Journal: Nucleic acids research

Article Title: Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

doi: 10.1093/nar/gkae002

Figure Lengend Snippet: Figure 1. Validation of K142 and K226 acetylation sites using custom-generated anti-Ac (K142 or K226) specific antibodies (Ab) in stable cell lines. ( A ) PNKP was IP’d using the anti-AcK142 Ab from the chromatin fraction of GO-treated WT (lane 1) and K142R mutant (lane 2) PNKP expressing cells and probed with the anti-FLAG Ab (upper panel). L o w er panel: Input shows similar ectopic expression of PNKP-FLAG in the cell lines, detected with the anti-FLAG Ab. ( B ) Indirect immunofluorescence was conducted to detect nuclear localization of AcK142 PNKP in GO-treated WT vs. mutant (K142R and K226R) cell lines. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( C ) PNKP was IP’d using the anti-AcK226 Ab from the chromatin fraction of mock (lanes 1, 2) or Bleo-treated (lanes 3, 4) WT (lanes 1, 3) and K226R mutant (lanes 2, 4) PNKP expressing cells and probed with anti-FLAG Ab (Upper Panel). L o w er panel sho ws the inputs, indicating similar ectopic e xpression of PNKP-FLA G in the cell lines detected with anti-FLA G Ab. ( D ) Mock- or Bleo-treated WT and mutant K226R PNKP expressing cells were stained with an anti-AcK226 Ab. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( E ) Indirect immunofluorescence w as perf ormed to assess the status of AcK226 PNKP in mock vs. Bleo-treated WT-PNKP expressing cells at 3 h, 6 h and 9 h post-Bleo treatment. Scale bar: 40 μm. All the immunofluorescence experiments were performed following depletion of endogenous PNKP by 3 ′ -UTR specific siRNA.

Article Snippet: List of antibodies used in this study Primary antibodies Source Catalog# Dilution FLAG (M2) Millipore Sigma F 1804 1:500 Acetyl lysine K142 PNKP Ez Biolabs Custom-generated 1:200 Acetyl lysine K226 PNKP Ez Biolabs Custom-generated 1:200 PNKP Bio-Bharati Life Sciences BB AB0105 1:500 CBP Santa Cruz Biotechnology Sc-7300 1:1000 RNA polymerase II (H5) Biolegend 920 202 1:500 p300 Active Motif 61 401 1:500 GAPDH GeneTex GTX100118 1:7000 HDAC2 GeneTex GTX109642 1:500 XRCC1 GeneTex GTX111712 1:500 XRCC4 GeneTex GTX109632 1:500 DNA ligase III In house ( 18 ) 1:500 DNA ligase IV GeneTex GTX108820 1:500 Ku 70 GeneTex GTX101820 1:500 Histone H2A.XS139ph (phosphoserine 139) Cell Signaling Technology 2577 1:500 phospho-53BP1 Cell Signaling Technology 2675 1:500 53BP1 Santa Cruz Biotechnology Sc-517 281 1:500 NEIL2 In house ( 18 ) 1:500 DNA polymerase η Cell signaling Technology 13848 1:500 G F b F w t i S t w m n s c p s a S S T a s w p a d n i E P d T A s t d t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae002/7547335 by guest on 16 January 2024 eneration of PNKP expressing stable cell lines or generating the WT and mutant PNKP expressing stale cell lines, HEK293 cells were transfected with PNKPLAG (WT and mutants) expressing vector (pcDNA3) in 6- ell plates using Lipofectamine 2000 (ThermoFisher Scienific) according to the manufacturer’s protocol.

