Journal: Burns & Trauma

Article Title: Macrophage-derived exosome piR-50971 exacerbates sepsis-induced myocardial injury by inhibiting autophagy through the upregulation of N6-Methyladenosine modification of mTOR

doi: 10.1093/burnst/tkaf045

Figure Lengend Snippet: PIWIL2 is highly expressed in myocardial cells and tissue in mice with SIMI. ( a ) qPCR results indicated that PIWIL2 expression was significantly elevated in the myocardial tissue under septic conditions. ( b – c ) Western blot analysis verified increased PIWIL2 expression in myocardial tissue of rats with SIMI. ( d ) Immunofluorescence assay showed enhanced expression of PIWIL2 in the myocardial tissue of rats with SIMI. ( e ) Immunofluorescence assay showed that incubation with LPS-Exo markedly increased PIWIL2 expression in AC16 cells compared with that in the CTL-Exo incubation. ( * P < 0.05, ** P < 0.01, *** P <0.001, **** P < 0.0001). SIMI sepsis-induced myocardial injury, PIWIL2 piwi-like protein 2, LPS lipopolysaccharide, Exo exosome, qPCR quantitative polymerase chain reaction

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against mTOR, PIWIL2, and WTAP (all from Proteintech, USA), as well as p62 and LC3 I/II (both from Cell Signaling Technology, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Incubation, Real-time Polymerase Chain Reaction

Journal: Biochemical and biophysical research communications

Article Title: Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells.

doi: 10.1016/j.bbrc.2010.05.022

Figure Lengend Snippet: Fig. 1. Selective expression of PiwiL2 in adult and aged bone marrow MSCs. (A) RT-PCR quantification of PiwiL2 transcripts: RT-PCR was implemented on RNA from passage 12 bone marrow MSCs from mice at the indicated age groups as described in Section 2 (n = 3; p < 0.05). (B) FACS quantification of PiwiL2 expression: MSCs from each age group at passage 14 were analyzed by FACS as described in Section 2, results are representative of three separate experiments. (C) PiwiL2 expression in testis and MSCs: protein lysates were prepared from mouse testis or MSCs from 8-month mice, immunoprecipitated and analyzed by Western blots as described in Section 2. (D) Identification of micro-RNAs: MSCs from 2-month mice were transfected with PiwiL2 cDNA; RNA extracted from 2 months ± transfection or 8 months was analyzed by Northern blot and probed with anti-piRNA oligonucleotide as described in Section 2. (E) Northern blot comparison of testis and MSC piRNA: RNA was purified from mouse testes or MSCs as indicated. Testis (20 lg) and MSC (200 lg) RNAs were electrophoresed on 15% denaturing PAGE gels and stained with SybrGold (Molecular Probes). Results are representative of at least three separate experiments.

Article Snippet: Equal amounts of protein were incubated with anti-PiwiL2 antibody and A/G agarose beads (Santa Cruz).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Transfection, Northern Blot, Comparison, Staining

Journal: Biochemical and biophysical research communications

Article Title: Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells.

doi: 10.1016/j.bbrc.2010.05.022

Figure Lengend Snippet: Fig. 2. Intracellular localization of PiwiL2 by immunostaining. Bone marrow MSCs from 8-month-old mice were cultured on chamber slides, fixed and stained as described in Section 2. No staining was evident when the primary antibody was replaced with irrelevant IgG antiserum (not shown).

Article Snippet: Equal amounts of protein were incubated with anti-PiwiL2 antibody and A/G agarose beads (Santa Cruz).

Techniques: Immunostaining, Cell Culture, Staining

Journal: Biochemical and biophysical research communications

Article Title: Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells.

doi: 10.1016/j.bbrc.2010.05.022

Figure Lengend Snippet: Fig. 3. PiwiL2 expression in metaphase synchronized cultures and effects of knockdown on proliferation. MSCs from 8-month mice were synchronized, labeled with PiwiL2- FITC antibody and analyzed by FACS as described in Section 2. (A) Metaphase cells before and after synchronization. (B) PiwiL2-FITC stain displays a rightward shift to higher PiwiL2 expression in synchronized cells. (C) MSCs from 8-month-old mice were transfected with an optimal concentration of PiwiL2 siRNA. PiwiL2 mRNA was undetectable in cells after siRNA knockdown (not shown). Cells were quantified at each time point as described in Section 2. Each point is the results of three separate experiments in triplicate (p < 0.05). (D) Cells were treated as described in (c) and cell cycle determined as described in Section 2. Results from three separate experiments (n = 3; p < 0.05).

Article Snippet: Equal amounts of protein were incubated with anti-PiwiL2 antibody and A/G agarose beads (Santa Cruz).

Techniques: Expressing, Knockdown, Labeling, Staining, Transfection, Concentration Assay

Journal: Biochemical and biophysical research communications

Article Title: Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells.

doi: 10.1016/j.bbrc.2010.05.022

Figure Lengend Snippet: Fig. 4. Microarray analyses and RT-PCR of putative PiwiL2-regulated genes. (A) MSCs from 8-month mice were synchronized, RNA extracted and subjected to microarray as described in Section 2. Significantly expressed genes were clustered using GeneSpring software (Silicon Genetics, Redwood City, CA). Arrows in (A) indicate down-regulated genes common to unsynchronized and synchronized cells. (B) Overlapping down and up-regulated genes common to synchronized and unsynchronized cells. (C) Gene expression quantified by microarray or RT-PCR of RNA from 8-month MSCs transfected with PiwiL2 siRNA or random sequence (RS) siRNA. Transcript changes for each gene shown were significant (n = 3; p < 0.05). indicates quantification by RT-PCR.

Article Snippet: Equal amounts of protein were incubated with anti-PiwiL2 antibody and A/G agarose beads (Santa Cruz).

Techniques: Microarray, Reverse Transcription Polymerase Chain Reaction, Software, Gene Expression, Transfection, Sequencing

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 1: Characteristic Piwil2 expression in cervical cancer and its precursor stages. a. Piwil2 expression detected by IHC in CIN lesion and cervical cancer tissue. Scale bar, 100 μm. b. Immunoreactive score of Piwil2 staining in CIN lesions and cervical cancer tissue. c. Western blot analysis of Piwil2 expression in CIN lesions and cervical cancer tissue. GAPDH expression is presented as a loading control. d. Western blot analysis of Piwil2 expression in cervical cancer cell lines.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques: Expressing, Staining, Western Blot, Control

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 2: Piwil2 knockdown affects cervical cancer cell line proliferation, invasion, and in vivo tumorigenicity. a. HeLa, SiHa, and CaSki cells were stably transfected with control shRNA or Piwil2 shRNA, and cell viability was measured daily. b. Numbers of invading cells in clones stably transfected with control shRNA and Piwil2 shRNA. c. Equal amounts of lysates from cancer cell lines stably transfected with control shRNA or Piwil2 shRNA were separated by SDS-PAGE, and proteins were analyzed by western blotting with specific antibodies against Piwil2 and molecules regulating cell proliferation. d. Tumor growth over time was measured after the subcutaneous injection of 5×106 of SiHa cells stably transfected with shPiwil2 control shRNA and Piwil2 shRNA. Tumor volume was monitored by caliper measurements twice weekly, and tumor weight was measured after sacrifice at the end of the experiment. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student’s t-test.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques: Knockdown, In Vivo, Stable Transfection, Transfection, Control, shRNA, Clone Assay, SDS Page, Western Blot, Injection

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 3: Piwil2 overexpression induces HaCaT cell malignant transformation. a. HaCaT cells were transfected with lentivirus containing Piwil2 or lentiviral vector, and cell numbers were plotted daily. b.-c. Colony formation numbers and invading HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. d. Proteins were analyzed by western blotting for c-Myc, E-cadherin, and molecules regulating cell proliferation and apoptosis. e.-f. The expression of EMT markers was determined by qRT-PCR and western blotting in HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student’s t-test.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques: Over Expression, Transformation Assay, Transfection, Plasmid Preparation, Infection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 4: Piwil2 initiates tumorigenicity of HaCaT cells. a. Approximately 5×106 HaCaT cells transfected with Piwil2 or Vector were injected subcutaneously into the oxters of nude mice. Four weeks after injection, tumorigenesis in nude mice was observed. b. Tumor volume was monitored by caliper measurements twice a week, and tumor weight was measured after sacrifice at the end of the experiment. The data are presented as the mean±SD. ** P < 0.01 by Student’s t-test.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques: Transfection, Plasmid Preparation, Injection

