotic death of chondroprogenitors. (A) Cells were electroporated with PRTG /pCAGGS (PRTG) construct in the absence or presence of miR-9 precursor (miR-9) and electroporation efficiency was confirmed by immunoblotting (left upper panel). Precartilage condensation and chondrogenic differentiation were analyzed by PA staining at day 3 and Alcian blue staining at day 5 of culture, respectively (left lower panel) and chondrogenesis was quantified by measuring the absorbance of bound Alcian blue at 600 nm at day3 and day5 of culture (right panel). (B) Cells were electroporated with PRTG construct and the number of viable cells was determined at 1, 2, and 3 day of culture (left panel), treated with JNK inhibitor or miR-9, electroporated with PRTG or PRTG siRNA, or in the combination of JNK inhibitor and PRTG siRNA or PRTG and miR-9 and the number of viable cells were determined at day 2 and 3 of culture (right panel). (C) Apoptotic cells were analyzed by FACS analysis (left panel) and changes in the cleaved form of caspase-3 were analyzed by Western blotting (right panel). (D) Cells were treated with JNK inhibitor in the combination of miR-9 or PRTG-specific siRNA, or introduced with miR-9 in the combination of PRTG. Precartilage condensation and chondrogenic differentiation were analyzed by PA staining at day 3. The diameter of typical standard culture is 5 mm. (E) HH stage 18 chick embryos (wing bud) were treated with JNK inhibitor in the presence or absence of miR-9 precursor and incubated for additional 2 days (HH stage23). The number of embryos used for each experiment is represented as a table (upper panel), and a representative image of each limb is shown (lower panel). The mean is plotted and the error bars represent 95% CI (lower/upper limit). *, statistically different from control cells (p < 0.001).

Article Snippet: Activities of caspase-3 and caspase-7 were determined using a caspase colorimetric assay kit (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Construct, Electroporation, Western Blot, Staining, Incubation, Control

Journal: Cell Communication and Signaling : CCS

Article Title: MicroRNA-9 regulates survival of chondroblasts and cartilage integrity by targeting protogenin

doi: 10.1186/1478-811X-11-66

Figure Lengend Snippet: MiR-9 is involved in pathogenesis of OA. Articular chondrocytes were isolated form cartilage (upper panel) that was divided into 3 classes depending on the progression of OA pathology (A: healthy zone, B: intermediate zone, and C: severe zone). (A) Images of the cultures were captured using light microscopy and human cartilages were stained with safranin O and Alcian blue. (B) The expressions of MMP-2, MMP-9, MMP-12, and MMP-13 were measured with real-time PCR. (C) Cell viability (right panel) and caspase-3/7 activity (left panel) were analyzed. (D) Changes in the protein level of Type II collagen and PRTG were analyzed by Western blotting. GAPDH was used as control (upper left panel). PRTG expression was analyzed by immunocytochemisty (upper right panel). The expressions of type II collagen (Col II), PRTG, and miR-9 were analyzed by real-time PCR (lower panel). (E) Mouse cartilages with OA induced by destabilization of the medial meniscus (DMM), were infected with miR-9 or si-miR-9 lentiviruses and stained with safranin O, propium iodide, and Tunnel. PRTG level was analyzed by immunohistochemistry (left panel). Inserted number in safranin-O photomicrographs indicated the averages of semi-quantitative score for the degree of cartilage destruction in MFC (first score) and MTP (second score) view. Each histological score for the degree of cartilage destruction (n = 5 mice/group) in MFC (first score) and MTP (second score) view were graphed (right panel). Sham-operated (Sham) cartilage was used as control. *, statistically different from control cells (p < 0.001). The error bars represent average of data from each human sample. Scale bar, 200 μm.

Article Snippet: Activities of caspase-3 and caspase-7 were determined using a caspase colorimetric assay kit (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Isolation, Light Microscopy, Staining, Real-time Polymerase Chain Reaction, Activity Assay, Western Blot, Control, Expressing, Infection, Immunohistochemistry