Techniques: Biomarker Discovery, Generated, Stable Transfection, Mutagenesis, Expressing, Immunofluorescence, Staining

Journal: Nucleic acids research

Article Title: Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

doi: 10.1093/nar/gkae002

Figure Lengend Snippet: Figure 2. Identification of Histone Acetyl Transferases (HATs) responsible for acetylation of PNKP. ( A ) Detection of K142 specific PNKP acetylation in control (Con) siRNA vs. CBP / p300 siRNA transfected and mock / GO-treated HEK293 cells by indirect immunofluorescence using anti-AcK142 PNKP Ab. The three separate panels show nuclear DAPI st aining , Texas red Ab st aining , and the overlay. ( B ) K226-specific PNKP acetylation in control siRNA vs. CBP / p300 siRNA transfected ± Bleo-treated HEK293 cells was determined by fluorescence microscopic imaging using anti-AcK226 PNKP Ab. The scale bars for images of (A) and (B) were 40 μm.

Article Snippet: List of antibodies used in this study Primary antibodies Source Catalog# Dilution FLAG (M2) Millipore Sigma F 1804 1:500 Acetyl lysine K142 PNKP Ez Biolabs Custom-generated 1:200 Acetyl lysine K226 PNKP Ez Biolabs Custom-generated 1:200 PNKP Bio-Bharati Life Sciences BB AB0105 1:500 CBP Santa Cruz Biotechnology Sc-7300 1:1000 RNA polymerase II (H5) Biolegend 920 202 1:500 p300 Active Motif 61 401 1:500 GAPDH GeneTex GTX100118 1:7000 HDAC2 GeneTex GTX109642 1:500 XRCC1 GeneTex GTX111712 1:500 XRCC4 GeneTex GTX109632 1:500 DNA ligase III In house ( 18 ) 1:500 DNA ligase IV GeneTex GTX108820 1:500 Ku 70 GeneTex GTX101820 1:500 Histone H2A.XS139ph (phosphoserine 139) Cell Signaling Technology 2577 1:500 phospho-53BP1 Cell Signaling Technology 2675 1:500 53BP1 Santa Cruz Biotechnology Sc-517 281 1:500 NEIL2 In house ( 18 ) 1:500 DNA polymerase η Cell signaling Technology 13848 1:500 G F b F w t i S t w m n s c p s a S S T a s w p a d n i E P d T A s t d t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae002/7547335 by guest on 16 January 2024 eneration of PNKP expressing stable cell lines or generating the WT and mutant PNKP expressing stale cell lines, HEK293 cells were transfected with PNKPLAG (WT and mutants) expressing vector (pcDNA3) in 6- ell plates using Lipofectamine 2000 (ThermoFisher Scienific) according to the manufacturer’s protocol.

Techniques: Control, Transfection, Immunofluorescence, Fluorescence, Imaging

Journal: Nucleic acids research

Article Title: Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

doi: 10.1093/nar/gkae002

Figure Lengend Snippet: Figure 3. Assessment of the effect of acetylation on PNKP’s 3 ′ -phosphatase and 5 ′ -kinase activities. ( A ) The Western blot shows the expression of γH2AX (upper panel) in the chromatin fraction of WT (lanes 1, 2), K142R (lanes 3, 4), K226R (lanes 5, 6) and K142R / 226R (lanes 7, 8) PNKP expressing cells post mock (–) or Bleo (+) treatment. HD A C2 is used as the loading control for the chromatin fraction (lo w er panel). ( B ) IP’d and purified FLAG-PNKP from WT and mutant PNKP expressing cells ( ± Bleo) was immunoblotted with anti-FLAG Ab (upper panel). 10 μg of the IP’d chromatin fraction from WT and K-R mutant PNKP expressing cells was used as input (lower panel). ( C ) A schematic representation of the PNKP’s 3 ′ -phosphatase assay. ( D ) The 3 ′ -phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lanes 2–3), K142R (lane 4–5), K226R (lane 6–7) and K142R / 226R (lanes 8–9) PNKP-expressing cells post mock (–) or Bleo (+) treatment. Lane 1: No protein (NP), substrate only. Lower panel represents the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, * P < 0.05 between mock (–) and Bleo (+) treatment in WT and K142R; ns: non-significant, P > 0.05). ( E ) The phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lane 2), K142Q (lane 3), K226Q (lane 4) and K142Q / 226Q (lane 5). Lane 1: no protein (NP), substrate only. L o w er panel shows the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, ns: non-significant, P > 0.05). ( F ) 5 ′ -kinase assay was conducted with IP’d FLAG-PNKP from WT (lane 2–3), K142R (lanes 4–5), K226R (lane 6–7) and K142R / 226R (lanes 8–9) post mock (–) or Bleo (+) treatment. Lane 10: radiolabeled 32 nt marker (M). Lane 1: No protein (NP), substrate only. The quantitation of the products (% phosphorylated product) is represented in the bar diagram (Error bars show ± SD of the mean; n = 3, ns = non-significant, P > 0.05). In each case of (D–F), S: substrate and P: product.