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 5: Piwil2 facilitates the establishment of a cancer stem-like cell gene-set enrichment pattern. a. Gene-set enrichment in HaCaT cells transfected with lentivirus containing Piwil2 compared with cells transfected with lentiviral vector. Red, gene- set enrichment; black, no significant enrichment. b. GSEA analyses demonstrating the gene set expression and the relationship between target gene expression and factor occupancy. c.-d. The expression of 5 transcription factors associated with cell reprogramming was determined by qRT-PCR and western blotting in HaCaT cells transfected with lentivirus containing Piwil2 or lentiviral vector. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student’s t-test.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques: Transfection, Plasmid Preparation, Expressing, Targeted Gene Expression, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 6: The HR-HPV oncoproteins E6 and E7 reactivate Piwil2 expression in HaCaT cells. a.-c. qRT-PCR, RT-PCR and western blotting reveal upregulated expression of Piwil2 in HaCaT cells transfected with lentivirus containing complete HPV16 E6 and E7 sequences compared with those transfected with lentiviral vector. d. Gene-set enrichment pattern of HaCaT cells transfected with lentivirus containing complete HPV16 E6 and E7 sequences. Red, gene-set enrichment; black, no significant enrichment. e. GSEA analyses demonstrating the gene set expression and the relationship between target gene expression and factor occupancy. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student’s t-test.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Targeted Gene Expression

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 7: Piwil2 initiates cell reprogramming by regulating the balance between the acetylation and trimethylation of H3K9. a. Western blot showing significantly induced H3K9 acetylation but reduced H3K9 trimethylation in HaCaT cells transfected with Piwil2 or E6 and E7 compared with those only transfected with vector. b. Co-immunoprecipitation showing that Piwil2, either overexpressed or induced by E6 and E7, interacted with acetylated H3K9. c. EMT markers upregulated in SiHa cells exhibiting Piwil2 overexpression but downregulated in those cells in which Piwil2 was knocked down, as verified by qRT-PCR. d. The proportion of ALDH- , MSCA-1-, and ABCG2-positive cells, determined by FACS in cells with Piwil2 overexpression or knockdown. e. The LD50 dose of cisplatin, detected by CCK8 assay in cells with Piwil2 overexpression or knockdown. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student’s t-test.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Over Expression, Quantitative RT-PCR, Knockdown, CCK-8 Assay

Journal: Oncotarget

Article Title: Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

doi: 10.18632/oncotarget.11810

Figure Lengend Snippet: Figure 8: Model of cell reprogramming and tumor-initiating cell formation secondary to Piwil2 induction.

Article Snippet: To knock down Piwil2 expression, the human cervical cancer cell lines HeLa, SiHa, CaSki, and C33a were transfected in 6-well plates with 1 μg Piwil2 shRNA expression plasmids (TG302470, Origene, Beijing), which contained four unique 29-mer shRNA constructs, respectively (1.

Techniques:

Journal: Nucleic Acids Research

Article Title: A piRNA utilizes HILI and HIWI2 mediated pathway to down-regulate ferritin heavy chain 1 mRNA in human somatic cells

