Journal: PLoS ONE

Article Title: Epigenetic Disruption of the PIWI Pathway in Human Spermatogenic Disorders

doi: 10.1371/journal.pone.0047892

Figure Lengend Snippet: Bisulfite sequencing of the piRNA processing genes PIWIL1 (A), PIWIL2 (B), TDRD1 (C) and TDRD9 (D). Black and white squares indicate CpG methylation and unmethylated sites, respectively. One representative sample of a large difference between testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and in Sertoli cell-only syndrome (SCO) are displayed. Sample number corresponding to that in is also indicated. (E) Methylation level of gene promoters of PIWIL1, PIWIL2 , TDRD1 and TDRD9 in testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and in Sertoli cell-only syndrome (SCO) samples. Independent data are also shown in and . Significant differences compared to CS samples are indicated (*).

Article Snippet: Quantitative real-time PCR (qPCR) reactions were performed on an ABI 7300 real-time PCR system (Applied Biosystems) using gene-specific TaqMan Assays ( PIWIL2 : Hs00216263_m1; TDRD1 : Hs00229805_m1; PGM1 : Hs00160062_m1) and custom-designed small RNA TaqMan Assays (Applied Biosystems).

Techniques: Methylation Sequencing, CpG Methylation Assay, Methylation

Journal: PLoS ONE

Article Title: Epigenetic Disruption of the PIWI Pathway in Human Spermatogenic Disorders

doi: 10.1371/journal.pone.0047892

Figure Lengend Snippet: Methylation levels of PIWIL2 (A) and TDRD1 (B) in mature spermatozoa (sperm), testis with a conserved spermatogenic pattern (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages, Sertoli cell-only syndrome (SCO) and somatic tissue measured by pyrosequencing. The black bar indicates the mean methylation level. Methylation per cell profiling of PIWIL2 and TDRD1 , displayed as methylation per somatic cell (x100) (C, D) and methylation per germ cell (x100) (E, F) in testis with conserved spermatogenesis (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages and Sertoli cell-only syndrome (SCO). The horizontal bar displays the mean cellular expression level. Significant differences from the control are indicated: *p<0.05; **p<0.01.

Article Snippet: Quantitative real-time PCR (qPCR) reactions were performed on an ABI 7300 real-time PCR system (Applied Biosystems) using gene-specific TaqMan Assays ( PIWIL2 : Hs00216263_m1; TDRD1 : Hs00229805_m1; PGM1 : Hs00160062_m1) and custom-designed small RNA TaqMan Assays (Applied Biosystems).

Techniques: Methylation, Expressing, Control

Journal: PLoS ONE

Article Title: Epigenetic Disruption of the PIWI Pathway in Human Spermatogenic Disorders

doi: 10.1371/journal.pone.0047892

Figure Lengend Snippet: Tissular expression profiling of PIWIL2 (A) and TDRD1 (B) by quantitative real-time qPCR in testis with conserved spermatogenesis (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages and Sertoli cell-only syndrome (SCO). Expression levels relative to PGM1 are shown. Expression per cell profiling of PIWIL2 , displayed as expression ratio per spermatogonia/spermatocyte (x1000) (C) and expression per cell of TDRD1 , displayed as expression ratio per spermatocyte (X1000) (D) in testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF), the round spermatid (rsMF), and the spermatogonia (sgMF) stages. The horizontal bar displays the mean cellular expression level. Significant differences from the control are indicated: *p<0.05; **p<0.01.

Article Snippet: Quantitative real-time PCR (qPCR) reactions were performed on an ABI 7300 real-time PCR system (Applied Biosystems) using gene-specific TaqMan Assays ( PIWIL2 : Hs00216263_m1; TDRD1 : Hs00229805_m1; PGM1 : Hs00160062_m1) and custom-designed small RNA TaqMan Assays (Applied Biosystems).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Epigenetic Disruption of the PIWI Pathway in Human Spermatogenic Disorders

doi: 10.1371/journal.pone.0047892

Figure Lengend Snippet: Pearson correlation coefficients and adjusted p values (r;p) between the molecular and the histological parameters for all the samples analysed.

Article Snippet: Quantitative real-time PCR (qPCR) reactions were performed on an ABI 7300 real-time PCR system (Applied Biosystems) using gene-specific TaqMan Assays ( PIWIL2 : Hs00216263_m1; TDRD1 : Hs00229805_m1; PGM1 : Hs00160062_m1) and custom-designed small RNA TaqMan Assays (Applied Biosystems).

Techniques:

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: Scientific Reports

Article Title: PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells

doi: 10.1038/s41598-018-34861-2

Figure Lengend Snippet: Silencing of PIWIL2 and PIWIL4 decreases piRNA expression during ER stress. ( A ) 16HBE14o- cells were transfected with siRNA against PIWIL2 (siPIWIL2), PIWIL4 (siPIWIL4) or co-transfected with both (siPIWL2/4) and treated with TM (2.5 µg/ml) or ALLN (100 µM) for 6 hours, and the corresponding changes in PIWIL2 and PIWIL4 protein levels were detected with SDS-PAGE and Western Blot and normalized to the β-Actin (2 µg of total protein per lane). The experiments were repeated twice and the error bars represent standard errors. Significant changes (p < 0.05) are marked with an asterisk. The original blots and gels are provided in Supplemental Fig. . ( B ) Reduction of PIWIL2 and PIWIL4 relative expression with siRNAs (siPIWIL2/4) lowers piRNAs levels after 6 hours of ER stress. The piRNAs levels were accessed by next generation small RNA sequencing and expressed as fold change relative to the no stress control. The groups (ALLN and ALLN siPIWIL2/4; TM and TM siPWIL2/4) were compared using ANOVA on ranks. Significant changes (p < 0.05) are marked with an asterisk.