Article Snippet: List of antibodies used in this study Primary antibodies Source Catalog# Dilution FLAG (M2) Millipore Sigma F 1804 1:500 Acetyl lysine K142 PNKP Ez Biolabs Custom-generated 1:200 Acetyl lysine K226 PNKP Ez Biolabs Custom-generated 1:200 PNKP Bio-Bharati Life Sciences BB AB0105 1:500 CBP Santa Cruz Biotechnology Sc-7300 1:1000 RNA polymerase II (H5) Biolegend 920 202 1:500 p300 Active Motif 61 401 1:500 GAPDH GeneTex GTX100118 1:7000 HDAC2 GeneTex GTX109642 1:500 XRCC1 GeneTex GTX111712 1:500 XRCC4 GeneTex GTX109632 1:500 DNA ligase III In house ( 18 ) 1:500 DNA ligase IV GeneTex GTX108820 1:500 Ku 70 GeneTex GTX101820 1:500 Histone H2A.XS139ph (phosphoserine 139) Cell Signaling Technology 2577 1:500 phospho-53BP1 Cell Signaling Technology 2675 1:500 53BP1 Santa Cruz Biotechnology Sc-517 281 1:500 NEIL2 In house ( 18 ) 1:500 DNA polymerase η Cell signaling Technology 13848 1:500 G F b F w t i S t w m n s c p s a S S T a s w p a d n i E P d T A s t d t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae002/7547335 by guest on 16 January 2024 eneration of PNKP expressing stable cell lines or generating the WT and mutant PNKP expressing stale cell lines, HEK293 cells were transfected with PNKPLAG (WT and mutants) expressing vector (pcDNA3) in 6- ell plates using Lipofectamine 2000 (ThermoFisher Scienific) according to the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Control, Purification, Mutagenesis, Phosphatase Assay, Quantitation Assay, Kinase Assay, Marker

Journal: Nucleic acids research

Article Title: Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

doi: 10.1093/nar/gkae002

Figure Lengend Snippet: Figure 4. The role of lysine 142 acetylated PNKP on SSB repair. ( A ) (Upper panels) Representative agarose gel images showing each long amplicon (10–12 kb) and a short amplicon ( ∼20 0–30 0 bp) of the transcribed (HPRT, Pol Beta, and RNA polII) genes and non-transcribed (NANOG, OCT4 and MyH2) from the genomic DNA isolated from WT (lanes 1–3), K142R (lanes 4–6), K226R (lanes 7–9) and K142R / 226R (lanes 10–12) PNKP expressing cells either mock (C), GO-treated (G), or at 4 h post-GO treatment (R). (L o w er panels) The bar diagrams represent the normalized (with short PCR amplicon) relative band intensity with the mock-treated control (C) set as 100 (Error bars show ± SD of the mean; n = 3, *** P < 0.005; ns = non-significant, P > 0.05) for each cell line. ( B ) Similar LA-qPCR assay of the transcribed (HPRT, Pol Beta, and RNA polII) genes from the genomic DNA isolated from WT (lanes 1–3), K142Q (lanes 4–6), K226Q (lanes 7–9) and K142Q / 226Q (lanes 10–12) PNKP expressing cells under similar treatment conditions as described in (A). All LA-qPCR experiments were performed following the depletion of endogenous PNKP in WT and mutant cells by 3 ′ -UTR-specific siRNA.