doi: 10.1093/nar/gky728

Figure Lengend Snippet: piR-FTH1 associates with HIWI and HILI and utilizes the HIWI pathway for its silencing function. ( A ) The relative level of Fth1 mRNA level to Gapdh 24 hrs after piR-FTH1(NM) treatment in MDA-MB-231 cells was quantified by RT-qPCR. The cells treated with scramble piR-FTH1(NM) act as a control for this experiment. ( B ) Histogram representing the relative Hiwi, Hili, Hiwi2 and Ago2 mRNA levels to Gapdh mRNA 24 hrs after the HIWI, HILI and HIWI2 and AGO2 siRNA treatment were generated by RT-qPCR. The control cells were treated with control siRNAs. ( C ) Western blot analysis confirmed HIWI, HILI, HIWI2 and AGO2 knockdown after transfection with HIWI, HILI, HIWI2 and AGO2 siRNAs in MDA-MB-231 cell lines. ( D ) Histogram representing the relative Fth1 mRNA level to Gapdh mRNA 24 hrs after the piR-FTH1(50 nM) and piR-FTH1(NM) (300 nM) transfection in the HIWI, HILI, HIWI2 and AGO2 depleted MDA-MB-231 cell lines were generated by RT-qPCR. The cells treated with scramble piR-FTH1(50 nM) and scramble piR-FTH1(NM) (300 nM) considered as a control for piR-FTH1 and piR-FTH1(NM) respectively. The HILI and HIWI2 knockdown rescued the Fth1 mRNA expression after piR-FTH1 confirmed the HIWI family protein dependent functional role of piR-FTH1. ( E ) Immunocytochemistry of FTH1 in control and HIWI2 depleted MDA-MB-231 cell, DAPI (4′,6-diamidino-2-phenylindole) in the first panel from left, FTH1 stained with green in the second panel from left, HIWI2 stained with red in the third panel from left and merged in the right panel. The presence of clear FTH1 staining signal in HIWI2 depleted cells after piR-FTH1 treatment confirmed the importance of HIWI2 in piR-FTH1 function. ( F ) The relative piR-FTH1 and piR-58320 in HILI and HIWI2 immunoprecipitated RNA from HEK-293 cells was quantified by TaqMan small RNA assay. The levels of piR-FTH1, piR-58320 and miR-382 were normalized to exogenous piR-48164. 10 pmol of a known synthetic piRNA (piR-48164) was added to the IP as an exogenous RNA control. The levels of piR-FTH1, piR-58320 and miR-382 in total RNA pool were used as controls to calculate the relative amount of those RNA level in the IPs. This was done by considering each of the values of piR-FTH1, piR-58320 and miR-382 in total RNA pool as 1 and normalizing the measured amount of those RNAs in the IP samples to calculate the relative percentage of each of the RNAs. The piR-58230 was used as a positive control and miR-382 was used as a negative control for this experiment. The endogenous piR-FTH1 loaded into HILI and HIWI2 proteins. ( G ) The synthetically made transfected piR-FTH1 and scramble piR-FTH1 were detected in HILI (left panel) and HIWI2 (right panel) IPs from MDA-MB-231 cell lysate. piR-FTH1(NM) served as a negative control. The input denotes the amount of radiolabeled RNAs (piR-FTH1, scramble piR-FTH1 and piR-FTH1(NM)) were transfected into the cells. The results are presented as the mean ± SEM ( n = 3) of three independent experiments. The statistical significance was calculated by t- test analysis. (*** P < 0.005).

Article Snippet: For knockdown studies, siRNAs for HIWI (sc-40677), HILI (sc-62456), HIWI2 (sc-62458) and AGO2 (sc-44409) were purchased from Santa Cruz.

Techniques: Quantitative RT-PCR, Control, Generated, Western Blot, Knockdown, Transfection, Expressing, Functional Assay, Immunocytochemistry, Staining, Immunoprecipitation, Positive Control, Negative Control

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Decreased Expression Levels Of PIWIL2, PIWIL3 and PIWIL4 Are Associated with Poor Prognosis and Worse Survival in Bladder Cancer Patients

doi: 10.31557/APJCP.2025.26.7.2467

Figure Lengend Snippet: a) section in a case of chronic cystitis showing high expression of PIWIL2 in the lining urothelium (black arrow), b) section in papillary transitional cell carcinoma showing lower expression of PIWIL2 in urothelial cells (black arrow) compared to non-malignant urothelium; c) section in case of invasive high grade UC showing focal positive (red arrow) and focal negative (yellow arrow) expression of PIWIL2 in malignant cells.; d) sections in invasive poorly differentiated squamous cell carcinoma showing low expression of PIWIL2 in malignant cells (black arrow) (Haematoxylin and eosin stain, X200).

Article Snippet: Tissue sections (4 μM thick) were cut from each block and stained using the PIWIL2 antibody (Novus Biologicals, LLC., USA), NBP2-24590 PIWIL2/HILI, following the manufacturer’s protocol.

Techniques: Expressing, H&E Stain

Journal: Nature

Article Title: Differential LINE-1 regulation in pluripotent stem cells of humans and other great apes

doi: 10.1038/nature12686

Figure Lengend Snippet: List of primers used in this study.

