Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: IL‐17A NAb inhibited EMT in SHR renal tissues. (A) Representative IHC images showing the expression of E‐cadherin, α‐SMA, and Collagen III, with quantitative analysis of their positive areas. (B) Representative immunoblots and relative expression levels of E‐cadherin, α‐SMA, and Collagen III proteins. (C) mRNA expression levels of E‐cadherin, α‐SMA, and Collagen III. Data are expressed as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Expressing, Western Blot

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 4 ch5E6 treatment leads to a substantial decrease in the growth of organoids derived from PC patients or pancreatic and lung cancer genetically engineered mouse models. a PC patient and b KPC mouse organoid staining with ch5E6 indicating MUC16 expression (green) and specific binding of mAb compared to no binding with isotype control mAb huIgG1. The binding of ch5E6 to the ductal cells (yellow) was illustrated by colocalization with CK-19 (red) staining in human and mouse pancreatic tumor organoids. Scale bar 10 µm.The representative images for ch5E6 treated. c Human PC patient. d KPC and (e) KPA mouse-derived organoids obtained by real-time kinetics using Incucyte live cell imaging system. The data were quantitated for organoid counts using essence incucyte software, plotted as a graph of change in organoid counts or area over time (3–5 days) for both ch5E6 and huIgG1 treatments, and is shown in parallel. Error bars indicate SEM. *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Derivative Assay, Staining, Expressing, Binding Assay, Control, Live Cell Imaging, Software

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 6 Inhibition of EMT by ch5E6 is validated in PC and NSCLC cell line-derived xenografts. a Immunofluorescence analysis showing a decrease in pFAK(Y397) and N-cadherin expression in xenograft tumors of SW1990 cells treated with ch5E6 compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: three animals). The data was plotted for changes in fluorescence intensity using GraphPad Prism 9 and is shown in parallel. Nuclei were stained with DAPI. b Immunofluorescence analysis of ch5E6 treated SW1573 cell line-derived xenografts showing a reduction in pFAK(Y397) and N-cadherin levels compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: 3 animals). Scale bar, 10 µm; magnified images, 2 µm. No significant changes in the intensity of MUC16 were seen in the ch5E6 treated versus isotype control tumors derived from both cancers. c, d Immunoblot analysis of ch5E6 treated PDAC and NSCLC tumor lysates showing a substantial decrease in phosphorylated levels of FAK(Y397), p70S6K(T389) and N-cadherin as compared to huIgG1 treatment. Error bars indicate SEM. Scale bar, 400 μm; magnified images, 100 μm; *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Inhibition, Derivative Assay, Expressing, Control, Staining, Western Blot

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 7 MUC16 and N-cadherin are clinically correlated in patient tumors. a, b Representative images and quantitative illustration of immunohistochemical analyses demonstrate a strong positive correlation between MUC16 and N-cadherin (R = 0.84) in both primary PC tumors (n = 10) and liver metastasis (R = 0.99) samples (n = 8). Scale bar, 400 µm; magnified images, 100 µm. *P < 0.05; **P < 0.01. c Representative images of immunofluorescence analysis showing coexpression of MUC16 (green) and N-cadherin (red) in primary PDAC tumors compared to no MUC16 and N-cadherin in normal pancreatic sections. Scale bar, 20 µm; magnified images, 5 µm. d Schematic diagram representing ch5E6 induced downregulation of MUC16 mediated EMT resulting in its anti-tumor potential in PC and NSCLC. Overall, anti- MUC16 chimeric mAb5E6 (ch5E6) binds to the cell surface-tethered domain of MUC16, interferes with oncogenic pFAK/p70S6K/N-cadherin signaling associated with MUC16-mediated EMT, and reduces tumor burden in both PC and NSCLC models.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Immunohistochemical staining

Journal: Veterinary Research

Article Title: PEDV infection in neonatal piglets through the nasal cavity is mediated by subepithelial CD3 + T cells

doi: 10.1186/s13567-020-00883-w

Figure Lengend Snippet: The distribution of PEDV in nasal cavity after intranasal inoculation in neonatal piglets. A For FACS analyses, neonatal piglets were nasally administered PEDV at indicated times. Then, individual cells isolated from the nasal mucosa were gated based on cytokeratin 18, and viral infection was detected by PEDV N protein staining, n = 3 from 3 piglets per group. B IHC results showed PEDV distribution in five cross-sections (I, II, III, IV and V) of the nasal cavity at 12 h post intranasal infection. The scale bar represents 20 μm. C Quantitative analysis of PEDV positive cells in the nasal cavity . All data shown are the mean results ± SD from three independent experiments. Statistical significance was tested using one-way ANOVA. ** P < 0.01.

Article Snippet: The anti-PEDV N protein mAb was purchased from Medgene labs. Anti-pig epithelial cell marker PE- Keratin 18 (CK18) mAb was purchased from Novus Biologicals.

Techniques: Isolation, Infection, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: EphA2 promotes the transcription of KLF4 to facilitate stemness in oral squamous cell carcinoma.

doi: 10.1007/s00018-024-05325-w

Figure Lengend Snippet: Fig. 5 EphA2 promotes the nuclear translocation of YAP by activating ERK pathway. A The correlation between YAP and EphA2 acquired by IHC. B Knockdown of EphA2 reduced the expression of YAP. C, D Knockdown of EphA2 reduced the nuclear expression of YAP detected by confocal micrographs (C) and western blotting (D). Scale bars: 20 μm. E Knockdown of EphA2 reduced the pERK protein expression. F, G Total (F) and nuclear (G) expression of YAP were upregulated after adding ERK activator (TBHQ, 5 µM) in shEphA2-OSCC cells. H, I Total (H) and nuclear (I) expression of YAP were decreased after adding ERK inhibitor (SCH772984, 10 µM) in OE- EphA2-OSCC cells

Article Snippet: EphA2 (#66736-1-Ig, 1:5,000), CD133 (#18470-1-AP, 1:2,000), CD44 (#15675-1-AP, 1:2,000), ALDH1A1 (#15910-1-AP, 1:1,000) and YAP (#13584-1-AP, 1:2,000 and #66900- 1-Ig, 1:5,000) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Translocation Assay, Knockdown, Expressing, Western Blot

Journal: Cellular and molecular life sciences : CMLS

Article Title: EphA2 promotes the transcription of KLF4 to facilitate stemness in oral squamous cell carcinoma.

doi: 10.1007/s00018-024-05325-w

Figure Lengend Snippet: Fig. 7 EphA2 inhibits the growth and stemness of OSCC cells in vivo. A, B The suppression of CSC-related markers expression by knockdown of EphA2 could be reversed by overex pression of YAP (A) or KLF4 (B). C Tumor images of cells pretreated with knockdown of EphA2 or overexpres sion of YAP or overexpression of KLF4 (cell dose = 3 × 106, n = 7). D Growth curves of tumors that measured every 7 days. E-G Representative IHC images of Ki67 and CD44 in excised tumor tissues (E), and their histochem istry score (F, G). Scale bars: 20 μm

Article Snippet: EphA2 (#66736-1-Ig, 1:5,000), CD133 (#18470-1-AP, 1:2,000), CD44 (#15675-1-AP, 1:2,000), ALDH1A1 (#15910-1-AP, 1:1,000) and YAP (#13584-1-AP, 1:2,000 and #66900- 1-Ig, 1:5,000) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: In Vivo, Expressing, Knockdown, Over Expression

Journal: Cellular and molecular life sciences : CMLS

Article Title: EphA2 promotes the transcription of KLF4 to facilitate stemness in oral squamous cell carcinoma.

doi: 10.1007/s00018-024-05325-w

Figure Lengend Snippet: Fig. 8 A schematic model of this study. EphA2 activates the ERK pathway, which leads to nuclear translocation of YAP. YAP subsequently binds to TEAD3 to form a transcriptional complex, which activates the transcrip tion of KLF4, a key transcription factor for CSCs. This ultimately enhances OSCC cell stemness