Article Snippet: TaqMan probes ids used were: TBP - Hs4332659_m1; BIP - Hs00607129_gH; CHOP - Hs00358796_g1; PIWIL2 - Hs01032720_m1; PIWIL4 – Hs00381509_m1;; NOXA Hs00560402_m1.

Techniques: Expressing, Transfection, SDS Page, Western Blot, RNA Sequencing, Control

Journal: Scientific Reports

Article Title: PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells

doi: 10.1038/s41598-018-34861-2

Figure Lengend Snippet: ER stressors have different effects on the expression of PIWIL2 and PIWIL4 mRNA and protein. ( A ) 16HBE14o- cells were treated with TM (2.5 µg/ml) or ALLN (100 µM) for time points specified and PIWIL2 and PIWIL4 mRNA levels from 2 independent experiments (n = 8) are plotted normalized to TBP mRNA levels and expressed as a fold-change over the no stress controls. Error bars represent standard deviations. Significant changes (p < 0.05) are marked with an asterisk. ( B ) The corresponding changes of PIWIL2 and PIWIL4 protein levels were detected with SDS-PAGE and Western Blot and normalized to the β-Actin and 2 individual samples (2 µg of total protein per lane) were tested for each treatment and the experiments were repeated twice. The original blots and gels are provided in Supplemental Fig. .

Article Snippet: TaqMan probes ids used were: TBP - Hs4332659_m1; BIP - Hs00607129_gH; CHOP - Hs00358796_g1; PIWIL2 - Hs01032720_m1; PIWIL4 – Hs00381509_m1;; NOXA Hs00560402_m1.

Techniques: Expressing, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells

doi: 10.1038/s41598-018-34861-2

Figure Lengend Snippet: Silencing of PIWIL2 and PIWIL4 decreases caspase 3/7 activity. ( A ) 16HBE14o- cells were transfected with control siRNA (Ctrl); PIWIL2 siRNA (siPWIL2); PIWIL4 siRNA (siPWIL4); or both PIWIL2 and PIWIL4 siRNAs (siPWIL2/4), and treated with ER stressors (TM (2.5 µg/ml) - left panel, ALLN (100 µM) - right panel) for time points specified. The caspase 3/7 activity was monitored by luminescence and expressed in Relative Light Units (R.L.U). Cells for each time point were seeded in triplicate, and the experiments repeated twice. Error bars represent standard derivation. ( B ) Corresponding real time analysis of 16HBE14o- cell survival during ER stress during PIWIL mRNA silencing. The cell conductances (expressed as normalized cell index) were accessed every 15 minutes following 24 hours of treatment with TM (2.5 µg/ml), ALLN (100 µM) or DMSO (CTRL). The conductances were normalized to the last value prior to experimental start. The results from 3 measurements (n = 3) are plotted as slopes obtained from the cell growth curves. Error bars represent standard deviations. The experiments were repeated twice. Significant changes (p < 0.05) are marked with an asterisk.

Article Snippet: TaqMan probes ids used were: TBP - Hs4332659_m1; BIP - Hs00607129_gH; CHOP - Hs00358796_g1; PIWIL2 - Hs01032720_m1; PIWIL4 – Hs00381509_m1;; NOXA Hs00560402_m1.

Techniques: Activity Assay, Transfection, Control

Journal: Scientific Reports

Article Title: PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells

doi: 10.1038/s41598-018-34861-2

Figure Lengend Snippet: Silencing of PIWIL2/4 decreases CHOP and NOXA mRNA expression levels during ER stress. 16HBE14o- cells were transfected with control siRNA or both PIWIL2 and PIWIL4 siRNAs (siPWIL2/4), and treated with ER stressors (TM (2.5 µg/ml) - left panels, ALLN (100 µM) - right panels) for time points specified. The corresponding mRNA levels of ( A ) BIP , ( B ) CHOP , and ( C ) NOXA from 2 independent experiments (n = 8) are plotted normalized to TBP mRNA levels and expressed as a fold-change over the no stress siRNA controls. Error bars represent standard deviations. Significant changes (p < 0.05) are marked with an asterisk.

Article Snippet: TaqMan probes ids used were: TBP - Hs4332659_m1; BIP - Hs00607129_gH; CHOP - Hs00358796_g1; PIWIL2 - Hs01032720_m1; PIWIL4 – Hs00381509_m1;; NOXA Hs00560402_m1.

Techniques: Expressing, Transfection, 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: 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

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: 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 5×10 6 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: Stable Transfection, Transfection, 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: 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: 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: a. Approximately 5×10 6 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: 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, 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: 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: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation

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: 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, 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: 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: 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

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: 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

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: 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 5×10 6 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: Stable Transfection, Transfection, 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: 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: 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: a. Approximately 5×10 6 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: 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, 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: 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: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation

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: 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, 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: 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: