Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Western blot immunoreactivity of the polyclonal anti-MPV17 antibody Ab 93374 ( left ) and the monoclonal anti- human MPV17 antibodies 6F5 and 5D2 on extracts of human and murine cells of different MPV17 genotypes. Left extracts from human U2OS cells, untransfected or transfected with the MPV17 expression clone SC118652 (origene), were probed with the antibody ab 93374 (abcam). Center untransfected U2OS cells with MPV17 +/+ endogenous genotype probed with the monoclonal antibodies 6F5 and 5D2. Right Murine embryo fibroblast (MEF) cells of MPV17 +/+ or MPV17 -/- genotype probed with the monoclonal antibodies 6F5 and 5D2. Indicated marker sizes were transferred from Ponceau stained marker bands run in parallel. Loading was controlled as indicated by reaction of the filters with an anti-ß-actin antibody. 10 μg of protein were separated on SDS gels and analysed by Western blot according to ( left panel ), and Kinkley et al. respectively

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Western Blot, Transfection, Expressing, Marker, Staining

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Ab93374 (Abcam) recognizes MPV17 specific structures in U2OS cells transiently transfected with an MPV17 expression construct in immunofluorescence analysis. Ab93374 signal ( green ) is largely missing in nontransfected cells. The immunoreactivity does colocalise with peroxisomal (anti-catalase, mouse polyclonal, Abcam ab88650) and mitochondrial (anti-complex IV mitochondrial subunit I, mouse monoclonal, Invitrogen 459600) markers ( red ). Immunofluorescence (IF) method was according to Pircher et al. using a MicroRadiance confocal scanning system (Bio-Rad) in combination with a Zeiss Axiophot microscope. Colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Transfection, Expressing, Construct, Immunofluorescence, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: In U2OS cells, 5D2 antibody detects a punctate pattern colocalising with a peroxisomal (PMP70) but not with a mitochondrial (MitoTracker) marker. Top Rabbit anti PMP70 antibody ( decorated green ) was a gift from W. Just, Heidelberg. Secondary antibodies: Alexa Fluor 555 anti-mouse and Alexa Fluor 488 anti-rabbit. Bottom Mitotracker 7510 (Invitrogen) was used as recommended by the supplier. Secondary antibody: Alexa 488 anti-mouse. IF method and microscopy according to Kinkley et al. using a Zeiss LSM confocal microscope. Mathematical colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: 5D2 colocalisation with a marker of early endosomes (EEA1) and lysosomes (LAMP1) in U2OS cells. Top Partial colocalisation of 5D2 with EEA1 (rabbit anti-human). Bottom colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig.

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Colocalisation of 5D2 and LAMP1 mediated immunofluorescence in a U2OS single cell. IF and analysis of 5D2 and Colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig. . In addition a merge of the two immunofluorescence pictures is depicted

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Immunofluorescence

Journal: Nature genetics

Article Title: Gene regulation by convergent promoters.

doi: 10.1038/s41588-024-02025-w

Figure Lengend Snippet: Fig. 6 | Characteristics of convergent promoters. a, Schematics (left) highlight the TSSs that have been used to identify the extended set of convergent promoters and that have been compared for their log2FC (Nutlin-3a compared with DMSO control). Spearman correlation in MCF-7 (left panel), U2OS (middle panel) and RPE-1 cells (right panel). Linear regression with 95% confidence intervals. Spearman correlation with two-tailed significance. b, Summary profile (top panel) and heatmap with individual convergent promoter regions (bottom panels) starting from the host TSS display CpG island signals. The extended set of MCF-7 convergent promoters is length-sorted in descending order. c,d,f,g, ENCODE data from MCF-7 cells57. Summary profiles of H3K4me3 (left panels c and f), H3K27ac (middle panel c, right panel f), H3K4me1 (right panel c) and Pol II (d,g) signals for the extended set of scale-adjusted MCF-7 convergent promoters. e, Violin plots summarize the read counts at GENCODE-annotated TSS-overlapping

Article Snippet: Cell culture, drug treatment and transfection MCF-7 (ATCC, cat. no. HTB-22) and U2OS (DSMZ, cat. no. ACC 785) cells were grown in high glucose Dulbecco’s modified Eagle’s media (DMEM) with pyruvate (ThermoFisher Scientific).