Article Snippet: EphA2 (#66736-1-Ig, 1:5,000), CD133 (#18470-1-AP, 1:2,000), CD44 (#15675-1-AP, 1:2,000), ALDH1A1 (#15910-1-AP, 1:1,000) and YAP (#13584-1-AP, 1:2,000 and #66900- 1-Ig, 1:5,000) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Translocation Assay

Journal: Science advances

Article Title: A cytoskeletal function for PBRM1 reading methylated microtubules.

doi: 10.1126/sciadv.abf2866

Figure Lengend Snippet: Fig. 1. PBRM1 binds -tubulin and colocalizes with the SETD2 methyl mark on spindle microtubules during mitosis. (A) Representative images of HEK293T cells ectopically expressing GFP-PBRM1 (green) and stained with antibodies specific for the SETD2 methyl mark (-TubK40me3 red), -tubulin (far red/purple), and 4′,6-diamidino- 2-phenylindole (DAPI) to visualize chromosomes (blue) showing PBRM1 colocalization with -TubK40me3 and -tubulin at mitotic spindle and spindle pole (lower panels). (B) To demonstrate the colocalization of PBRM1 (green) and -TubK40me3 (red), line profiles were obtained between the two spindle poles using deconvolution microscope NIS elements software, which showed that green and red signals are aligned at spindle pole and PBRM1 and -TubK40me3 are colocalized. Scale bars, 5 m (n = 3 biological replicates). (C) Representative images from deconvolution microscopy showing localization of PBRM1 to the mitotic spindle in SETD2+/+ and SETD2−/− 786-O cells during mitosis stained for PBRM1 (purple) and -tubulin (red) and DAPI to visualize chromosomes (blue). Scale bars, 5 m (n = 3 biological replicates). (D) Representative images of line intensity profiles and intensity measurement of PBRM1 on mitotic spindle in SETD2+/+ and SETD2−/− 786-O cells using deconvolution microscope NIS elements software. Scale bars, 5 m (n = 3). (E) Quantification of intensity at peak spindle pole localization of PBRM1 from (D) in SETD2+/+ and SETD2−/− 786-O cells. Data are represented as means ± SEM. P value was determined by t test (n = 45 mitotic cells per condition). (F) Immunoblot (IB) analysis showing coimmunoprecipitation (IP) of endogenous -tubulin and PBRM1 and respective input lysates from HEK293T cells and PBRM1 CRISPR-KO HEK293T cells. COX5A serves as a negative control and shows the specificity of the interaction of PBRM1 with -tubulin. (G) IB analysis showing coimmunoprecipitation of endogenous -tubulin and ectopically expressed PBRM1 and respective input lysates from HEK293T cells. (H) IB analysis showing coimmunoprecipitation of endogenous PBRM1 and ectopically expressed -tubulin and respective input lysates from HEK293T cells. Representative blots (n = 3).

Article Snippet: Details of the antibodies used for immunoblotting (IB) and immunofluorescence (IF) are as follows: PBRM1 (Bethyl Laboratories, A301-591A; 1:1000 IB), lactate dehydrogenase (Abcam, ab47010; 1:5000 IB), lamin A/C (Cell Signaling, 2032S; 1:1000 IB), COX5A (Abcam, ab180129; 1:500 IB), DM1A (Santa Cruz Biotechnology, sc32293; 1:4000 IB, IF), GFP (Santa Cruz Biotechnology, sc-9996; 1:5000 IB), p150Glued (BD Biosciences, 610474; 1:400 IB), MAP4 (Abcam, ab89650; 1:800 IB), -TubK40me3 (custom-made rabbit polyclonal antibody from Covance; 1:1000 IB), SETD2 (SigmaAldrich, HPA-042451-100; 1:1000 IB), H3K36me3 (Active Motif, 39915; 1:1000 IB), H3 (Cell Signaling, D2B12; 1:5000 IB), BRG1 (Santa Cruz Biotechnology, sc-17796; IB), SNF5 (Sigma-Aldrich, SAB4200202; 1:1000 IB), ARID2 (Santa Cruz Biotechnology, sc-166117; 1:500 IB), PBRM1 (Boster Bio, M01130; 1:200 IF), BRG1 (Thermo Fisher Scientific, 720129; 1:200 IF), -tubulin (Sigma-Aldrich, T6199; 1:4000 IB), -tubulin (Thermo Fisher Scientific, PA5-19489; 1:5000 IF), and ARID2 (Novus Biologicals, NBP-2-57220; 1:250 IF) (GFPTrap magnetic agarose bead, ChromoTek, gtma20).

Techniques: Expressing, Staining, Microscopy, Software, Western Blot, CRISPR, Negative Control

Journal: Science advances

Article Title: A cytoskeletal function for PBRM1 reading methylated microtubules.

doi: 10.1126/sciadv.abf2866

Figure Lengend Snippet: Fig. 2. PBRM1 interacts with -tubulin via its BAH methyl-binding domain. (A) Schematic of PBRM1 protein showing its various domains including six bromo- domains, two BAH domains, and an HMG domain. (B) GST pull-down assay for -tubulin using GST fusion proteins for each of six bromodomains (BD) and two BAH do- mains incubated with 1 g of porcine brain microtubule protein and immunoblotted using -tubulin antibody (upper panel). GST-PBRM1 BD and BAH domain construct expression was assessed using Coomassie blue staining (lower panel). Representative blots and gels (n = 3). (C) IB analysis following coimmunoprecipitation of PBRM1 and ectopically expressed wild-type (WT) or mutant -tubulin (K40R) or acetylation mimic (K40Q) and respective input lysates from HEK293T cells. Representative blots (n = 3). (D) IB analysis following peptide pull-down using biotin-labeled K40 -tubulin peptides that were unmodified (K40-UN), acetylated (K40-Ac), or trimethylated (K40-me3) using lysates from PBRM1-proficient (HEK293T) or PBRM1-deficient (RCC4) cells. Representative blots are shown (n = 3). (E) IB analysis showing coimmunoprecipitation of endogenous PBRM1 and -tubulin, and their respective input lysates from SETD2+/+ and SETD2−/− 786-O cells. Residual signal detected with -TubK40me3 may be due to cross-reactivity with dimethylated K40, other methyl marks on -tubulin, or redundancy with other -tubulin methyltransferases acting at K40. Representative blots (n = 3).

Article Snippet: Details of the antibodies used for immunoblotting (IB) and immunofluorescence (IF) are as follows: PBRM1 (Bethyl Laboratories, A301-591A; 1:1000 IB), lactate dehydrogenase (Abcam, ab47010; 1:5000 IB), lamin A/C (Cell Signaling, 2032S; 1:1000 IB), COX5A (Abcam, ab180129; 1:500 IB), DM1A (Santa Cruz Biotechnology, sc32293; 1:4000 IB, IF), GFP (Santa Cruz Biotechnology, sc-9996; 1:5000 IB), p150Glued (BD Biosciences, 610474; 1:400 IB), MAP4 (Abcam, ab89650; 1:800 IB), -TubK40me3 (custom-made rabbit polyclonal antibody from Covance; 1:1000 IB), SETD2 (SigmaAldrich, HPA-042451-100; 1:1000 IB), H3K36me3 (Active Motif, 39915; 1:1000 IB), H3 (Cell Signaling, D2B12; 1:5000 IB), BRG1 (Santa Cruz Biotechnology, sc-17796; IB), SNF5 (Sigma-Aldrich, SAB4200202; 1:1000 IB), ARID2 (Santa Cruz Biotechnology, sc-166117; 1:500 IB), PBRM1 (Boster Bio, M01130; 1:200 IF), BRG1 (Thermo Fisher Scientific, 720129; 1:200 IF), -tubulin (Sigma-Aldrich, T6199; 1:4000 IB), -tubulin (Thermo Fisher Scientific, PA5-19489; 1:5000 IF), and ARID2 (Novus Biologicals, NBP-2-57220; 1:250 IF) (GFPTrap magnetic agarose bead, ChromoTek, gtma20).

