Journal: Genome biology

Article Title: BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

doi: 10.1186/s13059-024-03175-0

Figure Lengend Snippet: Fig. 7 Opening of chromatin by BORIS facilitates binding of other transcriptional factors. a Scatter plot displaying the enrichment of 190 TF motifs at CTCF/BORIS binding sites that reprogrammed into active promoters, compared to transcriptionally silent CTCF/BORIS sites in NIH3T3 + BORIS (clone#2) cells. b MAZ and MXI1 motifs are significantly enriched at CTCF/BORIS binding sites converted into active promoters. c Genome browser view illustrates the recruitment of TBP, HCFC1, MXI1, and MAZ proteins at the CTCF site within the Rbpjl promoter, activated by BORIS binding in NIH3T3 + BORIS (clone#2) cells. The activated promoter is highlighted by a red open box. d Left panel: Scatter plot of normalized read counts (log10) for HCFC1 occupancy at the combined set of HCFC1 binding sites (46,287) in NIH3T3 + BORIS (clone#2) cells compared to the same genomic sites in EV cells. Right panel: Heatmap of CTCF (red), BORIS (blue), and HCFC1 (brown) occupancy at the 19,519 HCFC1 sites from the left panel (connected by red arrow). e TF motifs enriched at HCFC1 peaks (60 bp around the summit of peak) in NIH3T3 + EV versus NIH3T3 + BORIS (clone#2) cells. f Heatmap of BORIS (blue), TBP (purple), HCFC1 (brown), MXI1 (orange), and MAZ (green) occupancy at the 5871 CTCF/BORIS binding sites converted into active promoters in NIH3T3 + BORIS (clone#2) cells compared to NIH3T3 + EV cells from Fig. 4h. g Summary of epigenetic reprogramming: BORIS binding recruits SRCAP, which replaces H2A histone with H2A.Z, leading to the opening of chromatin around CTCF sites. This, in turn, attracts other TFs to bind and stimulate transcription, resulting in the conversion of transcriptionally inert CTCF sites into active promoters

Article Snippet: Rabbit polyclonal antibody against SRCAP (Kerafast, ESL103), rabbit polyclonal against HCFC1 (Novus Biologicals, NB100-68209), goat affinity-purified polyclonal antibody against Mxi1 (R&D Systems, AF4185), rabbit monoclonal to TATA-binding protein TBP (Abcam, ab220788), rabbit polyclonal anti-PHF8 antibody (Bethyl Laboratories, A301-772A), rabbit polyclonal to MAZ (Abcam, ab85725), rabbit polyclonal antibody against the region of histone H3 containing the trimethylated lysine 27 (H3K27me3) (Diagenode, C15410195), rabbit polyclonal antibody against histone H3, trimethylated at lysine 36 (H3K36me3) (Diagenode, C15410058), rabbit polyclonal antibody against histone H2A.Z (Abcam, ab4174), rabbit polyclonal to Histone H3 (acetyl K27) (Abcam, ab4729), rabbit polyclonal to Histone H3 (tri methyl K4) (Abcam, ab8580), anti-RNA Polymerase II Antibody, CTD Antibody, clone 8WG16 (Millipore, 05–952-I-100UG).

Techniques: Binding Assay

Journal: Scientific reports

Article Title: The effects of WWP1 overexpression on the golgi apparatus stress response and proteoglycan production in adipocytes.

doi: 10.1038/s41598-024-79114-7

Figure Lengend Snippet: Fig. 2. WWP1 is required for maintaining the structure of the Golgi apparatus in differentiated 3T3L1 adipocytes. (A) Representative images of the differentiated mock-transfected, WWP1 OE, WWP1-Δ1, and WWP1-Δ2 3T3L1 cells stained for Golgin97 (green) and DAPI (blue). Cells were treated with each concentration of monensin for 6 h. Scale bar is 10 μm. (B–C) Relative Golgi size was determined based on Golgin97 staining. Each dot represents a single cell. N = 17–42 cells. Data are means ± SD. Differences between each value were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (***: p < 0.001, ****: p < 0.0001). (D) WWP1 protein abundance in WWP1 knockdown (KD) 3T3L1 cells was analyzed by immunoblotting with anti-WWP1 (n = 3). Lamin B1 was used as a loading control. (E) Representative images of the differentiated mock-transfected and WWP1 KD differentiated 3T3L1 stained for Golgin97 (green) and DAPI (blue). Cells were treated with each concentration of monensin for 6 h. Scale bar is 10 μm. (F–G) Relative Golgi size was determined based on Golgin97 staining. Each dot represents a single cell. N = 17–42 cells. Data are means ± SD. Differences between each value were analyzed by one-way ANOVA with Tukey multiple comparisons test (*: p < 0.05).

Article Snippet: Therefore, to avoid non-specific detection, we used another commercially available WWP1 antibody (Proteintech, 13567-1-AP 1:500) in 3T3L1 adipocytes and WAT; however, this did not accurately detect WWP1 (data not shown).

Techniques: Transfection, Staining, Concentration Assay, Quantitative Proteomics, Knockdown, Western Blot, Control

Journal: Scientific reports

Article Title: The effects of WWP1 overexpression on the golgi apparatus stress response and proteoglycan production in adipocytes.

doi: 10.1038/s41598-024-79114-7

Figure Lengend Snippet: Fig. 3. WWP1 suppresses monensin-induced increases in Golgi stress markers. (A–B) The mRNA level of Gcp60 and Giantin in differentiated mock-transfected, WWP1 OE, WWP1-Δ1, and WWP1-Δ2 overexpressing (OE) 3T3L1 cells were treated with DMSO or 10 µM monensin for 6 h and analyzed by real-time PCR. Rps18 was used as a reference control. (C–D) The mRNA level of Gcp60 and Giantin in subcutaneous white adipose tissue (WAT) derived from normal diet (ND)-fed or high-fat diet (HFD)-fed Wwp1 KO mice of 13–15 weeks of age analyzed by real-time PCR. Rps18 was used as a reference control. Differences between each value were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ***: p < 0.0001).

Article Snippet: Therefore, to avoid non-specific detection, we used another commercially available WWP1 antibody (Proteintech, 13567-1-AP 1:500) in 3T3L1 adipocytes and WAT; however, this did not accurately detect WWP1 (data not shown).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Derivative Assay

Journal: Scientific reports

Article Title: The effects of WWP1 overexpression on the golgi apparatus stress response and proteoglycan production in adipocytes.

doi: 10.1038/s41598-024-79114-7

Figure Lengend Snippet: Fig. 4. WWP1 contributes to the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) in 3T3L1 adipocytes and white adipose tissue (WAT). (A, B) Effect of WWP1 overexpression (OE) on the disaccharide composition and amount of HS in differentiated 3T3L1 cells. (C, D) Effect of WWP1 knockdown (KD) on the disaccharide composition and amount of HS in differentiated 3T3L1 cells. (E, F) Effect of WWP1 OE on the disaccharide composition and amount of CS in differentiated 3T3L1 cells. (G, H) Effect of WWP1 KD on the disaccharide composition and amount of CS in differentiated 3T3L1 cells. (I, J) Disaccharide composition and levels of HS in subcutaneous WAT derived from normal diet (ND)-fed or high-fat diet (HFD)-fed Wwp1 KO mice of 13–15 weeks of age. (K, L) Disaccharide composition and levels of CS in subcutaneous WAT derived from ND-fed or HFD-fed Wwp1 KO mice of 13–15 weeks of age. Extraction of glycosaminoglycans and analysis of their unsaturated disaccharides was performed as described. Differences between each value were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (*: p < 0.05, **: p < 0.01).

Article Snippet: Therefore, to avoid non-specific detection, we used another commercially available WWP1 antibody (Proteintech, 13567-1-AP 1:500) in 3T3L1 adipocytes and WAT; however, this did not accurately detect WWP1 (data not shown).

Techniques: Over Expression, Knockdown, Derivative Assay, Extraction

Journal: Analytical chemistry

Article Title: Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry

doi: 10.1021/acs.analchem.2c02709

Figure Lengend Snippet: Workflows showing absolute quantitation for a) multiple proteins in a protein mixture, b) HCPs in mAb , and c) mAb deamidation by CMS.

Article Snippet: Host cell proteins (HCPs) are a major class of impurities derived from biotherapeutics production processes.

Techniques: Quantitation Assay