Journal: International Journal of Molecular Sciences

Article Title: Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

doi: 10.3390/ijms232415921

Figure Lengend Snippet: Western blot analysis of ANP32a in zebrafish embryos undergoing either knockdown or overexpression of Anp32a . ( A ) Diagrams were used to depict base-pairings among Anp32a mRNA, antisense morpholino oligonucleotides (MO) of Anp32a ( Anp32a -MO) and wobble nucleotides of Anp32a -flag mRNA (wobble- Anp32a -flag mRNA). ( B ) Western blot analysis. Zebrafish embryos at one-cell stage were microinjected with Anp32a -MO (MO), wobble- Anp32a -flag mRNA (wb-RNA) and MO plus wobble mRNA (MO + wb-RNA) as indicated, followed by extraction of total embryonic proteins at 48 hpf. Protein levels of endogenous ANP32a and exogenous ANP32a -flag were detected using antibodies against ANP32a and reporter protein flag. α-tubulin served as an internal control. Protein levels relative to each internal control are presented below each lane.

Article Snippet: Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [ ], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used.

Techniques: Western Blot, Knockdown, Over Expression, Extraction, Control

Journal: International Journal of Molecular Sciences

Article Title: Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

doi: 10.3390/ijms232415921

Figure Lengend Snippet: ANP32a is required for the neuronal regeneration of zebrafish larvae after SCI. ( A ) The experimental flowchart showing the time course of microinjection of either Anp32a mRNA or Anp32a morpholinos, SCI performance and observation of spinal cord regeneration. Embryos at the one-cell stage were injected with either wobble- Anp32a -flag mRNA or antisense morpholino oligonucleotides (MO) of Anp32a and SCI at 48 hpf, followed by analysis of the lesion gap at 0, 18 and 24 hpi. ( B ) Time course image of spinal cord regeneration in control embryos and Anp32a -overexpressed embryos at 0, 18 and 24 hpi. ( C ) Quantification of temporal regenerative response in SCI-control and wobble- Anp32a -flag mRNA-injected embryos at 18 hpi (left; control: n = 25; RNA + SCI: n = 19) and 24 hpi (right; control: n = 28; RNA + SCI: n = 16). ( D ) Time course image of spinal cord regeneration in control embryos and Anp32a -morphants at 0, 18 and 24 hpi. ( E ) Quantification of temporal regenerative response in SCI control embryos and Anp32a -morphants at 18 hpi (left; control: n = 31; MO + SCI: n = 30) and 24 hpi (right; control: n = 32; MO + SCI: n = 34). Scale bar: 100 μm; The Student’s t -test was used to perform statistical analysis (*, p < 0.05; p < 0.01; ***, p < 0.001; error bars indicate mean ± SD).

Article Snippet: Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [ ], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used.

Techniques: Microinjection, Injection, Control

Journal: International Journal of Molecular Sciences

Article Title: Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

doi: 10.3390/ijms232415921

Figure Lengend Snippet: Defective swimming activity was partially recovered in the SCI larvae with Anp32a overexpression. ( A ) Experimental flowchart showing the time course of microinjection of either Anp32a mRNA (RNA) or Anp32a morpholinos (MO), SCI performance and behavior test. Embryos at the one-cell stage were microinjected with either wobble- Anp32a -flag mRNA or Anp32a -MO. Following SCI at 48 hpf, we recorded the swimming trajectory route at 24 hpi. ( B ) To carry out gain-of-function and loss-of function studies, the swimming trajectory route of each fish from three groups was examined, including untreated control (neither injection nor SCI), SCI only (SCI), both wobble- Anp32a -flag mRNA injection and SCI (RNA + SCI), injection of Anp32a -MO and SCI (MO + SCI), and co-injected Anp32a -MO with wobble- Anp32a -flag mRNA injection and SCI (MO + RNA + SCI). ( C ) Quantification and statistical analysis. Swimming distances (cm) were calculated based on the recorded trajectory route shown in ( B ). The data in each group were averaged from all examined larvae (untreated control: n = 30; SCI: n = 66; RNA + SCI: n = 35; MO + SCI: n = 39; MO + RNA + SCI: n = 35). One-way ANOVA, followed by Tukey’s multiple comparison test, was used to perform statistical analysis (***, p < 0.001; error bars indicate mean ± SD).

Article Snippet: Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [ ], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used.

Techniques: Activity Assay, Over Expression, Microinjection, Control, Injection, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

doi: 10.3390/ijms232415921

Figure Lengend Snippet: Overexpression of Anp32a could promote cell proliferation in SCI embryos during recovery. ( A ) The experimental flowchart. Anp32a mRNA was injected into embryos from zebrafish transgenic line Tg(mnx1:GFP) at the one-cell stage, followed by SCI at 48 hpf and staining with anti-PH3 at 18 and 24 hpi. ( B ) Proliferation patterns around the lesion site of embryos treated as indicated at 18 and 24 hpi were presented by immunofluorescence through antibody against phosphorylated histone H3 (PH3; Red) and visualized by laser confocal microscopy. Arrow indicates the SCI site, while white dotted box indicates the calculation area. ( C ) Statistical count of PH3-positive cells between SCI- Anp32a -overexpressed embryos ( n = 11 at 18 hpi; n = 10 at 24 hpi) and SCI embryos without overexpression of Anp32a ( n = 11 at 18 hpi; n = 10 at 24 hpi). A) Scale bar: 100 μm. The Student’s t -test was used to perform statistical analysis (*, p < 0.05; p < 0.01; ***, p < 0.001; error bars indicate mean ± SD).

Article Snippet: Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [ ], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used.

Techniques: Over Expression, Injection, Transgenic Assay, Staining, Immunofluorescence, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

doi: 10.3390/ijms232415921

Figure Lengend Snippet: The overexpression of Anp32a in SCI embryos of zebrafish could induce an increase in the cell number of motor neuron progenitor cells (pMN) around the SCI site. Fluorescence-activated cell sorting (FACS) and a cell cycle analysis of pMN in Anp32a -overexpressing SCI embryos of zebrafish were performed. The Anp32a mRNA was microinjected into the one-cell stage of embryos from wild-type zebrafish, followed by SCI at 48 hpf. After deheading, suspended cells were stained with antibodies against Olig2 and DAPI at 24 hpi. ( A ) Scatter profiles of Anp32a -overexpressed SCI-zebrafish embryonic cells. All analytic cells were gated at P1; RFP-labelled Olig2-positive pMN cells were gated at P2; DAPI-labeled-positive cells were gated at P4; Olig2-negative cells were gated at P5. ( B ) Typical FACS dataset. The number shown in the right corner represents the percentage of cells at each gate relative to the total number of examined cells in each group. ( C ) Cell cycle analysis from DAPI-labeled-positive cells shown on P4.

Article Snippet: Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [ ], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used.

Techniques: Over Expression, Fluorescence, FACS, Cell Cycle Assay, Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

doi: 10.3390/ijms232415921

Figure Lengend Snippet: Overexpression of Anp32a in SCI embryos of zebrafish could induce the active proliferation of radial glial cells around the SCI site. Fluorescence-activated cell sorting (FACS) and a cell cycle analysis of radial glial cells in Anp32a -overexpressing SCI embryos of zebrafish were performed. Anp32a mRNA was microinjected into one-cell stage embryos from Tg(gfap:GFP) zebrafish, followed by SCI at 48 hpf and DAPI staining at 24 hpi. ( A ) Scatter profiles of Anp32a -overexpressing Tg(gfap:GFP) SCI-embryonic cells. All analytic cells were gated at P1; GFP-positive cells were gated at P2; DAPI-labeled-positive cells were gated at P6; GFP-negative cells were gated at P4. ( B ) Typical FACS dataset. The number shown in the right corner is the percentage of proliferated cells at each gate relative to the total examined cells in each group. ( C ) Cell cycle analysis from DAPI-labeled-positive cells shown on P6.

Article Snippet: Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [ ], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used.

Techniques: Over Expression, Fluorescence, FACS, Cell Cycle Assay, Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

doi: 10.3390/ijms232415921

Figure Lengend Snippet: Proliferation and cell number of radial glial cells and motor neurons were increased in the Anp32a -overexpression SCI embryos. The Anp32a mRNA was microinjected into the one-cell stage of embryos from zebrafish transgenic lines ( A , B ) Tg(gfap:GFP) and ( C – F ) the crossed strain of Tg(gfap:GFP) and Tg(mnx1:TagRFP) , followed by BrdU injection, SCI at 48 hpf, and finally, staining with PH3 at 24 hpi. ( A ) Proliferation patterns at the lesion site of Tg(gfap:GFP) embryos treated as indicated at 24 hpi were displayed by immunofluorescence through reaction with antibodies against phosphorylated histone H3 (PH3; Red) and observation under laser confocal microscopy. ( B ) Statistical analysis showed a significantly increased number of PH3-positive cells in Anp32a -overexpressed SCI embryos ( n = 10) compared to untreated embryos ( n = 3) and SCI-control embryos ( n = 14) at 24 hpi. One-way ANOVA followed by Tukey’s multiple comparison test was used to perform statistical analysis (*, p < 0.05; ** p < 0.01; error bars indicate mean ± SD). ( C ) Proliferation patterns at the lesion site of transgenic cross strain Tg(gfap:GFP) X Tg(mnx1:TagRFP) embryos treated as indicated at 24 hpi showing GFAP-positive cells (green) colocalized with BrdU-positive cells (purple) around the lesion area. ( D ) Statistical analysis of GFAP +/BrdU+ colocalized cells in SCI-control and Anp32a -overexpressing SCI embryos. ( E ) Proliferation patterns at the lesion site of Tg(gfap:GFP) X Tg(mnx1:TagRFP) embryos treated as indicated at 24 hpi showing red-labelled motor neurons colocalized with BrdU-positive cells (purple) around the lesion area. The specimens in C, F originated from the same embryos. ( F ) Statistical analysis of motor neuron +/BrdU + colocalized cells in SCI-control and Anp32a -overexpression SCI embryos. Scale bar: 100 μm; ( D , F ) Student’s t -test was used to perform statistical analysis (* p < 0.05; ** p < 0.01; error bars indicate mean ± SD).

Article Snippet: Total proteins extracted from embryos were analyzed on a 15% SDS-PAGE, followed by Western blot analysis, according to the procedures described by Lee et al. [ ], except that the antibodies against ANP32a (1:1000; Aviva Systems Biology; ARP40204_T100, San Diego, CA, USA), α-tubulin (1:5000; Sigma; RRID:AB_477579, St. Louis, MO, USA), Flag (1:1000; Abcam; RRID:AB_446355, Cambridge, UK), goat anti-rabbit-HRP (1:10,000; Cell Signaling; RRID:AB_2099233, Frankfurt, Germany) and goat anti-mouse-HRP (1:10,000; Abcam; RRID:AB_955439, Cambridge, UK) were used.

Techniques: Over Expression, Transgenic Assay, Injection, Staining, Immunofluorescence, Confocal Microscopy, Control, Comparison

Journal: Science advances

Article Title: Avian ANP32A incorporated in avian influenza A virions promotes interspecies transmission by priming early viral replication in mammals.

doi: 10.1126/sciadv.adj4163

Figure Lengend Snippet: Fig. 7. avANP32A transferred by avian influenza A virus accelerates the process of obtaining adaptive mutations. (A) H9N2 virus produced from HEK293T cells overexpressing Flag-tagged ANP32A or with an empty vector were purified by ultracentrifugation with a prior HAd step. The cell lysates and purified virions were then subjected to Western blotting. (B) Viral replication in MDCK cells or MDCK-avANP32A cells infected with H9N2 (huANP32A) virus and H9N2 (avANP32A) virus at an MOI of 0.1. Error bars represent mean ± SD from n = 3 independent biological replicates; unpaired t test; **P < 0.01 and ****P < 0.0001. (C) Model for the effect of avANP32A transferred by avian influenza A virus on viral replication and adaptive mutation acquisition when jumping from avian hosts to mammalian hosts. (D) H9N2 virus pack- aged with either avANP32A or huANP32A was blind passaged six times in MDCK cells. Viral RNAs were extracted and the C terminus of PB2 was amplified and deep- sequenced to monitor the residue phenotype of PB2 627 and 701 positions during passages in MDCK cells. Bar graph represents the percentage of adaptive mutations including PB2-E627K/V and D701N in each passage. Error bars represent mean ± SEM from n = 4 independent biological replicates, unpaired t test; *P < 0.05. (E) Sche- matic representation of the protocol for the experiments shown in (F). (F) The lungs of infected mice were collected for viral RNA extraction, and the C terminus of PB2 was amplified and cloned into T vectors. Nine molecular clones from each sample were randomly picked and sequenced for determination of the residue phenotype of PB2 627 and 701 positions. Samples with nine clone negatives for the emergence of PB2-E627K/V and D701N were recognized as no occurrence of adaptive mutations. In each group, the percentage of mice with emergence of adaptive mutations was calculated.

Article Snippet: Immunoblotting analysis was carried out using the following primary antibodies: ANP32A rabbit polyclonal (15810- 1- AP, Proteintech), ANP32B mouse monoclonal (66160- 1- Ig, Proteintech), ANP32E rabbit polyclonal (A17220, Abclonal), Flag mouse monoclonal (F1804, SigmaAldrich), Flag rabbit polyclonal (F7425, Sigma- Aldrich), HA mouse monoclonal (H9658, Sigma- Aldrich), ACTB rabbit monoclonal (AC026, Abclonal), His mouse monoclonal (66005- 1, Proteintech), V5 mouse monoclonal (ab27671, Abcam), glutathione S- transferase (GST) rabbit polyclonal (10000- 0- AP, Proteintech), CD81 mouse monoclonal (66866- 1- Ig, Proteintech), and IBV NP rabbit polyclonal (GTX128538, GeneTex).

Techniques: Virus, Produced, Plasmid Preparation, Purification, Western Blot, Infection, Mutagenesis, Amplification, Residue, RNA Extraction, Clone Assay

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) PANTHER pathway analysis of upstream transcriptional regulators identified in microarray data comparing articular cartilage of 8-week old male Anp32a -deficient to wild-type mice (n = 4 per group) using Ingenuity Pathway Analysis (IPA). (B) Real-time PCR analysis of direct Wnt target genes Tcf1 , Ccnd1 , cMyc and Axin2 in control and Anp32a knockdown (KD) ATDC5 cells (Pillai = 0.964, F 2,3 = 40.34, P = 0.0068 by MANOVA; Ccnd1 and Axin2 showed > 0.9 correlation with Tcf1 and were not included in the model, mean + SD of 3 technical replicates per condition). (C) Real-time PCR analysis of chondrogenic differentiation markers collagen 2 ( Col2a1 ), aggrecan ( Acan ) and collagen 10 ( Col10a1 ) in control and Anp32a KD ATDC5 cells [F 3,12 = 25.321, P < 0.0001 ( Col2a1 ), F 1.05,4.21 = 24.378 P = 0.007 ( Acan ), F 3,12 = 42.775 P < 0.0001 ( Col10a1 ) by 2-way ANOVA for interaction between silencing and time, mean + SD of 3 technical replicates per condition]. (D) Alcian blue (AB), safranin O (SO), picrosirius red (SR) at day 14 (D14) and alizarin red (AR) staining at day 21 (D21) showing reduced proteoglycan deposition (AB, SO), collagen content (SR) and mineralization (AR) in Anp32a KD ATDC5 cells during chondrogenesis. (E) AB staining demonstrating rescue of chondrogenic differentiation in Anp32a KD ATDC5 cells by treatment with Wnt inhibitor XAV939 (XAV). (F) Real-time PCR analysis of Wnt target gene expression in articular cartilage from 8-week old male wild-type (WT) and Anp32a -deficient ( Anp32a -/- ) mice (Pillai = 0.788, F 3,11 = 13.61, P = 0.0005 by MANOVA – cMyc showed > 0.9 correlation with Tcf1 and was not included in the model, n = 8 and 7 mice per group). (G-I) Immunohistochemical staining for TCF1 protein in male 8-week old (G), osteoarthritic (H) and female ageing WT and Anp32a -/- mice (I) (representative images of n = 3 different mice per group). Scale bar 50 µm. (J) Real-time PCR analysis of Wnt target gene expression in human articular chondrocytes transfected with siRNA targeting ANP32A (siANP32A) or scrambled siRNA (siSCR) (n = 2 different donors - mean ± SD of 3 technical replicates). (K) TOP/FOP reporter assay in human articular chondrocytes transfected with siANP32A, siRNA targeting β-catenin (siβCAT) or siSCR, and then treated with recombinant WNT3A (n = 2 biologically independent experiments, mean ± SD of 3 technical replicates). (L-M) TCF1 expression (L) and negative correlation with ANP32A expression (M) by RNA sequencing in paired preserved and damaged cartilage from hips (o) and knees (Δ) from osteoarthritis patients [log2-fold change (Log2FC) of damaged (D) vs. preserved (P)) (n = 21, P < 0.0001, Benjamini-Hochberg adjusted paired t -test (l), Pearson’s correlation = -0.40 - P = 0.0084 (m)].

Article Snippet: The sheared chromatin was incubated with antibodies against ANP32A (Santa Cruz, sc-100767) and RNA polymerase II (Thermo Scientific, MA1-46093), and recovered by binding to protein A/G agarose.

Techniques: Microarray, Real-time Polymerase Chain Reaction, Control, Knockdown, Staining, Targeted Gene Expression, Immunohistochemical staining, Transfection, Reporter Assay, Recombinant, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A, B) Real-time PCR analysis of Anp32a (A) and Atm (B) in control and Anp32a knockdown (KD) ATDC5 cells. Error bars indicate mean ± SD of three technical replicates per condition [F 1,4 = 69.892, P = 0.001 ( Anp32a ), F 1,4 = 24.841 P = 0.009 ( Atm ), by 2-way ANOVA for control versus KD cells, mean + SD of 3 technical replicates per condition].

Article Snippet: The sheared chromatin was incubated with antibodies against ANP32A (Santa Cruz, sc-100767) and RNA polymerase II (Thermo Scientific, MA1-46093), and recovered by binding to protein A/G agarose.

Techniques: Real-time Polymerase Chain Reaction, Control, Knockdown

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A, B) Real-time PCR analysis of chondrogenic differentiation markers collagen 2 ( Col2a1 ), aggrecan ( Acan ) and collagen 10 ( Col10a1 ) at day 7 in control and Anp32a knockdown (KD) ATDC5 cells after treatment with antioxidant N-acetylcysteine (NAC) (A) or Wnt inhibitor XAV939 (XAV) (B) at the indicated concentrations. Error bars indicate mean ± SD of three technical replicates per condition.

Article Snippet: The sheared chromatin was incubated with antibodies against ANP32A (Santa Cruz, sc-100767) and RNA polymerase II (Thermo Scientific, MA1-46093), and recovered by binding to protein A/G agarose.

Techniques: Real-time Polymerase Chain Reaction, Control, Knockdown

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A, B) Expression of Wnt target genes CCND1, CMYC and AXIN2 (A) and correlation with ANP32A expression (B) by RNA sequencing in paired preserved (P) and damaged (D) cartilage from hips (o) and knees (Δ) from osteoarthritis patients (log2-fold change (Log2FC) of damaged vs. preserved) (n=21, P < 0.0001 for CCND1 and AXIN2 , P =0.0809 for CMYC by Benjamini-Hochberg adjusted paired t -test (A), Pearson’s correlation R = -0.34 – P = 0.027, R = 0.03 – P = 0.853, R = -0.39 – P = 0.112 for CCND1, CMYC and AXIN2 respectively (B)).

Article Snippet: The sheared chromatin was incubated with antibodies against ANP32A (Santa Cruz, sc-100767) and RNA polymerase II (Thermo Scientific, MA1-46093), and recovered by binding to protein A/G agarose.

Techniques: Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) Co-immunoprecipitation (Co-IP) using an anti-ANP32A antibody showing the binding between ANP32A and AXIN1 in untreated human articular chondrocytes, which is abolished upon Wnt activation by recombinant WNT3A protein. Wnt activation increases the binding of ANP32A with β-catenin and with histone 3. Silencing of ANP32A (siANP32A) is shown to assess the specificity of the ANP32A antibody used in the immunoprecipitation. The image is representative of three independent experiments. (B) Immunoblot analysis of ANP32A, active β-catenin and total β-catenin protein amounts (with actin as loading control) in human articular chondrocytes transfected with ANP32A (siANP32A) or scrambled siRNA (siSCR). (C) Immunofluorescent staining of ANP32A (green) and counterstaining of nuclei with Hoechst dye (blue) in human articular chondrocytes upon vehicle or recombinant WNT3A treatment (4 hours). Representative images are shown (n = 2). Scale bar 10 µm. (D) Chromatin-immunoprecipitation quantitative PCR (ChIP-qPCR) analysis of ANP32A binding to chromatin on Wnt target ( Tcf1 , Cnnd1,cMyc and Axin2 ) gene promoter regions in human articular chondrocytes with high endogenous Wnt signaling (cells were expanded two passages). (E) ChIP-qPCR analysis of acetylated H3K9 (H3K9Ac) on Wnt target ( Tcf1 , Ccnd1, cMyc and Axin2 ) and Atm gene promoter regions in human articular chondrocytes with high endogenous Wnt signaling that were transfected with siANP32A or siSCR. Data are expressed as ratio of H3K9Ac and total Histone 3 (H3) in siANP32A cells normalized to siSCR cells. Data points are from two biologically independent experiments (D-E). (F) Scheme summarizing how ANP32A regulates the Wnt transcriptional response. In basal conditions (left panel), ANP32A interacts with AXIN1 in the destruction complex. Upon Wnt activation (right panel), ANP32A dissociates from AXIN1, associates with β-catenin, and translocates to the nucleus. Within the nucleus, ANP32A represses Wnt target genes via blocking histone acetylation.

Article Snippet: The sheared chromatin was incubated with antibodies against ANP32A (Santa Cruz, sc-100767) and RNA polymerase II (Thermo Scientific, MA1-46093), and recovered by binding to protein A/G agarose.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Activation Assay, Recombinant, Western Blot, Control, Transfection, Staining, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Blocking Assay

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) Schematic outline of in vivo pharmacological interventions against osteoarthritis in Anp32a -deficient ( Anp32a -/- ) male mice. 8-week old mice were subjected to destabilization of the medial meniscus (DMM) surgery. One week after injury, mice were injected intra-articularly with vehicle or Wnt inhibitor XAV939 (XAV) every 10 days for a total of 7 times. Mice were treated orally with vehicle, XAV or NAC alone or in combination. Knee joints were collected 12 weeks after surgery. (B-C) Hematoxylin-safranin-O-stained sections (B) and quantification by OARSI severity grade (C) demonstrating that NAC protects against articular cartilage damage in osteoarthritis: [F 1,28 =8.48 P = 0.007 for main effect NAC by two-way ANOVA, n = 8 per group]. Scale bar 200 µm. (D) Immunohistochemical staining for TCF1 protein in articular cartilage of Anp32a -/- mice in the DMM model treated or not with XAV or NAC (representative images of n = 5 different mice per group). Scale bar 50 µm. ( E-F) Hematoxylin-safranin-O-stained sections (E) and quantification (F) of osteophytes demonstrating that Wnt inhibition protects against osteophyte formation in osteoarthritis [F 1,28 =7.69 P = 0.0098 for main effect XAV by two-way ANOVA, n = 8 per group]. Scale bar 200 µm. (G) Immunohistochemical staining for TCF1 protein in developing osteophytes in Anp32a -/- mice in the DMM model treated or not with XAV or NAC (representative images of n = 5 different mice per group). Scale bar 50 µm.

Article Snippet: The sheared chromatin was incubated with antibodies against ANP32A (Santa Cruz, sc-100767) and RNA polymerase II (Thermo Scientific, MA1-46093), and recovered by binding to protein A/G agarose.

Techniques: In Vivo, Injection, Staining, Immunohistochemical staining, Inhibition

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) Selected Protein Atlas data showing the inverse relationship between ANP32A and Wnt target gene TCF1 in brain and muscle tissues compared to other systems. Data are presented as scaled variables. (B) Real-time PCR analysis of Wnt target gene expression (n = 8) in heart from 20-week old male WT and Anp32a -/- mice (Pillai = 0.492, F 3,12 = 3.877, P =0.038 by MANOVA - Axin2 data did not show homogeneity of variance and were not included in the model). (C) Immunohistochemical staining for TCF1 in 20-week old male WT and Anp32a -/- mice hearts. Scale bar 50 µm. (D) Macroscopic and Hematoxylin-Eosin stained hearts demonstrating cardiac hypertrophy in Anp32a -/- compared to WT mice. Scale bar 2 mm. ( E ) Heart weight/body weight ratio in 20-week old male WT and Anp32a -/- mice (t 27 = 10.45, P < 0.0001). ( F ) Real-time PCR analysis of hypertrophy markers natriuretic peptide precursor A (Nppa) and skeletal muscle α- actin (Acta1) (n = 8) in heart from 20-week old male WT and Anp32a -/- mice (t 14 = 4.010, P = 0.0011 ( Nppa ), t 14 = 2.87, P = 0.0125 ( Acta1 ) by Student’s t-test). ( G-H ) Picrosirius red staining (G) showing increased amounts of fibrotic tissue in hearts from 20-week old Anp32a -/- mice compared to WT (H) (t 6 = 6.82, P = 0.0005 by Student’s t-test). Scale bar 50 µm. (I) Real-time PCR analysis of Wnt target gene expression (n = 9) in brain from 8-week old male WT and Anp32a -/- mice (Pillai = 0.666, F 4,10 = 4.978, P =0.018 by MANOVA – 3 extreme outliers were excluded from the model as indicated in cyan color). (J) Immunohistochemical staining for TCF1 in 16-week old male hippocampus from WT and Anp32a -/- mice. Scale bar 250 µm.

Article Snippet: The sheared chromatin was incubated with antibodies against ANP32A (Santa Cruz, sc-100767) and RNA polymerase II (Thermo Scientific, MA1-46093), and recovered by binding to protein A/G agarose.

Techniques: Real-time Polymerase Chain Reaction, Targeted Gene Expression, Immunohistochemical staining, Staining

Journal: Journal of translational medicine

Article Title: A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma.

doi: 10.1186/s12967-017-1312-7

Figure Lengend Snippet: Fig. 1 Pharmacophore model for p32. a 3D structure of the peptide-SIVSSSRGQVRRS from the p32-binding part of protein C1q. This structure is extracted from the known PDB structure of C1q. b 3D structure of LyP-1 cyclic peptide (CGNKRTRGC) modeled with the program InsightII (Accelrys, San Diego). c Superposition of above mentioned tertiary structures. d Assignment of six-feature pharmacophore centers. Both peptides have super- imposable main features: 3 Donor|Cationic centers (violet: F1, F2, and F3), 2 Hydrophobic centers (green: F5 and F6), and 1 Acceptor|Anionic center (cyan: F4)

Article Snippet: Protein thermal shift assay Human recombinant p32 (OriGene) was tested in a reaction volume of 20 μL with 100 μM of p32 inhibitor.

Techniques: Binding Assay

Journal: Journal of translational medicine

Article Title: A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma.

doi: 10.1186/s12967-017-1312-7

Figure Lengend Snippet: Fig. 2 Primary assay screening p32 inhibitors for ability to rescue cells in glutamine free media. a Stable SF188 shControl cells or SF188 cells with genetic knockdown of p32 (shP32) were grown in glutamine free media for 3 days then allowed to recover 1 day in glutamine containing media. Cells were stained with crystal violet. b Potential p32 inhibitors were tested at 50 µM with SF188 glioma cells incubated in glutamine free DMEM (+ 10% dialyzed FBS), or c in complete media [DMEM with glutamine (+ 10% dialyzed FBS)], and cell viability was determined by Alamar Blue assay after 4 days. P32 inhibition promotes cell growth in glutamine free media and inhibits growth in complete media. Gray bar is DMSO control; green bar shows potential p32 inhibitor compound (M36). d Structure of small molecule M36

Article Snippet: Protein thermal shift assay Human recombinant p32 (OriGene) was tested in a reaction volume of 20 μL with 100 μM of p32 inhibitor.

Techniques: Knockdown, Staining, Incubation, Alamar Blue Assay, Inhibition, Control

Journal: Journal of translational medicine

Article Title: A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma.

doi: 10.1186/s12967-017-1312-7

Figure Lengend Snippet: Fig. 3 Fluorescence polarization assay demonstrating M36 binding to p32. a 5-FAM labeled cyclic LyP-1 peptide was incubated with increas- ing concentrations of recombinant p32 for 2 h. Increase in fluorescence polarization (mFP) indicates binding to the peptide. b 100 nM p32 was pre-incubated 1 h with increasing concentrations of unlabelled LyP-1 peptide in a competition assay before incubation with 0.5 nM labeled cyclic LyP-1 peptide. c 100 nM p32 was incubated with increasing concentrations of M36 for 30 min and then incubated with 5-FAM labeled cyclic LyP-1 peptide for 2 h. Direct binding of the hit compound M36 to p32 inhibits peptide binding resulting in decreased fluorescence polarization (mFP). Error bars represent standard deviation

Article Snippet: Protein thermal shift assay Human recombinant p32 (OriGene) was tested in a reaction volume of 20 μL with 100 μM of p32 inhibitor.

Techniques: Fluorescence, Binding Assay, Labeling, Incubation, Recombinant, Competitive Binding Assay, Standard Deviation

Journal: Journal of translational medicine

Article Title: A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma.

doi: 10.1186/s12967-017-1312-7

Figure Lengend Snippet: Fig. 6 M36 selectively targets high p32 expressing cells. a Immunoblot for p32 for indicated cell lines. b U373 and UW426 cells were incubated in low glucose (2 mM) DMEM (+ 10% dialyzed FBS) with increasing concentrations of M36. Cell viability was determined by Alamar Blue assay and IC50 calculated using Graphpad Prism and was compared to the IC50 values for SF188 and patient-derived neurospheres

Article Snippet: Protein thermal shift assay Human recombinant p32 (OriGene) was tested in a reaction volume of 20 μL with 100 μM of p32 inhibitor.

Techniques: Expressing, Western Blot, Incubation, Alamar Blue Assay, Derivative Assay

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) PANTHER pathway analysis of upstream transcriptional regulators identified in microarray data comparing articular cartilage of 8-week old male Anp32a -deficient to wild-type mice (n = 4 per group) using Ingenuity Pathway Analysis (IPA). (B) Real-time PCR analysis of direct Wnt target genes Tcf1 , Ccnd1 , cMyc and Axin2 in control and Anp32a knockdown (KD) ATDC5 cells (Pillai = 0.964, F 2,3 = 40.34, P = 0.0068 by MANOVA; Ccnd1 and Axin2 showed > 0.9 correlation with Tcf1 and were not included in the model, mean + SD of 3 technical replicates per condition). (C) Real-time PCR analysis of chondrogenic differentiation markers collagen 2 ( Col2a1 ), aggrecan ( Acan ) and collagen 10 ( Col10a1 ) in control and Anp32a KD ATDC5 cells [F 3,12 = 25.321, P < 0.0001 ( Col2a1 ), F 1.05,4.21 = 24.378 P = 0.007 ( Acan ), F 3,12 = 42.775 P < 0.0001 ( Col10a1 ) by 2-way ANOVA for interaction between silencing and time, mean + SD of 3 technical replicates per condition]. (D) Alcian blue (AB), safranin O (SO), picrosirius red (SR) at day 14 (D14) and alizarin red (AR) staining at day 21 (D21) showing reduced proteoglycan deposition (AB, SO), collagen content (SR) and mineralization (AR) in Anp32a KD ATDC5 cells during chondrogenesis. (E) AB staining demonstrating rescue of chondrogenic differentiation in Anp32a KD ATDC5 cells by treatment with Wnt inhibitor XAV939 (XAV). (F) Real-time PCR analysis of Wnt target gene expression in articular cartilage from 8-week old male wild-type (WT) and Anp32a -deficient ( Anp32a -/- ) mice (Pillai = 0.788, F 3,11 = 13.61, P = 0.0005 by MANOVA – cMyc showed > 0.9 correlation with Tcf1 and was not included in the model, n = 8 and 7 mice per group). (G-I) Immunohistochemical staining for TCF1 protein in male 8-week old (G), osteoarthritic (H) and female ageing WT and Anp32a -/- mice (I) (representative images of n = 3 different mice per group). Scale bar 50 µm. (J) Real-time PCR analysis of Wnt target gene expression in human articular chondrocytes transfected with siRNA targeting ANP32A (siANP32A) or scrambled siRNA (siSCR) (n = 2 different donors - mean ± SD of 3 technical replicates). (K) TOP/FOP reporter assay in human articular chondrocytes transfected with siANP32A, siRNA targeting β-catenin (siβCAT) or siSCR, and then treated with recombinant WNT3A (n = 2 biologically independent experiments, mean ± SD of 3 technical replicates). (L-M) TCF1 expression (L) and negative correlation with ANP32A expression (M) by RNA sequencing in paired preserved and damaged cartilage from hips (o) and knees (Δ) from osteoarthritis patients [log2-fold change (Log2FC) of damaged (D) vs. preserved (P)) (n = 21, P < 0.0001, Benjamini-Hochberg adjusted paired t -test (l), Pearson’s correlation = -0.40 - P = 0.0084 (m)].

Article Snippet: Antibody binding to the column was performed using 75 mg of either a mock antibody (donkey anti-goat IgG) as a control or ANP32A antibody (Novus Biologicals, NBP1-97576, clone RJ1).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Control, Knockdown, Staining, Targeted Gene Expression, Immunohistochemical staining, Transfection, Reporter Assay, Recombinant, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A, B) Real-time PCR analysis of Anp32a (A) and Atm (B) in control and Anp32a knockdown (KD) ATDC5 cells. Error bars indicate mean ± SD of three technical replicates per condition [F 1,4 = 69.892, P = 0.001 ( Anp32a ), F 1,4 = 24.841 P = 0.009 ( Atm ), by 2-way ANOVA for control versus KD cells, mean + SD of 3 technical replicates per condition].

Article Snippet: Antibody binding to the column was performed using 75 mg of either a mock antibody (donkey anti-goat IgG) as a control or ANP32A antibody (Novus Biologicals, NBP1-97576, clone RJ1).

Techniques: Real-time Polymerase Chain Reaction, Control, Knockdown

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A, B) Real-time PCR analysis of chondrogenic differentiation markers collagen 2 ( Col2a1 ), aggrecan ( Acan ) and collagen 10 ( Col10a1 ) at day 7 in control and Anp32a knockdown (KD) ATDC5 cells after treatment with antioxidant N-acetylcysteine (NAC) (A) or Wnt inhibitor XAV939 (XAV) (B) at the indicated concentrations. Error bars indicate mean ± SD of three technical replicates per condition.

Article Snippet: Antibody binding to the column was performed using 75 mg of either a mock antibody (donkey anti-goat IgG) as a control or ANP32A antibody (Novus Biologicals, NBP1-97576, clone RJ1).

Techniques: Real-time Polymerase Chain Reaction, Control, Knockdown

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A, B) Expression of Wnt target genes CCND1, CMYC and AXIN2 (A) and correlation with ANP32A expression (B) by RNA sequencing in paired preserved (P) and damaged (D) cartilage from hips (o) and knees (Δ) from osteoarthritis patients (log2-fold change (Log2FC) of damaged vs. preserved) (n=21, P < 0.0001 for CCND1 and AXIN2 , P =0.0809 for CMYC by Benjamini-Hochberg adjusted paired t -test (A), Pearson’s correlation R = -0.34 – P = 0.027, R = 0.03 – P = 0.853, R = -0.39 – P = 0.112 for CCND1, CMYC and AXIN2 respectively (B)).

Article Snippet: Antibody binding to the column was performed using 75 mg of either a mock antibody (donkey anti-goat IgG) as a control or ANP32A antibody (Novus Biologicals, NBP1-97576, clone RJ1).

Techniques: Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) Co-immunoprecipitation (Co-IP) using an anti-ANP32A antibody showing the binding between ANP32A and AXIN1 in untreated human articular chondrocytes, which is abolished upon Wnt activation by recombinant WNT3A protein. Wnt activation increases the binding of ANP32A with β-catenin and with histone 3. Silencing of ANP32A (siANP32A) is shown to assess the specificity of the ANP32A antibody used in the immunoprecipitation. The image is representative of three independent experiments. (B) Immunoblot analysis of ANP32A, active β-catenin and total β-catenin protein amounts (with actin as loading control) in human articular chondrocytes transfected with ANP32A (siANP32A) or scrambled siRNA (siSCR). (C) Immunofluorescent staining of ANP32A (green) and counterstaining of nuclei with Hoechst dye (blue) in human articular chondrocytes upon vehicle or recombinant WNT3A treatment (4 hours). Representative images are shown (n = 2). Scale bar 10 µm. (D) Chromatin-immunoprecipitation quantitative PCR (ChIP-qPCR) analysis of ANP32A binding to chromatin on Wnt target ( Tcf1 , Cnnd1,cMyc and Axin2 ) gene promoter regions in human articular chondrocytes with high endogenous Wnt signaling (cells were expanded two passages). (E) ChIP-qPCR analysis of acetylated H3K9 (H3K9Ac) on Wnt target ( Tcf1 , Ccnd1, cMyc and Axin2 ) and Atm gene promoter regions in human articular chondrocytes with high endogenous Wnt signaling that were transfected with siANP32A or siSCR. Data are expressed as ratio of H3K9Ac and total Histone 3 (H3) in siANP32A cells normalized to siSCR cells. Data points are from two biologically independent experiments (D-E). (F) Scheme summarizing how ANP32A regulates the Wnt transcriptional response. In basal conditions (left panel), ANP32A interacts with AXIN1 in the destruction complex. Upon Wnt activation (right panel), ANP32A dissociates from AXIN1, associates with β-catenin, and translocates to the nucleus. Within the nucleus, ANP32A represses Wnt target genes via blocking histone acetylation.

