Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Summary of protein expression losses in ccRCC tumors

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Expressing

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: ( A ) How a phylogenetic tree was constructed. A: ARID1A loss; M: SMARCA2 loss; P: PBRM1 loss, G: SMARCA4 loss; S: SETD2 loss. ( B ) Truncal losses of the markers at each stage, either alone or in combination, were presented. ( C ) Fisher's exact tests were performed to calculate the p values of the associations between the protein marker losses and stages.

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Construct, Marker

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: The survival curves were calculated based on SETD2 staining: positive (1) and negative (0). Associated log-rank p value was indicated. n: number of cases.

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Staining

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Univariate and multivariable analyses of indicated biomarker losses and their associations with overall survival

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Univariate and multivariable analyses of indicated biomarker losses and their associations with recurrence-free survival

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Biomarker Discovery

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 3. HIF1 can directly induce the expression of LBH under hypoxia a: LBH mRNA expression of T98G (left) and GSC4B (right) gradually increased during prolonged treatment under hypoxia as measured by qPCR. (T98G: p <0.0001, GSC4B: p <0.0001, One-Way ANOVA) b: LBH protein expression of T98G and GSC4B was gradually increased during prolonged treatment under hypoxia as measured by western blotting. c: Sequence motif representing the consensus HIF-1 binding motif (JASPAR database). d, e: Luciferase reporter assays showed hypoxia can upregulate the luciferase promoter activities of LBH in T98G (left) and GSC4B (right) cells. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) e: ChIP qPCR showed HIF-1 binding to the promoter of LBH under hypoxia. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) g, h: qPCR (g) and western blot (h) showed the expression of HIF-1 overexpression can upregulate the expression of LBH. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: Expressing, Western Blot, Sequencing, Binding Assay, Luciferase, ChIP-qPCR, Over Expression

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 5. LBH can activate VEGFA-mediated ERK signalling in hBMECs under hypoxia a, b: The VEGFA mRNA expression and secretion level of U118 and GSC2A cells was upregulated under hypoxia and further upregulated after LBH overexpression, as measured by qPCR (a) and ELISA (b). (qPCR: U118: p <0.0001, GSC2A: p <0.0001; ELISA: U118: p = 0.0019, GSC2A: p = 0.0011, One-Way ANOVA) c, d: The VEGFA mRNA expression and secretion level of T98G and GSC4B cells was upregulated under hypoxia and decreased after LBH knockdown, as measured by qPCR (c) and ELISA (d). (qPCR: T98G: p <0.0001, GSC4B: p = 0.0012; ELISA: T98G: p = 0.0023, GSC4B: p = 0.0029, One-Way ANOVA) e: The VEGFA protein expression of U118 and GSC2A cells was upregulated under hypoxia and further upregulated after LBH overexpression, as shown by western blotting. f: The VEGFA protein expression of T98G and GSC4B cells was upregulated under hypoxia and decreased after LBH knockdown, as shown by western blotting. g: The VEGFR-ERK signalling pathway in vascular endothelial cells following treatment with LBH-silenced T98G and GSC4B conditioned media under normoxia or hypoxia was measured by western blotting. All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 6. Anti-VEGFA treatment can abolish LBH-induced hBMECs proliferation, invasion and angiogenesis under hypoxia a, b: The induction of vascular endothelial cell viability following treatment with LBH-overexpressed U118 (a) and GSC2A (b) conditioned media was reversed following anti-VEGFA treatment, as measured by an MTS assay. (U118: p = 0.0016, GSC2A: p = 0.0011, One-Way ANOVA) c: The proliferation of vascular endothelial cells following treatment with LBH-overexpressed U118 and GSC2A conditioned media was reversed following anti-VEGFA treat- ment, as measured by an EDU incorporation assay. Scale bar = 100 μm. (U118: p = 0.0008, GSC2A: p < 0.0001, One-Way ANOVA) d: Representative transwell assay showing that treatment with LBH-overexpressed U118 and GSC2A conditioned media induced invasion of vascular endothelial cells that was reversed after anti-VEGFA treatment. Scale bar = 100 μm. (U118: p <0.0001, GSC2A: p = 0.0018, One-Way ANOVA) e: Representative tube formation assay showing that treatment with LBH-overexpressed U118 and GSC2A conditioned media induced tubulogenesis of vascular endothelial cells that was reversed after anti-VEGFA treatment. Scale bar = 100 μm. (number of branches: U118: p <0.0001, GSC2A: p < 0.0001, tubule length: U118: p = 0.0012, GSC2A: p = 0.0019, One-Way ANOVA) All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: MTS Assay, Transwell Assay, Tube Formation Assay

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 7. LBH regulates glioma tumorigenesis and angiogenesis in vivo a, c: Representative photographs showing the size of intracranial tumors in the coronal position. LBH overexpression in GSC2A cells increased the intracranial tumor size (a), whereas LBH knockdown in GSC4B cells decreased the intracranial tumor size (c). Scale bar = 10 mm. b, d: LBH-overexpressed GSC2A cells implanted into tumor-bearing mice showed shorter survival times as measured by Kaplan–Meier survival curves (b), compared with longer survival times when LBH-silenced GSC4B cells were implanted into tumor bearing mice (d). For each group, n = 5. e: Representative immunohistochemical staining showing the changes in LBH, VEGFA and CD31 in LBH overexpression and knockdown orthotopic xenograft models. Scale bar = 50 μm. f: Schematic diagram to illustrate that overexpression of LBH promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: In Vivo, Over Expression, Knockdown, Immunohistochemical staining, Staining

Journal: Science (New York, N.Y.)

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A) Cryo-EM density map of the hCENP-N1–286/CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel). The CENP-N construct used for the present structural analysis (hCENP-N1–286) and the regions of the sequence whose structure we report here [N-terminal domain: residues 1 to 81, and central domain: residues 101 to 185; hCENP-N(1–185)] are shown in the middle and bottom panels, respectively. (C) Cryo-EM density mapof the hCENP-N1–286/CENP-A nucleosome complex as viewed from the side, at an orientation 90° to the view shownin (A). This view also depicts the extra density connected to the N-terminal domain that we assign to MBP, shown with lighter shading. (D) Representative regions of the cryo-EM density mapto illustrate map quality (from left to right) for canonical histones H2A, H2B, and H4, centromere-specific H3 variant CENP-A, nucleosomal DNA, and CENP-N.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Cryo-EM Sample Prep, Functional Assay, Construct, Sequencing, Variant Assay

Journal: Science (New York, N.Y.)

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A) Cut-away view of the hCENP-N1–286/CENP-A nucleosome model to highlight interfaces involved in complex formation (see also fig. S11, A and B). For the CENP-N/DNA interface (labeled “1a” and “1b”), nucleosomal DNAis shown as a red ribbon, whereas positively charged residues of CENP-N that are proposed to interact with it are shown as blue spheres. Forthe CENP-N/CENP-A interface (labeled “2”), CENP-A residues (R80, G81, and V82) are marked by the short yellow ribbon, whereas interacting CENP-N residues (E3, T4, and E7) are shown as yellow spheres. (B) View of the CENP-N/DNA interface at different magnifications to highlight details of interactions between the nucleosomal DNA and positively charged residues of CENP-N. (C) Gel mobility shift experiment to examine the effects of CENP-N mutations (indicated atop the gel) on binding to the CENP-A nucleosome. Impaired binding is reflected by increased intensity of the free nucleosome (Nuc) band, concomitant with the disappearance of defined 1:1 and 2:1 bands. “N” indicates the migration position of the free CENP-A nucleosome; “1” and “2” denote the migration positions of CENP-A nucleosomes bound with either one or two molecules of CENP-N, respectively. WT, wild type. (D) Similar analysis to that in (C), carried out with a set of CENP-N mutations involving residues distal from the binding interface. (E) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-N and xCENP-L proteins containing the indicated mutations (with analogous human mutations in parentheses), stained with an antibody forMBP (green) and Hoechst (blue). (F) Centromeric MBP fluorescence intensity normalized as a percentage of that observed for wild-type MBP-xCENP-N. Error bars represent SEM (n > 200 centromeres). A.U., arbitrary units.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Labeling, Mobility Shift, Binding Assay, Migration, Staining, Fluorescence

Journal: Science (New York, N.Y.)

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A and B) Overall (A) and close-up (B) view of the hCENP-N1–286/CENP-A interface formed betweenR80, G81, and V82 on the L1 loop of CENP-A and E3, T4, and E7 on helix 1of CENP-N. (C) Gel mobility shift experiment to examine the effects of CENP-N mutations (indicated atopthe gel) on binding to the CENP-A nucleosome. (D) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-L andxCENP-L proteins containing the indicated mutations of xCENP-N residues E21 and E25 (correspondingto residues E3 and E7 in hCENP-N), stained with an antibody forMBP (green) and Hoechst (blue).(E) Centromeric MBP fluorescence intensity normalized as a percentageof that observed for wild-type MBP-xCENP-N. Error bars representSEM (n > 200 centromeres). (F) Alignment of human and Xenopus laevis sequences corresponding to the L1 loop of CENP-A and helix 1 of CENP-N. Closely interacting segments of the L1 loop of CENP-A and helix 1 of CENP-N are highlighted bythe shaded areas. The asterisks indicate conserved glutamic acid residues(black asterisks) and variability in the hydrophobic residue correspondingto position T4 (red asterisk) of human CENP-N. (G) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-N and xCENP-L proteins containing the indicated mutations of xCENP-N, as in (D).(H) Centromeric MBP fluorescence intensity, determined as in (E). (I) Gel mobility shift experiment to examine the effects of correlated amino acid substitutions between the L1 loop of CENP-A and helix 1 of CENP-N.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Mobility Shift, Binding Assay, Staining, Fluorescence

Journal: Science (New York, N.Y.)

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A) Sequence alignment between humanH3.1 and CENP-A to highlight distinct CENP-A motifs involvedin deposition and recognition of CENP-A at centromeric chromatin (see also fig. S11, C and D). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (B) Two different views of the CENP-A nucleosome bound to hCENP-N and a modeled CENP-C motif peptide (5) to highlight potential dual binding of full-length CENP-C and CENP-N proteins on the CENP-A nucleosome. The second CENP-N (shown with lighter shading) is modeled on the basis of the cryo-EM density map obtained in the presence of excess hCENP-N1–286 (fig. S4), whereas the CENP-C motif peptides (human numbering shown for clarity) on each face of the nucleosome are positioned according to the crystal structure of the nucleosome in complex with the rat CENP-C motif (5). (C) Schematic view to highlight recognition and possible enrichment of CENP-A nucleosomes by the CCAN proteins CENP-C, CENP-N, and CENP-L. Other kinetochore proteins and the dimerization of CENP-C have been omitted for clarity.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Sequencing, Binding Assay, Cryo-EM Sample Prep

Journal: Cell reports

Article Title: henn-1/HEN1 Promotes Germline Immortality in Caenorhabditis elegans

doi: 10.1016/j.celrep.2019.10.114

Figure Lengend Snippet:

Article Snippet: NEBNext Small RNA Library Prep Kit for Illumina , NEB , E7300S or E7580S.

Techniques: Recombinant, Protease Inhibitor, Hybridization, Magnetic Beads, Expressing, Plasmid Preparation, Software