Techniques: Control, Two Tailed Test

Journal: Cell Genomics

Article Title: Integrative high-throughput enhancer surveying and functional verification divulges a YY2-condensed regulatory axis conferring risk for osteoporosis

doi: 10.1016/j.xgen.2024.100501

Figure Lengend Snippet: YY2 preferentially binds to rs11202530-G to strengthen its enhancer effect on PAPSS2 expression (A) Bar shows comparison of allelic expression ratio of rs11202530 between STARR-seq output and input library samples evaluated by MPRAnalyze. (B) Graphic representation of rs11202530-G resides within YY2 DNA-binding motif predicted by FIMO from MEME Suite toolkit. (C) Comparison of significant Hi-C chromatin interactions linking rs11202530 to PAPSS2 promoter region in hMSCs or hMSC-differentiated osteoblasts or adipose tissue (no significant interaction). Genomic region surrounding rs11202530 was zoomed in with track of ChIP-seq signals on YY2 in HEK293 cell from ENCODE depicted below. (D) ChIP-qPCR results of YY2 binding at rs11202530 region in U2OS cells. Primers specifically targeting rs11202530 or RPL30 exon region (negative control [NC]) were used. The binding efficiency of YY2 is shown as fold enrichment over IgG. (E) Allele-specific ChIP-qPCR indicated allele-specific binding of YY2 binding at rs11202530 in MG63. Primers specifically targeting rs11202530-A or G were used. (F) RT-qPCR assay revealed the effect of YY2 knockdown on PAPSS2 expression by two independent shRNAs in U2OS cells. (G) Western blotting analysis showed the effect of YY2 knockdown on PAPSS2 expression in U2OS cells. GAPDH was used as loading control. (H) Dual-luciferase reporter assay comparing regulatory activity on PAPSS2 promoter inserted by rs11202530-G or rs11202530-A co-transfected with two independent shRNAs of YY2 or shRNA-NC in U2OS cells. (I) Chromosome conformation capture (3C) assay in U2OS cells. Normalized chromatin interaction frequencies between rs11202530 region as the anchor point (N7) and PAPSS2 promoter region (N4) or six other neighboring Hind III sites as negative controls (N1, N2, N3, N5, N6, and N8). (J) 3C assay shows comparison of normalized interaction frequency differences between rs11202530 region and PAPSS2 promoter region in YY2 -inhibited ( YY2 -shRNA-2) and control (shRNA-NC) U2OS cells. Data are presented as mean ± standard deviation in (D)–(F) and (H)–(J), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: not significant, two-tailed paired Student’s t test.

Article Snippet: We performed chromatin immunoprecipitation assay (ChIP) of YY2 with Simple ChIP Enzymatic Chromatin IP Kit (CST, Cat#9003, USA) in U2OS (homozygous GG of rs11202530) and MG63 (heterozygous AG of rs11202530) according to the manufacturer’s protocol, which was described in detail as previously.

Techniques: Expressing, Comparison, Binding Assay, Hi-C, ChIP-sequencing, ChIP-qPCR, Negative Control, Quantitative RT-PCR, Knockdown, Western Blot, Control, Luciferase, Reporter Assay, Activity Assay, Transfection, shRNA, Standard Deviation, Two Tailed Test