Article Snippet: List of antibodies used in this study Primary antibodies Source Catalog# Dilution FLAG (M2) Millipore Sigma F 1804 1:500 Acetyl lysine K142 PNKP Ez Biolabs Custom-generated 1:200 Acetyl lysine K226 PNKP Ez Biolabs Custom-generated 1:200 PNKP Bio-Bharati Life Sciences BB AB0105 1:500 CBP Santa Cruz Biotechnology Sc-7300 1:1000 RNA polymerase II (H5) Biolegend 920 202 1:500 p300 Active Motif 61 401 1:500 GAPDH GeneTex GTX100118 1:7000 HDAC2 GeneTex GTX109642 1:500 XRCC1 GeneTex GTX111712 1:500 XRCC4 GeneTex GTX109632 1:500 DNA ligase III In house ( 18 ) 1:500 DNA ligase IV GeneTex GTX108820 1:500 Ku 70 GeneTex GTX101820 1:500 Histone H2A.XS139ph (phosphoserine 139) Cell Signaling Technology 2577 1:500 phospho-53BP1 Cell Signaling Technology 2675 1:500 53BP1 Santa Cruz Biotechnology Sc-517 281 1:500 NEIL2 In house ( 18 ) 1:500 DNA polymerase η Cell signaling Technology 13848 1:500 G F b F w t i S t w m n s c p s a S S T a s w p a d n i E P d T A s t d t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae002/7547335 by guest on 16 January 2024 eneration of PNKP expressing stable cell lines or generating the WT and mutant PNKP expressing stale cell lines, HEK293 cells were transfected with PNKPLAG (WT and mutants) expressing vector (pcDNA3) in 6- ell plates using Lipofectamine 2000 (ThermoFisher Scienific) according to the manufacturer’s protocol.

Techniques: Agarose Gel Electrophoresis, Amplification, Isolation, Expressing, Control, Mutagenesis

Journal: Nucleic acids research

Article Title: Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

doi: 10.1093/nar/gkae002

Figure Lengend Snippet: Figure 5. Assessment of the effect of lysine 226 specific PNKP acetylation on the repair of Bleo-induced DSBs by long amplicon qPCR. ( A ) (Upper panels) R epresentativ e agarose gel images of the long amplicon (10–12 kb) and a short amplicon ( ∼20 0–30 0 bp) of the corresponding genes from WT (lanes 1–3), K142R (lanes 4–6), K226R (lanes 7–9) and K142R / 226R (lanes 10–12) PNKP expressing cells either mock (C), Bleo-treated (B) or at 16 h (R) f ollo wing Bleo-treatment. (L o w er panels) The bar diagrams represent the normalized (with short PCR amplicon) relative band intensity with the mock-treated control (C) set as 100 (error bars show ± SD of the mean; n = 3, *** P < 0.005; ns: non-significant, P > 0.05) for each cell line. ( B ) Similar LA-qPCR assay of the transcribed (HPRT, Pol Beta and RNA polII) genes from the genomic DNA isolated from WT (lanes 1–3), K142Q (lanes 4–6), K226Q (lanes 7–9) and K142Q / 226Q (lanes 10-12) cells under similar treatment conditions as described in (A). ( C ) The bar graph represents the percent LDH release in K142R, K226R and K142R / 226R mutant PNKP expressing cells compared to WT cells. Error bars show ± SD of the mean ( n = 3, ∗∗∗P < 0.005). All LA-qPCR / LDH assa y s w ere perf ormed f ollo wing the depletion of endogenous PNKP in WT and mutant cells b y 3 ′ -UTR-specific siRNA.

