The dermal papilla is a major component of hair, which signals

The dermal papilla is a major component of hair, which signals the follicular epithelial cells to prolong the hair growth process. results at the mRNA level, Western blot analysis revealed that Annexin A2 isoform 2 was up-regulated significantly in passage 3 DPC compared with passage 10 DPC. The Annexin Rabbit Polyclonal to RFA2 (phospho-Thr21) A2 isoform 2 siRNA was synthesized and transfected into passage 3 DPC. RT-PCR data showed the mRNA expression of Annexin A2 isoform 2 was suppressed in passage 3 DPC. Western blot results showed the expression level of Annexin A2 isoform 2 and PCNA were suppressed in passage 3 DPC. CCK-8 results showed that this proliferation of passage 3 DPC was suppressed (= 2) were from Affiliated Wuxi No. 2 Peoples Hospital of Nanjing (+)-JQ1 inhibitor Medical University or college with the informed consent of donors and approval of Wuxi second hospital ethical committee (20161209). Experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Statement. We used improved two-step enzyme method set up previously in our laboratory to isolate the dermal papillae [17]. Firstly, the skin was sterilized and digested in 0.5% (w/v) dispase (Sigma, U.S.A.) for 12C16 h at 4C and in 0.2% (w/v) collagenase D (Sigma, U.S.A.) for 6 h at 37C sequentially. The digested tissue was then centrifuged at 550C850 for 3C5 min. The dermal papillae deposited at the bottom of tube as a clump of cells, whereas other cells floated in the supernatant. After centrifuged for several times, the human dermal papillae were separated from other types of cells. The human dermal papillae were cultured in DMEM medium (Gibco, U.S.A.) containing 10% fetal calf serum (Gibco, U.S.A.) and incubated at 37C in an atmosphere of 95% air flow and 5% CO2. Different expression of Annexin A2 mRNA between passage 3 and passage 10 DPC Total RNA was extracted from passage 3 and passage 10 DPC by using Trizol (Invitrogen, U.S.A.) according to the manufacturers training. c-DNA was synthesized from 1 g total RNA using OligodT primers by reverse transcription reagent (Takara, Japan) based on the training (+)-JQ1 inhibitor of (+)-JQ1 inhibitor the manufacturer. DNA were amplified using Taq DNA Polymerase (Takara, Japan) with the following primers: -actin (618 bp), 5-CGG GAC CTG Take action GAC TAC CTC-3 and 5-CAA GAA AGG GTG TAA CGC AAC-3; Annexin A2 (329 bp), 5-TGAAGTCAGCCTTATCT GGC-3 and 5-ATTGACCAAGATGCTCGG-3. Amplified fragments were separated through electrophoresis on 1% agarose gels and visualized by ethidium bromide staining. The experiment was repeated three times. Different expression of Annexin A2 between passage 3 and passage 10 DPC The expression of Annexin A2 in DPC at passage 3 and passage 10 was measured through the Western blot assay. After cell lysis and centrifugation (14000?rpm??15?min), protein was subjected to a SDS-PAGE gel and then, transferred to polyvinylidene fluoride (PVDF) membrane. After blocking with TBST made up of 5% skim milk, the membranes were first incubated with mouse anti-human Annexin (+)-JQ1 inhibitor A2 mAb (diluted 1:1000, Proteintech Group, China) or anti-PCNA Mouse mAb (dilution 1:1000, Affinity Group, U.S.A.) at 4C overnight, and then with peroxidase-conjugated goat anti-mouse immunoglobulin (Beyotime, China) diluted 1:1000 in Tris-Buffered Saline Tween-20 (TBST) for 1 h. Finally, PVDF membranes were washed three times with TBST, and protein bands were visualized with ECL Substrates (Thermo, U.S.A.). The experiment was repeated three times. Design and synthesis of siRNA The siRNA oligonucleotide targeting Annexin A2 isoform 2 was designed and synthesized by Gene Pharma (Shanghai, China) based on the published sequence of Annxin A2 isoform 2 (sense 5-GCAUAGCAACUUCGGAUUUTT-3; antisense 5-AAAUCCGAAGUUGCUAUGCTT-3). In the mean time, siRNA for unfavorable control (sense 5-UUCUCCGAACGUGUCACGUTT-3; antisense 5-ACGUGACACGUUCGGAGAATT-3) was synthesized. Construction of recombinant plasmid PLJM-Annexin A2 isoform 2 Total RNA extracted from human DPC. cDNA was synthesized from mRNA by reverse transcription reagent. The full length cDNA encoding Annexin A2 isoform 2 was obtained through polymerase chain reaction (Promega, U.S.A.). The primers utilized for PCR amplification were: F 5-AATAATACCGGTAGTTCTACTGTTCACGAAATC-3 and R 5-AATAATTTCGAATCAGTCATCTCCACCACAC-3. PCR-amplified DNA fragments were separated by electrophoresis on 1% agarose. The PCR products were visualized on agarose gel by staining with ethidium bromide. cDNA of Annexin A2 was obtained from the gel used (+)-JQ1 inhibitor agarose gel DNA extraction kit (Takara, Japan), then cloned into PLJM (the expression plasmid) and placed between Age I and Bstb I restriction sites. The place was sequenced and validated to be in total agreement with the expected sequence. Down-regulate the expression of Annexin A2 isoform 2 in DPC Down-regulating the expression of Annexin A2 was performed by using Annexin A2 isoform 2 siRNA. Passage 3 DPC were diluted in new media without antibiotics 24 h before transfection and later transferred to six-well plates. When the DPC experienced produced to a confluence.

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