Article Snippet: Antibody binding to the column was performed using 75 mg of either a mock antibody (donkey anti-goat IgG) as a control or ANP32A antibody (Novus Biologicals, NBP1-97576, clone RJ1).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Activation Assay, Recombinant, Western Blot, Control, Transfection, Staining, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Blocking Assay

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) Schematic outline of in vivo pharmacological interventions against osteoarthritis in Anp32a -deficient ( Anp32a -/- ) male mice. 8-week old mice were subjected to destabilization of the medial meniscus (DMM) surgery. One week after injury, mice were injected intra-articularly with vehicle or Wnt inhibitor XAV939 (XAV) every 10 days for a total of 7 times. Mice were treated orally with vehicle, XAV or NAC alone or in combination. Knee joints were collected 12 weeks after surgery. (B-C) Hematoxylin-safranin-O-stained sections (B) and quantification by OARSI severity grade (C) demonstrating that NAC protects against articular cartilage damage in osteoarthritis: [F 1,28 =8.48 P = 0.007 for main effect NAC by two-way ANOVA, n = 8 per group]. Scale bar 200 µm. (D) Immunohistochemical staining for TCF1 protein in articular cartilage of Anp32a -/- mice in the DMM model treated or not with XAV or NAC (representative images of n = 5 different mice per group). Scale bar 50 µm. ( E-F) Hematoxylin-safranin-O-stained sections (E) and quantification (F) of osteophytes demonstrating that Wnt inhibition protects against osteophyte formation in osteoarthritis [F 1,28 =7.69 P = 0.0098 for main effect XAV by two-way ANOVA, n = 8 per group]. Scale bar 200 µm. (G) Immunohistochemical staining for TCF1 protein in developing osteophytes in Anp32a -/- mice in the DMM model treated or not with XAV or NAC (representative images of n = 5 different mice per group). Scale bar 50 µm.

Article Snippet: Antibody binding to the column was performed using 75 mg of either a mock antibody (donkey anti-goat IgG) as a control or ANP32A antibody (Novus Biologicals, NBP1-97576, clone RJ1).

Techniques: In Vivo, Injection, Staining, Immunohistochemical staining, Inhibition

Journal: bioRxiv

Article Title: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease

doi: 10.1101/2021.04.04.438364

Figure Lengend Snippet: (A) Selected Protein Atlas data showing the inverse relationship between ANP32A and Wnt target gene TCF1 in brain and muscle tissues compared to other systems. Data are presented as scaled variables. (B) Real-time PCR analysis of Wnt target gene expression (n = 8) in heart from 20-week old male WT and Anp32a -/- mice (Pillai = 0.492, F 3,12 = 3.877, P =0.038 by MANOVA - Axin2 data did not show homogeneity of variance and were not included in the model). (C) Immunohistochemical staining for TCF1 in 20-week old male WT and Anp32a -/- mice hearts. Scale bar 50 µm. (D) Macroscopic and Hematoxylin-Eosin stained hearts demonstrating cardiac hypertrophy in Anp32a -/- compared to WT mice. Scale bar 2 mm. ( E ) Heart weight/body weight ratio in 20-week old male WT and Anp32a -/- mice (t 27 = 10.45, P < 0.0001). ( F ) Real-time PCR analysis of hypertrophy markers natriuretic peptide precursor A (Nppa) and skeletal muscle α- actin (Acta1) (n = 8) in heart from 20-week old male WT and Anp32a -/- mice (t 14 = 4.010, P = 0.0011 ( Nppa ), t 14 = 2.87, P = 0.0125 ( Acta1 ) by Student’s t-test). ( G-H ) Picrosirius red staining (G) showing increased amounts of fibrotic tissue in hearts from 20-week old Anp32a -/- mice compared to WT (H) (t 6 = 6.82, P = 0.0005 by Student’s t-test). Scale bar 50 µm. (I) Real-time PCR analysis of Wnt target gene expression (n = 9) in brain from 8-week old male WT and Anp32a -/- mice (Pillai = 0.666, F 4,10 = 4.978, P =0.018 by MANOVA – 3 extreme outliers were excluded from the model as indicated in cyan color). (J) Immunohistochemical staining for TCF1 in 16-week old male hippocampus from WT and Anp32a -/- mice. Scale bar 250 µm.

Article Snippet: Antibody binding to the column was performed using 75 mg of either a mock antibody (donkey anti-goat IgG) as a control or ANP32A antibody (Novus Biologicals, NBP1-97576, clone RJ1).

Techniques: Real-time Polymerase Chain Reaction, Targeted Gene Expression, Immunohistochemical staining, Staining

Journal: Nucleic Acids Research

Article Title: PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4

doi: 10.1093/nar/gkx775

Figure Lengend Snippet: PP32 and SET/TAF-Iβ are components of the Complex Ib. ( A ) Scheme illustrating the purification procedure for obtaining Complex Ib from HeLa S100 extracts. ( B ) Western blots of fractions derived from the last DEAE-5PW column, as indicated. The corresponding salt concentration of the fractions and the elution of Complex Ib and Complex II are specified at the bottom. Complex Ib elutes in fractions 21–22 and Complex II in fractions 40–42. An additional uncharacterized histone H3 and H4 peak is observed from fraction 48. ( C ) Left, silver staining of the enriched Complex Ib, corresponding to the pool of fractions 19–23 derived from the last DEAE-5PW column. Right, mass spectrometry (MS) data of the enriched Complex Ib, indicating the number of peptides identified for each protein and the coverage. ( D ) Western blot of the enriched Complex Ib, corresponding to the pool of fractions 19–23 derived from the last DEAE-5PW column.

Article Snippet: Either 10 nM of human I2PP2A (SET/TAF-Iβ) siRNA duplex (Santa Cruz, sc-43856), human ANP32A (PP32) siRNA duplex (Santa Cruz, sc-40696), or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion) were transfected with Lipofectamine 2000 (Invitrogen) for 72 h, according to the manufacturer's instructions.

Techniques: Purification, Western Blot, Derivative Assay, Concentration Assay, Silver Staining, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4

doi: 10.1093/nar/gkx775

Figure Lengend Snippet: PP32 and SET/TAF-Iβ associates with newly synthesized histone H4. ( A ) Scheme illustrating the procedure utilized to label and isolate newly synthesized proteins. ( B ) Western blot analysis of 1% of the input material (left), purified Flag-immunoprecipitated proteins (middle), and Streptavidin–agarose pulled-down proteins derived from the Flag-immunoprecipitated material (right), as indicated. Top panels were developed with Streptavidin-HRP to visualize AHA-labeled proteins conjugated to biotin AHA–biotin. Bottom panels were developed as indicated. ( C ) Western blot analysis of 1% of the input material, purified Flag-immunoprecipitated proteins and Streptavidin–agarose pulled-down proteins derived from the Flag-immunoprecipitated material, with extracts in which the CLICK-IT procedure was performed with DMSO instead of AHA. Top panel was developed with Streptavidin–HRP to visualize AHA-labeled proteins conjugated to biotin AHA–biotin. Bottom panels were developed as indicated.

Article Snippet: Either 10 nM of human I2PP2A (SET/TAF-Iβ) siRNA duplex (Santa Cruz, sc-43856), human ANP32A (PP32) siRNA duplex (Santa Cruz, sc-40696), or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion) were transfected with Lipofectamine 2000 (Invitrogen) for 72 h, according to the manufacturer's instructions.

Techniques: Synthesized, Western Blot, Purification, Immunoprecipitation, Derivative Assay, Labeling

Journal: Nucleic Acids Research

Article Title: PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4

doi: 10.1093/nar/gkx775

Figure Lengend Snippet: PP32 and SET/TAF-Iβ proteins regulate newly synthesized H4 acetylation levels in vivo . Western blots of 10 and 30 μg of S100 extracts derived from either siControl and siPP32 ( A ) or siSET/TAF-Iβ ( B ) treated HeLa cells.

Article Snippet: Either 10 nM of human I2PP2A (SET/TAF-Iβ) siRNA duplex (Santa Cruz, sc-43856), human ANP32A (PP32) siRNA duplex (Santa Cruz, sc-40696), or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion) were transfected with Lipofectamine 2000 (Invitrogen) for 72 h, according to the manufacturer's instructions.

Techniques: Synthesized, In Vivo, Western Blot, Derivative Assay

Journal: Nucleic Acids Research

Article Title: PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4

doi: 10.1093/nar/gkx775

Figure Lengend Snippet: PP32 and SET/TAF-Iβ proteins block HAT1 mediated H4 acetylation in vitro . Acetylation assay performed using 0.128 nmol of recombinant histone H4 and purified HAT1 in the presence or absence of increasing amounts of recombinant PP32 ( A ) and recombinant SET/TAF-Iβ ( B ), followed by H4K12ac Dot-blot detection. Top: graph of the remaining HAT1 activity. 100% activity correspond to the HAT1 mediated H4 acetylation in the absence of PP32 and SET/TAF-Iβ. Bottom: a representative H4K12ac dot blot HAT1 assay. FT corresponds to the flow through material of the recombinant SET/TAF-Iβ Ni +2 -beads purification. ( C ) Top: Coomassie blue stained gel of the different recombinant proteins used in the acetylation assay. Bottom: acetylation assay performed as in (A), pre-incubating H4 as indicated. ( D ) Acetylation assay performed using recombinant histone H4 and purified HAT1 in the presence of 8 pmol of recombinant PP32, 12 pmol of recombinant SET/TAF-Iβ, and a mix of 8 pmol of recombinant PP32 and 12 pmol of recombinant SET/TAF-Iβ (Mix 1×), and 4 pmol of recombinant PP32 and 6 pmol of recombinant SET/TAF-Iβ (Mix 0.5×).

Article Snippet: Either 10 nM of human I2PP2A (SET/TAF-Iβ) siRNA duplex (Santa Cruz, sc-43856), human ANP32A (PP32) siRNA duplex (Santa Cruz, sc-40696), or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion) were transfected with Lipofectamine 2000 (Invitrogen) for 72 h, according to the manufacturer's instructions.

Techniques: Blocking Assay, In Vitro, Acetylation Assay, Recombinant, Purification, Dot Blot, Activity Assay, Staining

Journal: Nucleic Acids Research

Article Title: PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4

doi: 10.1093/nar/gkx775

Figure Lengend Snippet: PP32 knock-down affects the maturation of newly synthesized histone H4. ( A ) Western blot of HA-Hsp90 pull-down assay from cytosolic extracts derived from siControl and siHsp90 treated HeLa cells. ( B ) Graph of the tryptophan fluorescence spectroscopy of Hsp90 upon titrating full length histone H4 (left), or unmodified or acetylated amino acids 1–20 of histone H4 (right). Δ F is the difference between initial fluorescence ( F 0 ) and the fluorescence after ligand addition ( F Q ). K D is the dissociation constant of the ligand with Hsp90. ( C ) Left, Western blot analysis of thermal stability assay using HeLa S100 extracts. 20 μg of S100 extracts were heated for 20 min at the indicated temperatures and centrifuged at 10 000 × g for 5 min. Supernatant and pellet were analyzed by western blot. Right, graph of the percentage of either total or acetylated histone H4 found on the pellet, taken as 100% of the sum of the supernatant and pellet at each temperature. Standard deviations were taken from three independent experiments. * P < 0.05, Student′s t -test. ( D ) Thermal stability of S100 extracts derived from siControl and siPP32 HeLa cells, as described in (C). ( E ) Thermal stability of S100 extracts derived from siControl and siPP32 HeLa cells, as described in (C), in the absence or presence of recombinant PP32.

Article Snippet: Either 10 nM of human I2PP2A (SET/TAF-Iβ) siRNA duplex (Santa Cruz, sc-43856), human ANP32A (PP32) siRNA duplex (Santa Cruz, sc-40696), or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion) were transfected with Lipofectamine 2000 (Invitrogen) for 72 h, according to the manufacturer's instructions.

Techniques: Knockdown, Synthesized, Western Blot, Pull Down Assay, Derivative Assay, Fluorescence, Spectroscopy, Stability Assay, Recombinant

Journal: Nucleic Acids Research

Article Title: PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4

doi: 10.1093/nar/gkx775

Figure Lengend Snippet: PP32 knock-down affects cell cycle progression. ( A ) Cell cycle profiles of HeLa cells treated with siControl, siPP32, and siSET/TAF-Iβ. ( B ) Proposed model for the regulation of newly synthesized histone H4 acetylation. After synthesis, unacetylated histone H4 interacts with the heat shock proteins Hsp90 and Hsp70 and two members of the INHAT complex: PP32 and SET/TAF-Iβ. The binding of PP32 and SET/TAF-Iβ protects histone H4 from premature acetylation by HAT1, as well as for non-specific acetylation mediated by other HATs. When PP32 protein levels are diminished, histone H4 is acetylated at early steps of the maturation cascade, and it no longer can interact with Hsp90 affecting its stability and maturation.

Article Snippet: Either 10 nM of human I2PP2A (SET/TAF-Iβ) siRNA duplex (Santa Cruz, sc-43856), human ANP32A (PP32) siRNA duplex (Santa Cruz, sc-40696), or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion) were transfected with Lipofectamine 2000 (Invitrogen) for 72 h, according to the manufacturer's instructions.

Techniques: Knockdown, Synthesized, Binding Assay