Techniques: Binding Assay, Pull Down Assay, Incubation, Construct, Expressing, Staining, Mutagenesis, Labeling

Journal: Science advances

Article Title: A cytoskeletal function for PBRM1 reading methylated microtubules.

doi: 10.1126/sciadv.abf2866

Figure Lengend Snippet: Fig. 3. PBRM1 recruits SWI/SNF (PBAF) subunits to methylated microtubules. (A) IB analysis showing coimmunoprecipitation of BRG1 and ARID2 with -tubulin and their corresponding input lysates from HEK293T and HKC cells. Representative blot (n = 3). (B) Representative image using deconvolution microscopy of cells stained with antibodies directed against BRG1 (red, upper two panels) or ARID2 (red, lower two panels) and -tubulin (green), DAPI to visualize chromosomes (blue). Representative images show BRG1 and ARID2 localization at the mitotic spindle and spindle pole in PBRM1-proficient HEK293T cells (upper panel) and loss of mitotic spindle localization in PBRM1-depleted HEK293T cells (lower panel). Scale bars, 5 m (n = 3 biological replicates). (C) IB analysis showing coimmunoprecipitation of PBRM1 and -tubulin, coimmunoprecipitation of BRG1 and -tubulin, and their corresponding input lysates from PBRM1-proficient, PBRM1-depleted, and PBRM1-rescued HEK293T cells show- ing loss of interaction between BRG1 and -tubulin with loss of PBRM1. Representative blots (n = 3). (D) IB analysis following peptide pull-down using biotin-labeled K40 -tubulin peptides that were unmodified (K40-UN), acetylated (K40-Ac), or trimethylated (K40-me3) using lysates from PBRM1-proficient or PBRM1-depleted HEK293T cells showing interactions of BRG1, ARID2, and PBRM1 with K40 -tubulin peptides is dependent on PBRM1. Representative blots (n = 3). (E) IB analysis showing depen- dency on SETD2 for coimmunoprecipitation of BRG1 with -tubulin and their respective input lysates from SETD2+/+ and SETD2−/− 786-O cells. Representative blots (n = 3).

Article Snippet: Details of the antibodies used for immunoblotting (IB) and immunofluorescence (IF) are as follows: PBRM1 (Bethyl Laboratories, A301-591A; 1:1000 IB), lactate dehydrogenase (Abcam, ab47010; 1:5000 IB), lamin A/C (Cell Signaling, 2032S; 1:1000 IB), COX5A (Abcam, ab180129; 1:500 IB), DM1A (Santa Cruz Biotechnology, sc32293; 1:4000 IB, IF), GFP (Santa Cruz Biotechnology, sc-9996; 1:5000 IB), p150Glued (BD Biosciences, 610474; 1:400 IB), MAP4 (Abcam, ab89650; 1:800 IB), -TubK40me3 (custom-made rabbit polyclonal antibody from Covance; 1:1000 IB), SETD2 (SigmaAldrich, HPA-042451-100; 1:1000 IB), H3K36me3 (Active Motif, 39915; 1:1000 IB), H3 (Cell Signaling, D2B12; 1:5000 IB), BRG1 (Santa Cruz Biotechnology, sc-17796; IB), SNF5 (Sigma-Aldrich, SAB4200202; 1:1000 IB), ARID2 (Santa Cruz Biotechnology, sc-166117; 1:500 IB), PBRM1 (Boster Bio, M01130; 1:200 IF), BRG1 (Thermo Fisher Scientific, 720129; 1:200 IF), -tubulin (Sigma-Aldrich, T6199; 1:4000 IB), -tubulin (Thermo Fisher Scientific, PA5-19489; 1:5000 IF), and ARID2 (Novus Biologicals, NBP-2-57220; 1:250 IF) (GFPTrap magnetic agarose bead, ChromoTek, gtma20).

Techniques: Methylation, Microscopy, Staining, Labeling

Journal: Science advances

Article Title: A cytoskeletal function for PBRM1 reading methylated microtubules.

doi: 10.1126/sciadv.abf2866

Figure Lengend Snippet: Fig. 4. Structural analysis of human BAH1 (PDB 6OXB) and modeled human BAH2 domains of PBRM1. The structure of BAH1 and BAH2 is represented as cartoon, where selected amino acids are shown as sticks with oxygen depicted in red, nitrogen in blue, and sulfur in yellow. Interaction distances are shown by black dotted lines. (A) Surface representation of the BAH1 domain (cyan) showing P1048 (red) in a groove. Inset shows interactions of P1048 (red) with the surrounding residues. (B) Proline mutation to arginine (gray) at position 1048 causes steric clashes with the neighboring residues. (C) Surface representation of the modeled BAH2 domain (olive green) showing the location of C1233 (magenta) and its interactions with the neighboring residues. (D) Cysteine mutation to tryptophan (gray) at position 1233 causes steric clashes with the neighboring residues. (E) Surface representation of the modeled BAH2 domain (olive green) with T1202 (orange). The inset shows the interactions between T1202 (orange) with the neighboring residues. (F) Threonine mutation to lysine (gray) at position 1202 can be accommodated without causing any notable changes.

Article Snippet: Details of the antibodies used for immunoblotting (IB) and immunofluorescence (IF) are as follows: PBRM1 (Bethyl Laboratories, A301-591A; 1:1000 IB), lactate dehydrogenase (Abcam, ab47010; 1:5000 IB), lamin A/C (Cell Signaling, 2032S; 1:1000 IB), COX5A (Abcam, ab180129; 1:500 IB), DM1A (Santa Cruz Biotechnology, sc32293; 1:4000 IB, IF), GFP (Santa Cruz Biotechnology, sc-9996; 1:5000 IB), p150Glued (BD Biosciences, 610474; 1:400 IB), MAP4 (Abcam, ab89650; 1:800 IB), -TubK40me3 (custom-made rabbit polyclonal antibody from Covance; 1:1000 IB), SETD2 (SigmaAldrich, HPA-042451-100; 1:1000 IB), H3K36me3 (Active Motif, 39915; 1:1000 IB), H3 (Cell Signaling, D2B12; 1:5000 IB), BRG1 (Santa Cruz Biotechnology, sc-17796; IB), SNF5 (Sigma-Aldrich, SAB4200202; 1:1000 IB), ARID2 (Santa Cruz Biotechnology, sc-166117; 1:500 IB), PBRM1 (Boster Bio, M01130; 1:200 IF), BRG1 (Thermo Fisher Scientific, 720129; 1:200 IF), -tubulin (Sigma-Aldrich, T6199; 1:4000 IB), -tubulin (Thermo Fisher Scientific, PA5-19489; 1:5000 IF), and ARID2 (Novus Biologicals, NBP-2-57220; 1:250 IF) (GFPTrap magnetic agarose bead, ChromoTek, gtma20).

Techniques: Mutagenesis

Journal: Science advances

Article Title: A cytoskeletal function for PBRM1 reading methylated microtubules.