Article Snippet: Primary antibodies used in this study were: TRA-1-81 (1:100, Millipore, MAB4381), Nanog (1:500, R&D Systems, AF1997), APOBEC3B (D-15) (1:500, Santa Cruz, sc-86289), PIWIL2 (1:1000, R&D Systems, AF6558), GFP (1:200, Molecular Probes-Invitrogen, A-6455), Flag (1:1000 Sigma, F7425) and HA (1:1000 Sigma, H6908).

Techniques: Sequencing, Cloning, TaqMan Assay

Journal: Nature

Article Title: Differential LINE-1 regulation in pluripotent stem cells of humans and other great apes

doi: 10.1038/nature12686

Figure Lengend Snippet: Relative APOBEC3B and PIWIL2 mRNA levels in iPSC and fibroblasts. Relative expression of ( a ) A3B and ( b ) PIWIL2 in human and NHP iPSC lines, and the available source fibroblasts from, which iPSCs were derived from. mRNA levels were normalized to GAPDH and shown as relative to human iPSC1.

Article Snippet: Primary antibodies used in this study were: TRA-1-81 (1:100, Millipore, MAB4381), Nanog (1:500, R&D Systems, AF1997), APOBEC3B (D-15) (1:500, Santa Cruz, sc-86289), PIWIL2 (1:1000, R&D Systems, AF6558), GFP (1:200, Molecular Probes-Invitrogen, A-6455), Flag (1:1000 Sigma, F7425) and HA (1:1000 Sigma, H6908).

Techniques: Expressing, Derivative Assay

Journal: Cell Death & Disease

Article Title: PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation

doi: 10.1038/s41419-018-0462-8

Figure Lengend Snippet: a PIWIL2 can only interact with HDAC3 among class I HDACs. b Endogenous interaction between PIWIL2 and HDAC3. c PIWIL2 binds to HDAC3 in a TNT ® Quick Coupled Transcription/Translation System. d Co-localization of PIWIL2 and HDAC3 using immunofluorescence assays. e Schematics of PIWIL2 deletion mutants and HDAC3 deletion mutants. f PIWI domain is necessary for PIWIL2 binding with HDAC3. The “ *” indicates a non-specific bind. g C-terminal region of HDAC3 is necessary for HDAC3 binding with PIWIL2

Article Snippet: A set of deletion mutants and wide type PIWIL2 were constructed in our previous study . shRNA against PIWIL2, HDAC3, CK2 were synthesized by GenePharma company (Shanghai, China). shRNA for PIWIL2, HDAC3, and CK2 were synthesized and cloned into pGPU6/GFP/Neo, and the target sequences of these shRNA were as follows: PIWIL2 shRNA (shPIWIL2): 5′-CTATGAGATTCCTCAACTACAGAAG-3′ HDAC3 shRNA(shHDAC3): 5′-GCTGGTAGAAGAGGCCATTAG-3′ CK2α shRNA(shCK2α): 5′-ATTACCTGCAGGTGGAATATT-3′ CK2α’ shRNA(shCK2α’): 5′-CCTCACAATGTCATGATAGAT-3′ Antibodies against each named protein were: PIWIL2 (Santa Cruz, CA, USA); HDAC1, HDAC2, and HDAC3 (Cell signaling technology, MA, USA); HDAC8, P-HDAC3 (EnoGene, Nanjing, China); Siah2 (Proteintech, IL, USA); CK2α (Abgent, Suzhou, China); p53, HA, Myc (Santa Cruz, CA, USA); p21, GAPDH (Cell signaling technology, MA, USA).

Techniques: Immunofluorescence, Binding Assay

Journal: Cell Death & Disease

Article Title: PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation

doi: 10.1038/s41419-018-0462-8

Figure Lengend Snippet: a , b Expression of PIWIL2 and HDAC3 in tumor and adjacent normal tissues from 30 pairs of cervical cancer patients were examined. The magnification is ×400. * P < 0.05 and ** P < 0.01. c PIWIL2 up-regulates HDAC3 expression at protein level in Hela cells and HepG2 cells. d Knockdown of PIWIL2 specifically decreases expression of only HDAC3 among class I HDACs. e Immunofluorescent staining of HDAC3 in PIWIL2 shRNA transfected Hela and HepG2 cells. f PIWIL2 has no effect on HDAC3 mRNA expression (N.S., not significant, P > 0.05). g The degradation of HDAC3 is faster in cells transfected with PIWIL2 shRNA than in cells transfected with control plasmid. Cells transfected with shPIWIL2 or shNC were treated with cycloheximide (CHX) at 50 μM for indicated time

Article Snippet: A set of deletion mutants and wide type PIWIL2 were constructed in our previous study . shRNA against PIWIL2, HDAC3, CK2 were synthesized by GenePharma company (Shanghai, China). shRNA for PIWIL2, HDAC3, and CK2 were synthesized and cloned into pGPU6/GFP/Neo, and the target sequences of these shRNA were as follows: PIWIL2 shRNA (shPIWIL2): 5′-CTATGAGATTCCTCAACTACAGAAG-3′ HDAC3 shRNA(shHDAC3): 5′-GCTGGTAGAAGAGGCCATTAG-3′ CK2α shRNA(shCK2α): 5′-ATTACCTGCAGGTGGAATATT-3′ CK2α’ shRNA(shCK2α’): 5′-CCTCACAATGTCATGATAGAT-3′ Antibodies against each named protein were: PIWIL2 (Santa Cruz, CA, USA); HDAC1, HDAC2, and HDAC3 (Cell signaling technology, MA, USA); HDAC8, P-HDAC3 (EnoGene, Nanjing, China); Siah2 (Proteintech, IL, USA); CK2α (Abgent, Suzhou, China); p53, HA, Myc (Santa Cruz, CA, USA); p21, GAPDH (Cell signaling technology, MA, USA).

Techniques: Expressing, Knockdown, Staining, shRNA, Transfection, Control, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation

doi: 10.1038/s41419-018-0462-8

Figure Lengend Snippet: a MG132-treatment recovers HDAC3 protein level reduced by shPIWIL2. Cells were transfected with shPIWIL2 for 48 h and then treated with MG132 (10 μM) or DMSO (negative control) for 6 h. b PIWIL2 knockdown enhances the ubiquitin-mediated degradation of HDAC3. Hela cells were transfected with HA-ubiquitin vector, followed by treatment of MG132 for 6 h. Cell lysates were subjected to anti-HDAC3 antibody for WB. The “ *” indicates a non-specific bind. c PIWIL2 reduces Siah2-mediated degradation of HDAC3. Cells were transfected with PIWIL2 alone or in combination with Siah2, followed by a treatment with MG132 or DMSO as control for 6 h. d , e PIWIL2 reduces the interaction between Siah2 and HDAC3. Cells were transfected with PIWIL2 vector or PIWIL2 shRNA vector, and then analyzed by co-immuoprecipitation assays

Article Snippet: A set of deletion mutants and wide type PIWIL2 were constructed in our previous study . shRNA against PIWIL2, HDAC3, CK2 were synthesized by GenePharma company (Shanghai, China). shRNA for PIWIL2, HDAC3, and CK2 were synthesized and cloned into pGPU6/GFP/Neo, and the target sequences of these shRNA were as follows: PIWIL2 shRNA (shPIWIL2): 5′-CTATGAGATTCCTCAACTACAGAAG-3′ HDAC3 shRNA(shHDAC3): 5′-GCTGGTAGAAGAGGCCATTAG-3′ CK2α shRNA(shCK2α): 5′-ATTACCTGCAGGTGGAATATT-3′ CK2α’ shRNA(shCK2α’): 5′-CCTCACAATGTCATGATAGAT-3′ Antibodies against each named protein were: PIWIL2 (Santa Cruz, CA, USA); HDAC1, HDAC2, and HDAC3 (Cell signaling technology, MA, USA); HDAC8, P-HDAC3 (EnoGene, Nanjing, China); Siah2 (Proteintech, IL, USA); CK2α (Abgent, Suzhou, China); p53, HA, Myc (Santa Cruz, CA, USA); p21, GAPDH (Cell signaling technology, MA, USA).

Techniques: Transfection, Negative Control, Knockdown, Ubiquitin Proteomics, Plasmid Preparation, Control, shRNA

Journal: Cell Death & Disease

Article Title: PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation

doi: 10.1038/s41419-018-0462-8

Figure Lengend Snippet: a , b Hela and HepG2 cells were transfected with PIWIL2, shPIWIL2 or control vectors, respectively, followed by western blotting with antibody against P-HDAC3. c Treatment with CK2 inhibitor TBB abolishes the up-regulation of P-HDAC3 by PIWIL2. Hela cells transfected with PIWIL2 vector or control vector were treated with CK2 inhibitor TBB, PI3K inhibitor LY294002 or GSK3β inhibitor TWS119 for 6 h. d Knockdown of CK2 (shCK2α and shCK2α’) blocks PIWIL2 from upregulating P-HDAC3

Article Snippet: A set of deletion mutants and wide type PIWIL2 were constructed in our previous study . shRNA against PIWIL2, HDAC3, CK2 were synthesized by GenePharma company (Shanghai, China). shRNA for PIWIL2, HDAC3, and CK2 were synthesized and cloned into pGPU6/GFP/Neo, and the target sequences of these shRNA were as follows: PIWIL2 shRNA (shPIWIL2): 5′-CTATGAGATTCCTCAACTACAGAAG-3′ HDAC3 shRNA(shHDAC3): 5′-GCTGGTAGAAGAGGCCATTAG-3′ CK2α shRNA(shCK2α): 5′-ATTACCTGCAGGTGGAATATT-3′ CK2α’ shRNA(shCK2α’): 5′-CCTCACAATGTCATGATAGAT-3′ Antibodies against each named protein were: PIWIL2 (Santa Cruz, CA, USA); HDAC1, HDAC2, and HDAC3 (Cell signaling technology, MA, USA); HDAC8, P-HDAC3 (EnoGene, Nanjing, China); Siah2 (Proteintech, IL, USA); CK2α (Abgent, Suzhou, China); p53, HA, Myc (Santa Cruz, CA, USA); p21, GAPDH (Cell signaling technology, MA, USA).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Knockdown

Journal: Cell Death & Disease

Article Title: PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation

doi: 10.1038/s41419-018-0462-8

Figure Lengend Snippet: a Co-immunoprecipitation assays showed the interaction of PIWIL2, CK2α, and HDAC3. b Cell lysates were immunoprecipitated with anti-HA antibody, and then a secondary immunoprecipitation assay was performed with anti-PIWIL2 antibody. c Immunofluorescence assays showed the co-localization of PIWIL2, CK2α, and HDAC3. d , e PIWIL2 facilitates CK2α binding with HDAC3 to phosphorylate HDAC3

Article Snippet: A set of deletion mutants and wide type PIWIL2 were constructed in our previous study . shRNA against PIWIL2, HDAC3, CK2 were synthesized by GenePharma company (Shanghai, China). shRNA for PIWIL2, HDAC3, and CK2 were synthesized and cloned into pGPU6/GFP/Neo, and the target sequences of these shRNA were as follows: PIWIL2 shRNA (shPIWIL2): 5′-CTATGAGATTCCTCAACTACAGAAG-3′ HDAC3 shRNA(shHDAC3): 5′-GCTGGTAGAAGAGGCCATTAG-3′ CK2α shRNA(shCK2α): 5′-ATTACCTGCAGGTGGAATATT-3′ CK2α’ shRNA(shCK2α’): 5′-CCTCACAATGTCATGATAGAT-3′ Antibodies against each named protein were: PIWIL2 (Santa Cruz, CA, USA); HDAC1, HDAC2, and HDAC3 (Cell signaling technology, MA, USA); HDAC8, P-HDAC3 (EnoGene, Nanjing, China); Siah2 (Proteintech, IL, USA); CK2α (Abgent, Suzhou, China); p53, HA, Myc (Santa Cruz, CA, USA); p21, GAPDH (Cell signaling technology, MA, USA).

Techniques: Immunoprecipitation, Immunofluorescence, Binding Assay

Journal: Cell Death & Disease

Article Title: PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation

doi: 10.1038/s41419-018-0462-8

Figure Lengend Snippet: a PIWIL2 knockdown reduces the binding of HDAC3 and increases Ac-H3 level on P53 promoter. b HDAC3 overexpression recovers the up-regulation of p53 and p21 by PIWIL2 knockdown. c , d In Hela cells, HDAC3 inhibition eliminates the effect of PIWIL2 on cell proliferation while HDAC3 overexpression recovers the impact of PIWIL2 knockdown on cell proliferation ( P < 0.05). e , f HDAC3 inhibition eliminates the inhibition role of PIWIL2 on cell apoptosis. ( P < 0.05)

Article Snippet: A set of deletion mutants and wide type PIWIL2 were constructed in our previous study . shRNA against PIWIL2, HDAC3, CK2 were synthesized by GenePharma company (Shanghai, China). shRNA for PIWIL2, HDAC3, and CK2 were synthesized and cloned into pGPU6/GFP/Neo, and the target sequences of these shRNA were as follows: PIWIL2 shRNA (shPIWIL2): 5′-CTATGAGATTCCTCAACTACAGAAG-3′ HDAC3 shRNA(shHDAC3): 5′-GCTGGTAGAAGAGGCCATTAG-3′ CK2α shRNA(shCK2α): 5′-ATTACCTGCAGGTGGAATATT-3′ CK2α’ shRNA(shCK2α’): 5′-CCTCACAATGTCATGATAGAT-3′ Antibodies against each named protein were: PIWIL2 (Santa Cruz, CA, USA); HDAC1, HDAC2, and HDAC3 (Cell signaling technology, MA, USA); HDAC8, P-HDAC3 (EnoGene, Nanjing, China); Siah2 (Proteintech, IL, USA); CK2α (Abgent, Suzhou, China); p53, HA, Myc (Santa Cruz, CA, USA); p21, GAPDH (Cell signaling technology, MA, USA).

Techniques: Knockdown, Binding Assay, Over Expression, Inhibition

Journal: Cell Death & Disease

Article Title: PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation

doi: 10.1038/s41419-018-0462-8

Figure Lengend Snippet: a In WT cancer cells (at least Hela and HepG2), PIWIL2 stabilizes HDAC3 by inhibiting Siah2-mediated degradation. PIWIL2 binding enhances CK2-mediated phosphorylation of HDAC3. As a result, the expression of p53 is reduced followed by a decrease of p21. b In PIWIL2 knockdown cells, p53 has a relative high expression level and p21 as well

Article Snippet: A set of deletion mutants and wide type PIWIL2 were constructed in our previous study . shRNA against PIWIL2, HDAC3, CK2 were synthesized by GenePharma company (Shanghai, China). shRNA for PIWIL2, HDAC3, and CK2 were synthesized and cloned into pGPU6/GFP/Neo, and the target sequences of these shRNA were as follows: PIWIL2 shRNA (shPIWIL2): 5′-CTATGAGATTCCTCAACTACAGAAG-3′ HDAC3 shRNA(shHDAC3): 5′-GCTGGTAGAAGAGGCCATTAG-3′ CK2α shRNA(shCK2α): 5′-ATTACCTGCAGGTGGAATATT-3′ CK2α’ shRNA(shCK2α’): 5′-CCTCACAATGTCATGATAGAT-3′ Antibodies against each named protein were: PIWIL2 (Santa Cruz, CA, USA); HDAC1, HDAC2, and HDAC3 (Cell signaling technology, MA, USA); HDAC8, P-HDAC3 (EnoGene, Nanjing, China); Siah2 (Proteintech, IL, USA); CK2α (Abgent, Suzhou, China); p53, HA, Myc (Santa Cruz, CA, USA); p21, GAPDH (Cell signaling technology, MA, USA).

Techniques: Binding Assay, Phospho-proteomics, Expressing, Knockdown

Journal: PLoS ONE

Article Title: PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

doi: 10.1371/journal.pone.0097821

Figure Lengend Snippet: Differentially expressed genes in triple knockout iPS cells.

Article Snippet: Gene expression levels were determined using Applied Biosystems TaqMan Gene Expression Assays: Miwi : Mm00451649_m1; Mili : Mm01313483_m1; Miwi2 : Mm01144775_m1; HIWI : Hs01041737_m1; HILI : Hs00216263_m1; HIWI2 : Hs00381509_m1, and detected by iCycler iQ (Bio-Rad, Hercules, CA).

Techniques: Triple Knockout