Journal: Cell Genomics

Article Title: Integrative high-throughput enhancer surveying and functional verification divulges a YY2-condensed regulatory axis conferring risk for osteoporosis

doi: 10.1016/j.xgen.2024.100501

Figure Lengend Snippet: Rs11202530 acts as an allele-biased enhancer to regulate PAPSS2 expression and osteoblast differentiation (A) Comparison of H3K27ac histone modification in hMSCs or hMSC-differentiated osteoblasts or adipose tissue surrounding rs11202530. (B) Bar diagrams display all significant cis -QTL associations (p < 0.05 by linear model controlling for covariates) between genotypes of rs11202530 and PAPSS2 expression from GTEx (V8), and effect sizes are indicated by shade of colors. (C) Dual-luciferase reporter assay shows allelic-biased enhancer activity effect on rs11202530 compared with pGL3- PAPSS2 -promoter in U2OS cells. Luciferase signal was computed as the ratio of firefly luciferase activity to Renilla signal, and relative activity was normalized by pGL3- PAPSS2 -promoter. (D) RT-qPCR shows effect of deletion of the genomic region surrounding rs11202530 by CRISPR-Cas9 on PAPSS2 expression at mRNA level in U2OS or human primary osteoblasts isolated from healthy cancellous bone tissue . The upper cartoon shows relative position of targeting sites for different pairs of sgRNAs. The bottom gel showed the deletion efficiency of two paired sgRNAs by amplifying a flanking region of 1740 bp (rs11202530) and validated the truncated short regions by PCR. (E) RT-qPCR displays effect of deletion of the genomic region surrounding rs11202530 by CRISPR-Cas9-decreased expression of osteoblast differentiation marker gene expression ( ALP , OCN , OSX , RUNX2 , and COL1A1 ) at mRNA level in human primary osteoblasts. (F) Alkaline phosphatase (ALP) staining revealed the effect of deletion of the region surrounding rs11202530 by CRISPR-Cas9 on ALP activity in human primary osteoblasts. The ALP staining results were analyzed using ImageJ software. Data are presented as mean ± standard deviation in (C)‒(E), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: not significant, two-tailed paired Student’s t test.

Article Snippet: We performed chromatin immunoprecipitation assay (ChIP) of YY2 with Simple ChIP Enzymatic Chromatin IP Kit (CST, Cat#9003, USA) in U2OS (homozygous GG of rs11202530) and MG63 (heterozygous AG of rs11202530) according to the manufacturer’s protocol, which was described in detail as previously.

Techniques: Expressing, Comparison, Modification, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, CRISPR, Isolation, Marker, Gene Expression, Staining, Software, Standard Deviation, Two Tailed Test

Journal: Cell Genomics

Article Title: Integrative high-throughput enhancer surveying and functional verification divulges a YY2-condensed regulatory axis conferring risk for osteoporosis

doi: 10.1016/j.xgen.2024.100501

Figure Lengend Snippet: YY2 may play roles in promoting human osteoblast differentiation through positive regulation of PAPSS2 (A) RT-qPCR shows comparison of Yy2 and Papss2 expression between ovariectomy (OVX)-induced osteoporosis mice (females, n = 3) and control normal mice with parietal ovarian fat tissue removed (females, n = 3). (B) RT-qPCR shows expression change of YY2 and PAPSS2 in isolated human primary osteoblasts after differentiation for 3, 7, or 14 days . The upper ALP staining displays ALP activity change in osteoblasts after differentiation for different days. (C and D) RT-qPCR shows effect of YY2 overexpression (C) or knockdown (D) on PAPSS2 or expression of several osteoblast differentiation marker genes ( ALP , OCN , RUNX2 , and COL1A1 ) at mRNA level in human primary osteoblasts. (E and F) RT-qPCR shows effect of PAPSS2 overexpression (E) or knockdown (F) on expression of several osteoblast differentiation marker genes ( ALP , OCN , RUNX2 , and COL1A1 ) at mRNA level by two independent shRNAs in human primary osteoblasts. (G) Speculative regulatory model connecting different genotypes of rs11202530 to allele-preferable binding of YY2 and modified PAPSS2 expression, osteoblast differentiation, and osteoporosis risk. Data are presented as mean ± standard deviation in (A)–(F), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: not significant, two-tailed paired Student’s t test.