doi: 10.1126/sciadv.abf2866

Figure Lengend Snippet: Fig. 5. Oncogenic mutations in PBRM1 BAH domain disrupt interaction with -tubulin and localization to mitotic spindle. (A) IB analysis from PBRM1-deficient PBRM1 CRISPR-KO HEK293T cells rescued with GFP-tagged WT or mutant (P1048R, T1202K, or C1233W) PBRM1 constructs showing reduced coimmunoprecipitation of -tubulin with P1048R and C1233W mutants and respective input lysates. Representative blots (n = 4). (B) IB analysis cells in (A) using GFP-Trap agarose magnetic beads. Representative blots (n = 5). Bottom panels in (A) and (B) shows quantification of the IB analysis for interaction with tubulin (represented as fold change after normaliza- tion for tubulin) with means ± SEM. P value was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons against WT GFP-PBRM1. (C) Flowchart and graph showing analysis of stacked images of PBRM1-deficient PBRM1 CRISPR-KO HEK293T cells expressing WT or mutated GFP-PBRM1 constructs using MATLAB software. Data are represented as means ± SEM. P values are determined by one-way ANOVA followed by Kruskal-Wallis post hoc test for multiple comparisons against WT GFP-PBRM1. (D) Representative images showing localization of PBRM1 to the mitotic spindle using deconvolution microscopy of PBRM1-deficient PBRM1 CRISPR-KO HEK293T cells ectopically expressing WT or mutant P1048R, T1202K, and C1233W GFP-PBRM1 constructs (green) and stained for -tubulin (red) and chromosomes (DAPI).

Article Snippet: Details of the antibodies used for immunoblotting (IB) and immunofluorescence (IF) are as follows: PBRM1 (Bethyl Laboratories, A301-591A; 1:1000 IB), lactate dehydrogenase (Abcam, ab47010; 1:5000 IB), lamin A/C (Cell Signaling, 2032S; 1:1000 IB), COX5A (Abcam, ab180129; 1:500 IB), DM1A (Santa Cruz Biotechnology, sc32293; 1:4000 IB, IF), GFP (Santa Cruz Biotechnology, sc-9996; 1:5000 IB), p150Glued (BD Biosciences, 610474; 1:400 IB), MAP4 (Abcam, ab89650; 1:800 IB), -TubK40me3 (custom-made rabbit polyclonal antibody from Covance; 1:1000 IB), SETD2 (SigmaAldrich, HPA-042451-100; 1:1000 IB), H3K36me3 (Active Motif, 39915; 1:1000 IB), H3 (Cell Signaling, D2B12; 1:5000 IB), BRG1 (Santa Cruz Biotechnology, sc-17796; IB), SNF5 (Sigma-Aldrich, SAB4200202; 1:1000 IB), ARID2 (Santa Cruz Biotechnology, sc-166117; 1:500 IB), PBRM1 (Boster Bio, M01130; 1:200 IF), BRG1 (Thermo Fisher Scientific, 720129; 1:200 IF), -tubulin (Sigma-Aldrich, T6199; 1:4000 IB), -tubulin (Thermo Fisher Scientific, PA5-19489; 1:5000 IF), and ARID2 (Novus Biologicals, NBP-2-57220; 1:250 IF) (GFPTrap magnetic agarose bead, ChromoTek, gtma20).

Techniques: CRISPR, Mutagenesis, Construct, Magnetic Beads, Expressing, Software, Microscopy, Staining

Journal: Science advances

Article Title: A cytoskeletal function for PBRM1 reading methylated microtubules.

doi: 10.1126/sciadv.abf2866

Figure Lengend Snippet: Fig. 6. PBRM1 BAH domain participates in maintenance of genomic instability. (A) Representative images from deconvolution microscopy of PBRM1-deficient PBRM1 CRISPR-KO HEK293T cells showing various genomic instability phenotypes: lagging chromosomes, chromosome bridges, and micronuclei at cytokinesis. Scale bars, 5 m (n = 4 biological replicates). (B) Quantitation of genomic instability phenotypes seen in (A). Data are represented as means ± SEM for four independent biological replicates, with 500 cells counted per replicate. P value was determined by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons against control cells. (C) Representative image from deconvolution microscopy showing multipolar spindles observed in PBRM1 CRISPR-KO HEK293T cells. Scale bars, 5 m (n = 5 biological replicates). (D) Quantitation of multipolar spindles shown in (C). Data are represented as means ± SEM for five independent biological replicates, with 500 cells counted per replicate. P value was determined by one-way ANOVA with Dunnett’s post hoc test for multiple comparisons against control cells.

Article Snippet: Details of the antibodies used for immunoblotting (IB) and immunofluorescence (IF) are as follows: PBRM1 (Bethyl Laboratories, A301-591A; 1:1000 IB), lactate dehydrogenase (Abcam, ab47010; 1:5000 IB), lamin A/C (Cell Signaling, 2032S; 1:1000 IB), COX5A (Abcam, ab180129; 1:500 IB), DM1A (Santa Cruz Biotechnology, sc32293; 1:4000 IB, IF), GFP (Santa Cruz Biotechnology, sc-9996; 1:5000 IB), p150Glued (BD Biosciences, 610474; 1:400 IB), MAP4 (Abcam, ab89650; 1:800 IB), -TubK40me3 (custom-made rabbit polyclonal antibody from Covance; 1:1000 IB), SETD2 (SigmaAldrich, HPA-042451-100; 1:1000 IB), H3K36me3 (Active Motif, 39915; 1:1000 IB), H3 (Cell Signaling, D2B12; 1:5000 IB), BRG1 (Santa Cruz Biotechnology, sc-17796; IB), SNF5 (Sigma-Aldrich, SAB4200202; 1:1000 IB), ARID2 (Santa Cruz Biotechnology, sc-166117; 1:500 IB), PBRM1 (Boster Bio, M01130; 1:200 IF), BRG1 (Thermo Fisher Scientific, 720129; 1:200 IF), -tubulin (Sigma-Aldrich, T6199; 1:4000 IB), -tubulin (Thermo Fisher Scientific, PA5-19489; 1:5000 IF), and ARID2 (Novus Biologicals, NBP-2-57220; 1:250 IF) (GFPTrap magnetic agarose bead, ChromoTek, gtma20).

Techniques: Microscopy, CRISPR, Quantitation Assay, Control

Journal: Science advances

Article Title: A cytoskeletal function for PBRM1 reading methylated microtubules.

doi: 10.1126/sciadv.abf2866

Figure Lengend Snippet: Fig. 7. Graphical model showing dual chromatin and cytoskeletal function of PBRM1 in regulating gene expression and genomic stability via interactions with chromatin in the nucleus (via BD domains) and microtubules of the mi- totic spindle via BAH domain binding to -TubK40me3, respectively.

Article Snippet: Details of the antibodies used for immunoblotting (IB) and immunofluorescence (IF) are as follows: PBRM1 (Bethyl Laboratories, A301-591A; 1:1000 IB), lactate dehydrogenase (Abcam, ab47010; 1:5000 IB), lamin A/C (Cell Signaling, 2032S; 1:1000 IB), COX5A (Abcam, ab180129; 1:500 IB), DM1A (Santa Cruz Biotechnology, sc32293; 1:4000 IB, IF), GFP (Santa Cruz Biotechnology, sc-9996; 1:5000 IB), p150Glued (BD Biosciences, 610474; 1:400 IB), MAP4 (Abcam, ab89650; 1:800 IB), -TubK40me3 (custom-made rabbit polyclonal antibody from Covance; 1:1000 IB), SETD2 (SigmaAldrich, HPA-042451-100; 1:1000 IB), H3K36me3 (Active Motif, 39915; 1:1000 IB), H3 (Cell Signaling, D2B12; 1:5000 IB), BRG1 (Santa Cruz Biotechnology, sc-17796; IB), SNF5 (Sigma-Aldrich, SAB4200202; 1:1000 IB), ARID2 (Santa Cruz Biotechnology, sc-166117; 1:500 IB), PBRM1 (Boster Bio, M01130; 1:200 IF), BRG1 (Thermo Fisher Scientific, 720129; 1:200 IF), -tubulin (Sigma-Aldrich, T6199; 1:4000 IB), -tubulin (Thermo Fisher Scientific, PA5-19489; 1:5000 IF), and ARID2 (Novus Biologicals, NBP-2-57220; 1:250 IF) (GFPTrap magnetic agarose bead, ChromoTek, gtma20).

Techniques: Gene Expression, Binding Assay