Article Snippet: List of antibodies used in this study Primary antibodies Source Catalog# Dilution FLAG (M2) Millipore Sigma F 1804 1:500 Acetyl lysine K142 PNKP Ez Biolabs Custom-generated 1:200 Acetyl lysine K226 PNKP Ez Biolabs Custom-generated 1:200 PNKP Bio-Bharati Life Sciences BB AB0105 1:500 CBP Santa Cruz Biotechnology Sc-7300 1:1000 RNA polymerase II (H5) Biolegend 920 202 1:500 p300 Active Motif 61 401 1:500 GAPDH GeneTex GTX100118 1:7000 HDAC2 GeneTex GTX109642 1:500 XRCC1 GeneTex GTX111712 1:500 XRCC4 GeneTex GTX109632 1:500 DNA ligase III In house ( 18 ) 1:500 DNA ligase IV GeneTex GTX108820 1:500 Ku 70 GeneTex GTX101820 1:500 Histone H2A.XS139ph (phosphoserine 139) Cell Signaling Technology 2577 1:500 phospho-53BP1 Cell Signaling Technology 2675 1:500 53BP1 Santa Cruz Biotechnology Sc-517 281 1:500 NEIL2 In house ( 18 ) 1:500 DNA polymerase η Cell signaling Technology 13848 1:500 G F b F w t i S t w m n s c p s a S S T a s w p a d n i E P d T A s t d t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae002/7547335 by guest on 16 January 2024 eneration of PNKP expressing stable cell lines or generating the WT and mutant PNKP expressing stale cell lines, HEK293 cells were transfected with PNKPLAG (WT and mutants) expressing vector (pcDNA3) in 6- ell plates using Lipofectamine 2000 (ThermoFisher Scienific) according to the manufacturer’s protocol.

Techniques: Amplification, Agarose Gel Electrophoresis, Expressing, Control, Isolation, Mutagenesis

Journal: Nucleic acids research

Article Title: Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

doi: 10.1093/nar/gkae002

Figure Lengend Snippet: Figure 6. Characterization of AcK142 and AcK226 PNKP immunocomplexes and their association to a specific genomic region. Benzonase-treated chromatin fraction from the WT-PNKP-FLAG cells f ollo wing mock ( −) / GO(+) or mock(-) / Bleo(+) treatment was IP’d individually with ( A ) anti-AcK142 Ab or ( B ) anti-AcK226 Ab, respectively along with control IgG (rabbit) Ab and probed with respective Abs as indicated to the right of the panels. The binding to the e x onic regions of transcribed (POLR2A, HPR T, A ctin B) v ersus the non-transcribed (MyH2, Oct3, NanoG) genes w ere quantified b y quantitativ e PCR (qPCR) from IP’d DNA from PNKP-FLAG stable cells ± GO or ± Bleo treatment using AcK142 ( C ) or AcK226 ( D ) Abs, respectively. The data are represented as fold enrichment of % input over IgG with mock-treated samples considered as unity. Error bars represent ± SD of the mean ( n = 3). *** P < 0.005, ** P < 0.01 represent statistical significance between mock and GO / Bleo treatment for each gene (ns = nonsignificant, P > 0.05).