Article Snippet: We performed chromatin immunoprecipitation assay (ChIP) of YY2 with Simple ChIP Enzymatic Chromatin IP Kit (CST, Cat#9003, USA) in U2OS (homozygous GG of rs11202530) and MG63 (heterozygous AG of rs11202530) according to the manufacturer’s protocol, which was described in detail as previously.

Techniques: Quantitative RT-PCR, Comparison, Expressing, Control, Isolation, Staining, Activity Assay, Over Expression, Knockdown, Marker, Binding Assay, Modification, Standard Deviation, Two Tailed Test

Journal: Cell Genomics

Article Title: Integrative high-throughput enhancer surveying and functional verification divulges a YY2-condensed regulatory axis conferring risk for osteoporosis

doi: 10.1016/j.xgen.2024.100501

Figure Lengend Snippet:

Article Snippet: We performed chromatin immunoprecipitation assay (ChIP) of YY2 with Simple ChIP Enzymatic Chromatin IP Kit (CST, Cat#9003, USA) in U2OS (homozygous GG of rs11202530) and MG63 (heterozygous AG of rs11202530) according to the manufacturer’s protocol, which was described in detail as previously.

Techniques: Virus, Plasmid Preparation, Recombinant, Lysis, Gentle, Isolation, cDNA Synthesis, RNA HS Assay, Transfection, Luciferase, Reporter Gene Assay, Chromatin Immunoprecipitation, SYBR Green Assay, Functional Assay, Gene Expression, Sequencing, shRNA, Amplification, Marker, Software

Journal: Physiological Reports

Article Title: Abaloparatide exhibits greater osteoanabolic response and higher cAMP stimulation and β ‐arrestin recruitment than teriparatide

doi: 10.14814/phy2.14225

Figure Lengend Snippet: Activation of PTHR1 signaling by abaloparatide and teriparatide treatment: (A) Activation of intracellular cAMP production: MC3T3‐E1 cells were challenged with 0–100 nmol/L of abaloparatide or teriparatide for 40 min at 37°C in the presence of IBMX. The medium was disposed and cells were snap frozen on liquid N3 before storage at 80°C. Intracellular cAMP was extracted and measured using ELISA assay as described by the manufacturer protocol. Each dose treatment was performed in duplicate and the experiment was repeated three times. The EC50 was calculated using results from three independent experiments. Results are the means ± SD. Upper panel: Absolute values are expressed as pmol/well. Lower panel: Values are expressed as % stimulation/maximal response. (B) Activation of β ‐arrestin recruitment: PathHunter eXpress PTHR1 CHO‐K1 β ‐arrestin GPCR assay was described under the material and methods section. CHO‐K1 cells were treated with 0–100 nmol/L of teriparatide or abaloparatide for 60 min at 37°C. Teriparatide and abaloparatide stimulations of β ‐arrestin recruitment were determined by measuring light generation after adding β ‐gal enzyme substrate. The EC50 was calculated using results from three independent experiments. Results are the means ± SD. Upper panel: Absolute values are expressed as relative light units (RLU). Lower panel: Values are expressed as % stimulation/maximal response. (C) Stimulation of PTHR1 internalization: PathHunter eXpress PTHR1 U2OS activated GPCR internalization assay was described under the material and methods section. U2OS cells were treated with 0–100 nmol/L of teriparatide or abaloparatide for 60 min at 37°C. PTHR1 internalization was determined by measuring light generation after adding β ‐gal enzyme substrate. The EC50 was calculated using results from three independent experiments. Results are the means ± SD. Upper panel: Absolute values are expressed as RLU. Lower panel: Values are expressed as % stimulation/maximal response.

Article Snippet: To determine PTHR1 internalization, we used PathHunter eXpress PTHR1 U2OS Activated GPCR Internalization Assay (Cat number 93‐0770E3CP0S) from Eurofins DiscoverX (Fremont, CA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay