Journal: Cell proliferation

Article Title: A TALEN-based specific transcript knock-down of PIWIL2 suppresses cell growth in HepG2 tumor cell.

doi: 10.1111/cpr.12120

Figure Lengend Snippet: Figure 2. Screen and analysis of PIWIL2 mono-allelic mutations. (a) Sequencing and alignment of mutations. PCR products ampli- fied from wild type and mutants genomic DNA were sequenced. The TALEN pair tar- get sequences were coloured in blue. The deleted sequences of allele were indicated with black box on the other allele. The sizes of deletions were shown with 4. An unex- pected mutation (GT ? TG) in clone Mutant #3 was observed and underlined. (b) An allele analysis of clone Mutant #1. The PCR prod- ucts were cloned into pMD19-T vector. Eigh- teen positive clones were analysed by PCR amplification and Msc I digestion.

Article Snippet: Rabbit anti-PIWIL2 antibody was obtained from Santa Cruz Biotechnology (USA) as were goat anti-HSP90 antibody and rabbit antiSmad4 antibody.

Techniques: Sequencing, Mutagenesis, Clone Assay, Plasmid Preparation

Journal: Cell proliferation

Article Title: A TALEN-based specific transcript knock-down of PIWIL2 suppresses cell growth in HepG2 tumor cell.

doi: 10.1111/cpr.12120

Figure Lengend Snippet: Figure 3. Identification of PIWIL2 bi-alle- lic knockout cell lines. (a) Single strand con- formation polymorphism (SSCP) analysis to screen monoclonal bi-allele mutant cell lines. NC represents normal HepG2 cells. Control represents 14 bp deleted PIWIL2 mono-allelic knockout cell line. Compared to controls, lanes 6, 7 and 8 indicated second mutation. Remarkable differences with controls – marked with arrows. (b) Identification of PI- WIL2 bi-allelic knockout cell lines. Mutants in (a) were further identified by pMD19-T cloning and sequencing to analyse allelic mutations. Clones #7 and #8 respectively rep- resent cell lines chosen as mutants in lanes 7 and 8 in (a). Other mutants did not have frameshift mutations on one allele (data not shown). Nucleotide sequences of mutated alleles – indicated with dashes. (c)Further identification with PCR amplification and restriction endonuclease analysis of the two cell clones in (b). NC represents normal HepG2 cells. (d) Western bolt analysis of expression of PIWIL2 in normal HepG2, PI- WIL2+/ and PIWIL2/ cells.

Article Snippet: Rabbit anti-PIWIL2 antibody was obtained from Santa Cruz Biotechnology (USA) as were goat anti-HSP90 antibody and rabbit antiSmad4 antibody.

Techniques: Knock-Out, Mutagenesis, Control, Cloning, Sequencing, Clone Assay, Western Blot, Expressing

Journal: Cell proliferation

Article Title: A TALEN-based specific transcript knock-down of PIWIL2 suppresses cell growth in HepG2 tumor cell.

doi: 10.1111/cpr.12120

Figure Lengend Snippet: Figure 4. Loss of PIWIL2 reduced HepG2 cell proliferation. Cell proliferation levels of normal HepG2, PIWIL2+/ and PIWIL2/ cell lines. Clone #7 and clone #8 represent the two PIWIL2 knockout cell lines mentioned earlier. Respectively at days 3, 4 and 5, we used CCK-8 assay to evaluate cell proliferation levels.

Article Snippet: Rabbit anti-PIWIL2 antibody was obtained from Santa Cruz Biotechnology (USA) as were goat anti-HSP90 antibody and rabbit antiSmad4 antibody.

Techniques: Knock-Out, CCK-8 Assay

Journal: Cell proliferation

Article Title: A TALEN-based specific transcript knock-down of PIWIL2 suppresses cell growth in HepG2 tumor cell.

doi: 10.1111/cpr.12120

Figure Lengend Snippet: Figure 5. PIWIL2 promoted cell popula- tion growth by inhibiting TGF-b signalling in HepG2 cells. (a) Knockdown of PIWIL2 upregulated p21, TbRI, TbRII and phosphor- ylated Smad2/3 with or without TGF-b stimu- lation. Normal HepG2 and PIWIL2+/ cells were treated or not treated with TGF-b for 2 h before harvesting. (b) Ectopic expression of PIWIL2 reduced level of p21, TbRI, TbRII and prevented Smad2/3 activation. HepG2 cells were transfected with PIWIL2 over a concentration gradient and treated with TGF- b before cells were harvested. (c) and (d) PI- WIL2 attenuated TGF-b-induced population growth inhibition. HepG2 cells were respec- tively transfected with pcDNA3.1, PIWIL2, shControl and shPIWIL2, and cultured in complete medium with or without TGF-b stimulation for 96 h before cells were analy- sed with CCK-8. Each experiment was per- formed in triplicate. *Indicates P < 0.05. N.S. indicates P > 0.05.

Article Snippet: Rabbit anti-PIWIL2 antibody was obtained from Santa Cruz Biotechnology (USA) as were goat anti-HSP90 antibody and rabbit antiSmad4 antibody.

Techniques: Knockdown, Expressing, Activation Assay, Transfection, Concentration Assay, Inhibition, Cell Culture, CCK-8 Assay

Journal: Cell proliferation

Article Title: A TALEN-based specific transcript knock-down of PIWIL2 suppresses cell growth in HepG2 tumor cell.

doi: 10.1111/cpr.12120

Figure Lengend Snippet: Figure 6. PIWIL2 regulated degradation of TbRs via HSP90. (a) PIWIL2 promoted TbR degradation. HepG2 cells were transfected with Myc- PIWIL2, treated with MG132 for 6 h, and TGF-b for the last 2 h. (b) Reduction of TbRs and downstream events mediated by PIWIL2 was rescued by overexpression of HSP90. (c) Endogenous interactions between PIWIL2 and HSP90 in HepG2 cells. Co-IP was performed with anti- PIWIL2 or anti-HSP90, followed by western blotting. (d) Both PIWIL2 and HSP90 were co-localized mainly in cytoplasm. HepG2 cells were transfected with Myc-PIWIL2 and harvested for immunofluorescence with anti-Myc and anti-HSP90 antibodies. (e) PIWIL2 reduced interaction of HSP90 and TbRs. Normal and PIWIL2 knockdown cells were treated with MG132 before harvesting, and lysates were subjected to immunoprecipitation with anti-HSP90 antibody. (f) PIWIL2 attenuates interaction of HSP90 and TbRs. HepG2 cells were transfected with Myc-PIWIL2 over a concentration gradient. Forty eight hours later, cells were treated with MG132 before harvesting, and lysates were subjected to immunoprecipitation or western blot analysis. (g) PIWIL2 improved TbRI ubiquitination. HepG2 cells were co-transfected with Myc-PIWIL2 and HA-ubiquitin, then treated with MG132 and stimulated with TGF-b before harvesting. HA-ubiquitin was precipitated with anti-HA antibody, then ubiquitination and degradation of TbRI were determined by immunoblotting with anti-TbRI antibody.

Article Snippet: Rabbit anti-PIWIL2 antibody was obtained from Santa Cruz Biotechnology (USA) as were goat anti-HSP90 antibody and rabbit antiSmad4 antibody.

Techniques: Transfection, Over Expression, Co-Immunoprecipitation Assay, Western Blot, Knockdown, Immunoprecipitation, Concentration Assay, Ubiquitin Proteomics

Journal: Animals : an Open Access Journal from MDPI

Article Title: Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium

doi: 10.3390/ani14050803

Figure Lengend Snippet: Sequence of primers used for Q-PCR analysis.

Article Snippet: Samples were then washed and blocked with 3% bovine serum albumin (BSA) diluted in PBS (pH: 7.4) for 45 min. Proteins were immunodetected using anti- Wt1 rabbit polyclonal antibody (Cat. # ab89901, Abcam, Boston, MA, USA), anti- Oct4 mouse polyclonal antibody (Cat. # sc5279, Santa Cruz, CA, USA), anti- Nanog mouse polyclonal antibody (Cat. # sc293121, Santa Cruz), anti- Uchl1 mouse monoclonal antibody (Cat. # 480012, Thermo-Fisher, Waltham, MA, USA), anti- Cd73 rabbit monoclonal antibody (Cat. # ab137595, Abcam), anti- Piwil2 rabbit monoclonal antibody (Cat. # ab85084, Abcam), and anti- Dazl polyclonal rabbit (Cat. # ab34139, Abcam), diluted in 3% BSA diluted in PBS (pH: 7.4) ( Wt1 1:200, Oct4 , Nanog, Cd73, Piwil2 , and Dazl 1:50 and Uchl1 1:100).

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium

doi: 10.3390/ani14050803

Figure Lengend Snippet: Expression pattern of mesenchymal, pluripotency and germline markers in cultured cattle PB-MSCs/CM and SSCs/SCs compared to their controls for PB-MSCs and SSCs.

Article Snippet: Samples were then washed and blocked with 3% bovine serum albumin (BSA) diluted in PBS (pH: 7.4) for 45 min. Proteins were immunodetected using anti- Wt1 rabbit polyclonal antibody (Cat. # ab89901, Abcam, Boston, MA, USA), anti- Oct4 mouse polyclonal antibody (Cat. # sc5279, Santa Cruz, CA, USA), anti- Nanog mouse polyclonal antibody (Cat. # sc293121, Santa Cruz), anti- Uchl1 mouse monoclonal antibody (Cat. # 480012, Thermo-Fisher, Waltham, MA, USA), anti- Cd73 rabbit monoclonal antibody (Cat. # ab137595, Abcam), anti- Piwil2 rabbit monoclonal antibody (Cat. # ab85084, Abcam), and anti- Dazl polyclonal rabbit (Cat. # ab34139, Abcam), diluted in 3% BSA diluted in PBS (pH: 7.4) ( Wt1 1:200, Oct4 , Nanog, Cd73, Piwil2 , and Dazl 1:50 and Uchl1 1:100).

Techniques: Expressing, Cell Culture

Journal: Animals : an Open Access Journal from MDPI

Article Title: Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium

doi: 10.3390/ani14050803

Figure Lengend Snippet: Expression of GCs markers PIWIL2 and DAZL in PB-MSCs and SSCs treated with SCs/CM for 21 days. ( A ) Higher ( p < 0.05) gene expression of PIWIL2 was detected in the SSCs-SCs/CM on day 7. On day 14 of culture, gene expression of PIWIL2 was higher ( p < 0.05) in PB-MSCs-SCs/CM and SSCs-SCs/CM compared to SCs, PB-MSCs and SSCs controls. Over time the relative expression of PIWIL2 increased ( p < 0.05) in SSCs-SCs/CM from day 7 to day 14. ( B ) The relative expression of DAZL was higher ( p < 0.05) in SSCs-SCs/CM on days 7 and 14 compared with SCs and SSCs controls. In PB-MSCs or PB-MSCs-SCs/CM no gene expression of DAZL was detected. ( C ) Piwil2 protein expression was immunodetected in PB-MSCs-SCs/CM and SSCs-SCs/CM on day 14. ( D ) Dazl was immunodetected in SSCs and SSCs-SCs/CM on day 14. (*) Indicate mRNA levels of specific gene were not detected. Different superscripts (a, b, c) indicate differences ( p < 0.05) for the same marker between cell types and (1,2) for the same marker between days of culture.

Article Snippet: Samples were then washed and blocked with 3% bovine serum albumin (BSA) diluted in PBS (pH: 7.4) for 45 min. Proteins were immunodetected using anti- Wt1 rabbit polyclonal antibody (Cat. # ab89901, Abcam, Boston, MA, USA), anti- Oct4 mouse polyclonal antibody (Cat. # sc5279, Santa Cruz, CA, USA), anti- Nanog mouse polyclonal antibody (Cat. # sc293121, Santa Cruz), anti- Uchl1 mouse monoclonal antibody (Cat. # 480012, Thermo-Fisher, Waltham, MA, USA), anti- Cd73 rabbit monoclonal antibody (Cat. # ab137595, Abcam), anti- Piwil2 rabbit monoclonal antibody (Cat. # ab85084, Abcam), and anti- Dazl polyclonal rabbit (Cat. # ab34139, Abcam), diluted in 3% BSA diluted in PBS (pH: 7.4) ( Wt1 1:200, Oct4 , Nanog, Cd73, Piwil2 , and Dazl 1:50 and Uchl1 1:100).

Techniques: Expressing, Marker

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

Article Title: Roles for small noncoding RNAs in silencing of retrotransposons in the mammalian brain

doi: 10.1073/pnas.1609287113

Figure Lengend Snippet: Expression of the piRNA biogenesis machinery in the brain. (A) Transcript expression analyses by RNA-Seq. The dotted line represents baseline of expression based on TPM score for Piwil4, a transcript absent in adult testes (3). (B) Visualization of piwil2 (Mili) transcript alignment from poly(A) selected (green) and from ENCODE long RNA-Seq contigs (gray). Corresponding exons of the Mili gene were identified by numerical values from 1 to 23. Note, number of reads in exon 23 (noncoding) accounts for 61–63% of all reads mapping to the Mili transcript in all tissues. (C) Mili protein expression. Whole cell lysates from brain or testes, from either WT (+/+) or Mili−/− (−/−) mice were immunoprecipitated using the N-terminal anti-Mili antibody and Western blotted using the C-terminal anti-Mili antibody. The experiment was carried out two to three times. Note, brain lysates were used at 225-fold higher than testes for immunoprecipitation.

Article Snippet: Western blotting was carried out using a C-terminal (against amino acids 601–680 of Mili protein) anti-Mili antibody (polyclonal rabbit IgG; Santa Cruz Biotechnology).

Techniques: Expressing, RNA Sequencing, Immunoprecipitation, Western Blot

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

Article Title: Roles for small noncoding RNAs in silencing of retrotransposons in the mammalian brain

doi: 10.1073/pnas.1609287113

Figure Lengend Snippet: Expression of a putative truncated Mili isoform in the brain. (A) Expression of Piwil2-like protein 60 (PL2L60), a Mili variant in the adult testes. Five micrograms of testes whole cell lysates from 8-wk-old WT (+/+) or Mili−/− (−/−) mice were run on a SDS/PAGE followed by Western blotting using a C-terminal (601–680) and N-terminal (107–121) anti-Mili antibodies. Note that the 60- and 80-kDa species representing PL2L60 and a putative PL2L80 proteins were only detected in WT lysates when blotted with the C-terminal anti-Mili, confirming that these were N-terminally truncated products of Mili as previously described (29). The exact mechanism for the generation of PL2L60 is not clear. In the Mili−/− mice, the Mili gene was disrupted by homologous recombination within exon 2–5 (22), thus leading to the expression of exon 6–23 and a low level expression of exon 1–7 (29). Note that PL2L60 described as the major variant was only depleted but not completely absent from Mili−/− lysates due to transcription from a distal promoter located 3′ of the disrupted region (intron 10, a region still intact in Mili−/− mice) of the Mili gene and residual transcription at the 5′, thus encoding some PL2L60 protein with N-terminal truncation (encoding part of the N-terminal region and the PAZ domain) but with intact C-terminally located MID and PIWI domains (29). (B) Expression of PL2L60 in the brain. Cytoplasmic and nuclear fractions of whole 8-wk-old WT and Mili−/− brains were Western blotted using a C-terminal anti-Mili antibody. The band corresponding to PL2L60 was absent when reblotted with an N-terminal anti-Mili antibody, suggesting that PL2L60 could be a N-terminally truncated product of Mili. MAP2c and NeuN were respectively used as markers for cytoplasmic and nuclear fractions. Experiment was repeated two to three times.

Article Snippet: Western blotting was carried out using a C-terminal (against amino acids 601–680 of Mili protein) anti-Mili antibody (polyclonal rabbit IgG; Santa Cruz Biotechnology).

Techniques: Expressing, Variant Assay, SDS Page, Western Blot, Homologous Recombination

Journal: Genetics and Molecular Research

Article Title: Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and cell lines

doi: 10.4238/2015.june.26.15

Figure Lengend Snippet: Figure 1. Expression of PIWIL2 in normal bile duct tissue (A), well-differentiated adenocarcinoma (B), moderately differentiated adenocarcinoma (C), and poorly differentiated adenocarcinoma (D).

Article Snippet: A rabbit polyclonal antibody for PIWIL2, a SV-0001 2-step Immunostain SP Kit, a DAB color development kit, and an HE Staining Kit were provided by Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing

Journal: Genetics and Molecular Research

Article Title: Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and cell lines

doi: 10.4238/2015.june.26.15

Figure Lengend Snippet: Figure 2. Expression of PIWIL2 in QBC939 and HIBEpic cells detected by western blot.

Article Snippet: A rabbit polyclonal antibody for PIWIL2, a SV-0001 2-step Immunostain SP Kit, a DAB color development kit, and an HE Staining Kit were provided by Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Western Blot

Journal: Genetics and Molecular Research

Article Title: Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and cell lines

doi: 10.4238/2015.june.26.15

Figure Lengend Snippet: Figure 3. Expression of PIWIL2 in A) QBC939 cells and B) HIBEpic cells detected by immunocytochemistry.

Article Snippet: A rabbit polyclonal antibody for PIWIL2, a SV-0001 2-step Immunostain SP Kit, a DAB color development kit, and an HE Staining Kit were provided by Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Immunocytochemistry