Article Snippet: List of antibodies used in this study Primary antibodies Source Catalog# Dilution FLAG (M2) Millipore Sigma F 1804 1:500 Acetyl lysine K142 PNKP Ez Biolabs Custom-generated 1:200 Acetyl lysine K226 PNKP Ez Biolabs Custom-generated 1:200 PNKP Bio-Bharati Life Sciences BB AB0105 1:500 CBP Santa Cruz Biotechnology Sc-7300 1:1000 RNA polymerase II (H5) Biolegend 920 202 1:500 p300 Active Motif 61 401 1:500 GAPDH GeneTex GTX100118 1:7000 HDAC2 GeneTex GTX109642 1:500 XRCC1 GeneTex GTX111712 1:500 XRCC4 GeneTex GTX109632 1:500 DNA ligase III In house ( 18 ) 1:500 DNA ligase IV GeneTex GTX108820 1:500 Ku 70 GeneTex GTX101820 1:500 Histone H2A.XS139ph (phosphoserine 139) Cell Signaling Technology 2577 1:500 phospho-53BP1 Cell Signaling Technology 2675 1:500 53BP1 Santa Cruz Biotechnology Sc-517 281 1:500 NEIL2 In house ( 18 ) 1:500 DNA polymerase η Cell signaling Technology 13848 1:500 G F b F w t i S t w m n s c p s a S S T a s w p a d n i E P d T A s t d t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae002/7547335 by guest on 16 January 2024 eneration of PNKP expressing stable cell lines or generating the WT and mutant PNKP expressing stale cell lines, HEK293 cells were transfected with PNKPLAG (WT and mutants) expressing vector (pcDNA3) in 6- ell plates using Lipofectamine 2000 (ThermoFisher Scienific) according to the manufacturer’s protocol.

Techniques: Control, Binding Assay

Journal: iScience

Article Title: Bacterial host adaptation through sequence and structural variations of a single type III effector gene

doi: 10.1016/j.isci.2024.109224

Figure Lengend Snippet:

Article Snippet: acetylated-Lysine Mouse mAb (Ac-K-103) , Cell Signaling , Cat#9681; RRID: AB_331799.

Techniques: Protease Inhibitor, Western Blot, Reverse Transcription, DNA Purification, DNA Labeling, SYBR Green Assay, Plasmid Preparation, Imaging, Electroporation, Recombinant, Software

Journal: Nucleic Acids Research

Article Title: PCAF-mediated acetylation of transcriptional factor HOXB9 suppresses lung adenocarcinoma progression by targeting oncogenic protein JMJD6

doi: 10.1093/nar/gkw808

Figure Lengend Snippet: HOXB9 is acetylated by PCAF in cells. ( A ) HOXB9 is acetylated in H1299 cells. H1299 cells transfected with FLAG-HOXB9 expression vector and were treated with 3 μM TSA and 5 mM nicotinamide for 12 h, and then cell lysates were immunoprecipitated with an anti-acetylated-lysine (AcK) antibody or normal IgG, followed by immunoblotting with an anti-FLAG antibody. ( B ) PCAF acetylated HOXB9 in vivo . FLAG-HOXB9 expression vector was cotransfected separately with expression vectors containing a variety of acetyltransferases into H1299 cells. Forty-eight hours post transfection, cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by Western blot analysis with the AcK antibody. ( C ) PCAF acetylated HOXB9 in vitro . The in vitro acetylation assays were performed by using MBP, MBP-fusion proteins of HOXB9, and various amounts GST-fusion PCAF HAT domain. Then the reaction mixtures were subjected to SDS-PAGE, and followed by immunoblotting with the AcK Ab. The purified MBP and GST tagged fusion proteins were stained by Coomassie blue. Arrows showed the correct molecular masses of the indicated proteins.

Article Snippet: The following antibodies used in the experiments: Acetylated-Lysine and PCAF (Cell Signaling Technology; Catalogue #9441 and #3378, respectively), HOXB9 and JMJD6 (Santa Cruz; sc-398500, sc-133671 and sc-28348 respectively), SIRT1 and SIRT2 (abcam).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, In Vivo, In Vitro, SDS Page, Purification, Staining

Journal: Nucleic Acids Research

Article Title: PCAF-mediated acetylation of transcriptional factor HOXB9 suppresses lung adenocarcinoma progression by targeting oncogenic protein JMJD6

doi: 10.1093/nar/gkw808

Figure Lengend Snippet: HOXB9 is identified to be acetylated at lysine 27. ( A ) The spectrum of the charged ion (m/z 1384.3141) indicated that lysine 27 is acetylated (lower case Ac) in the peptide GSMSISGTLSSYYVDSIISHESEDAPPAKFPSGQYASSR. The b ions indicate the fragmentation ions containing the N-terminus of the peptide and the y ions are the fragmentation ions containing the C-terminus of the peptide. ( B ) Mutation of K27 significantly decreases HOXB9 acetylation. HEK-293T cells were co-transfected with different FLAG tagged HOXB9 mutants and PCAF. Cell lysates were co-immunoprecipitated with anti-FLAG antibody, immunoblotted with the AcK antibody. ( C ) Specificity of antibody against AcK27-HOXB9 was verified by dot blot assays. Nitrocellulose membrane was spotted by different amounts of HOXB9 unacetylated peptide (EDAPPAKFPSGQYAC), HOXB9 acetylated peptide (EDAPPA(AcK)FPSGQYAC), and detected with the anti-AcK27-HOXB9 antibody. ( D ) HOXB9-WT and HOXB9-K27R mutant expression vectors were co-transfected into HEK-293T cells, then subjected to SDS-PAGE, followed by immunoblotting with the anti-AcK27-HOXB9 antibody. ( E ) In vitro acetylation assays were performed using GST-PCAF HAT domain and different purified mutants of MBP-HOXB9. The reaction mixtures were subjected to SDS-PAGE, immunoblotted with the anti-AcK27-HOXB9 specific antibody. ( F ) The sequences adjacent to human HOXB9-K27 from different HOXB9 homologues species were aligned. ( G ) H1299 cells were transfected with FLAG-HOXB9. Endogenous PCAF was knocked down with small interfering RNA (siRNA). Cell lysates were subjected with SDS-PAGE, followed by immunoblotting with the indicated antibodies. ( H ) A variety of tissues from C57BL6 mouse were performed by immunohistochemical staining with anti-AcK27-HOXB9 and anti-HOXB9 total antibodies respectively.

Article Snippet: The following antibodies used in the experiments: Acetylated-Lysine and PCAF (Cell Signaling Technology; Catalogue #9441 and #3378, respectively), HOXB9 and JMJD6 (Santa Cruz; sc-398500, sc-133671 and sc-28348 respectively), SIRT1 and SIRT2 (abcam).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Dot Blot, Membrane, Expressing, SDS Page, Western Blot, In Vitro, Purification, Small Interfering RNA, Immunohistochemical staining, Staining

Journal: Nucleic Acids Research

Article Title: PCAF-mediated acetylation of transcriptional factor HOXB9 suppresses lung adenocarcinoma progression by targeting oncogenic protein JMJD6

doi: 10.1093/nar/gkw808

Figure Lengend Snippet: Elevated HOXB9-K27 acetylation is associated with longer overall survival in lung adenocarcinoma patients. ( A ) Immunohistochemical analysis for lung adenocarcinoma was performed using the anti-AcK27-HOXB9 antibody, and Kaplan–Meier analysis was performed in two groups of lung adenocarcinoma patients with strong staining (3+ and 4+) and weak staining (1+ and 2+) for HOXB9-K27 acetylation, with a log-rank at P = 0.0081. ( B–D ) The protein level of HOXB9-K27 were analyzed based on TNM classification ( B-C ) and AJCC category ( D ), the statistical analyses were performed by Student's t-test ( B ) and one way ANOVA analyses ( C-D ), * for P < 0.05, *** for P < 0.001. ( E ) Representative images of JMJD6 in normal lung tissue (a) and lung adenocarcinoma (b). ( F ) The expression level of JMJD6 was analyzed in normal lung tissue and lung adenocarcinoma. The statistical analyses were performed by Student's t-test, *** for P <0.001. ( G ) Kaplan-Meier analysis was performed in two groups of lung adenocarcinoma patients with strong staining and weak staining of JMJD6 separately (log-rank of P = 0.0212). ( H ) Representative images of immunohistochemical staining with AcK27-HOXB9 and JMJD6 antibodies separately in six lung adenocarcinoma patients. ( I ) Analyses of the expression level of AcK27-HOXB9 and JMJD6 in the two group (higher and lower expression). The statistical analyses were performed by Student's t -test, * for P < 0.05. ( J ) A working model depicts how HOXB9 is regulated by acetylation. In this model, we demonstrated that HOXB9 is acetylated by acetyltransferase PCAF, and deacetylated by deacetylase SIRT1. Acetylation of HOXB9 decreased its capacity in promoting lung cancer cell migration and tumor growth in mice by suppressing the transcription of its target gene Jumonji domain-containing protein 6 (JMJD6).

Article Snippet: The following antibodies used in the experiments: Acetylated-Lysine and PCAF (Cell Signaling Technology; Catalogue #9441 and #3378, respectively), HOXB9 and JMJD6 (Santa Cruz; sc-398500, sc-133671 and sc-28348 respectively), SIRT1 and SIRT2 (abcam).

Techniques: Immunohistochemical staining, Staining, Expressing, Histone Deacetylase Assay, Migration

Journal: Viruses

Article Title: Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

doi: 10.3390/v8070191

Figure Lengend Snippet: Effects of heat shock (HS) exposure on human T cell leukemia virus type-I (HTLV-I)-infected cell lines derived from acute adult T cell leukemia (ATL) patients: ( a ) Aliquots of ATL-026i and ATL-056i cells were incubated at various temperatures for 30 min and cultured for 24 h. The cells were then analyzed for the frequencies of trans-activator (Tax) + cells (left bar graphs) by flow cytometry (FCM) and the relative density (Mean Fluorescent Intensity, MFI) of heat shock protein 70 (HSP70) expression (middle bar graphs) and for cell viability using the CCK-8 cell counting kit (right bar graphs). ( b ) The kinetics of the up-regulation of Tax and HSP70 expression by the same two cell lines following exposure to 43 °C for various times is shown. The values denote the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Anti- HSP70 antibody (clone: C92F3A-5) was purchased from StressMarq Biosciences Inc (Victoria, BC, Canada).

Techniques: Infection, Derivative Assay, Incubation, Cell Culture, Flow Cytometry, Expressing, CCK-8 Assay, Cell Counting

Journal: Viruses

Article Title: Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

doi: 10.3390/v8070191

Figure Lengend Snippet: A comparison of time course of the expression among HSP70, Tax and gp46 antigens followed by exposure to HS: The ATL-026i (upper panel) and YT/cM1 (lower panel) cells were either exposed to HS (black square) or mock treated (open square), cultured for 12, 24 and 48 h, and then their phenotypes were analyzed by FCM. Data shown are representative of three independent experiments.

Article Snippet: Anti- HSP70 antibody (clone: C92F3A-5) was purchased from StressMarq Biosciences Inc (Victoria, BC, Canada).

Techniques: Expressing, Cell Culture

Journal: Viruses

Article Title: Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

doi: 10.3390/v8070191

Figure Lengend Snippet: The effects of the HSP70 inducing and inhibiting agents on the expression of Tax antigen: Triplicate cultures of YT/cM1 cells were either exposed to HS or mock treated, and then cultured in the presence or absence of either ( a ) 10 μM zerumbone (ZER, HSP70 inducer) or ( b ) 1 μM pifithlin-μ (PFT, HSP70 functional inhibitor) for 24 h. The Tax and HSP70 expression was analyzed by FCM. The values denote the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Anti- HSP70 antibody (clone: C92F3A-5) was purchased from StressMarq Biosciences Inc (Victoria, BC, Canada).

Techniques: Expressing, Cell Culture, Functional Assay