Journal: Experimental and Therapeutic Medicine

Article Title: Clinical characteristics and prognostic analysis of patients with HIV and glioma: A case series and literature review

doi: 10.3892/etm.2024.12380

Figure Lengend Snippet: Characteristics of HIV-positive patients with brain glial tumors treated at Beijing Ditan Hospital.

Article Snippet: The primary antibodies CD31 (cat. no. ZA-0568), CD68 (cat. no. ZM-0060), CD163 (cat. no. ZM-0428), IDH1 R132H mutation-specific (cat. no. ZM-0447) and Ki-67 (cat. no. ZM-0166) were purchased from ZSGB-BIO (OriGene Technologies, Inc.) and used at a dilution of 1:100.

Techniques:

Journal: eLife

Article Title: A nuclear-based quality control pathway for non-imported mitochondrial proteins

doi: 10.7554/eLife.61230

Figure Lengend Snippet: ( A ) 35 S-labeled Su9-DHFR was imported into mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. ( B ) Western blot showing protein levels in mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. ( C ) 35 S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. ( D ) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. ( E ) Yeast expressing Idh1-GFP and Tom70-mCherry in WT or Ilv2 (untagged) overexpressing strains ± FCCP. Bars = 2 μm. ( F ) Quantification of ( E ). ( G ) 35 S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP ± FCCP. ( H ) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP. For ( A ), ( C ), and ( G ), a mixture of antimycin A, oligomycin, and valinomycin (AVO) was used to dissipate the membrane potential. Non-imported proteins were proteolytically removed with proteinase K and the import was analyzed by SDS–PAGE and autoradiography. P = precursor and M = mature.

Article Snippet: Membranes were blocked and probed in blocking buffer (1× PBS, 0.05% Tween 20, 5% non-fat dry milk) using the primary antibodies for GFP (1814460001, Sigma Millipore), HA (11583816001, Sigma Millipore), Ilv2 (gift from Dr. Agnieszka Chacinska, International Institute of Molecular and Cell Biology), Tom70, Tom20, Tim54, Tim50, Tim18 (gifts from Dr. Nikolaus Pfanner, University of Freiburg), OM45 (gift from Dr. Carla Koehlar, UCLA), Idh1 (Ap31099PU-N, ORIGENE) or Pgk1 (22C5D8, Abcam), and HRP-conjugated secondary antibodies (715-035-150, Jackson Immunoresearch).

Techniques: Labeling, Isolation, Western Blot, Expressing, Membrane, SDS Page, Autoradiography

Journal: eLife

Article Title: A nuclear-based quality control pathway for non-imported mitochondrial proteins

doi: 10.7554/eLife.61230

Figure Lengend Snippet:

Article Snippet: Membranes were blocked and probed in blocking buffer (1× PBS, 0.05% Tween 20, 5% non-fat dry milk) using the primary antibodies for GFP (1814460001, Sigma Millipore), HA (11583816001, Sigma Millipore), Ilv2 (gift from Dr. Agnieszka Chacinska, International Institute of Molecular and Cell Biology), Tom70, Tom20, Tim54, Tim50, Tim18 (gifts from Dr. Nikolaus Pfanner, University of Freiburg), OM45 (gift from Dr. Carla Koehlar, UCLA), Idh1 (Ap31099PU-N, ORIGENE) or Pgk1 (22C5D8, Abcam), and HRP-conjugated secondary antibodies (715-035-150, Jackson Immunoresearch).

Techniques: Recombinant, Plasmid Preparation, Generated, Protease Inhibitor, Staining, Cloning, Software

Journal: The Journal of Biological Chemistry

Article Title: Antibody validation for Western blot: By the user, for the user

doi: 10.1074/jbc.RA119.010472

Figure Lengend Snippet: Validation of IDH1 antibody using purified recombinant protein in multicolor and chemiluminescent Western blotting. Multicolor and chemiluminescent Western blottings were performed using 10% Bis-Tris SDS-polyacrylamide gel and MOPS buffer system to validate the IDH1 antibody using a purified recombinant IDH1 protein (0.16 μg) containing a c-Myc tag in addition to HEK293T and HeLa whole-cell lysates. A, c-Myc protein tag present on the purified IDH1 recombinant protein is detected in the 700-nm channel ( red ) at 50 kDa via mouse anti-c-Myc antibody (ab32;1 μg/ml) using IRDye 680RD goat anti-mouse IgG (H + L) for detection. Some overspill of the recombinant protein into neighboring lanes is observed ( white box ). B, IDH1 recombinant protein and endogenous IDH1 protein, present in HEK293T and HeLa, is detected in the 800-nm channel ( green ) at 55 and 50 kDa, respectively, using rabbit anti-IDH1 antibody (ab172964; 1.2 μg/ml) and IRDye 800CW goat anti-mouse IgG (H + L) for detection. C, when both 700- and 800-nm channels are displayed, the signal from ab32 and ab172964 overlaps at 50 kDa, identifying the c-Myc–tagged IDH1 protein. No overlap is seen for the endogenous IDH1 present in HEK293T and HeLa whole-cell lysates. A–C , lysates loaded per lane are as follows: 20 μg of blocking buffer: Odyssey blocking buffer (TBS); imager: Odyssey® CLx; resolution: 169 μm; intensity: auto mode. Chameleon TM Duo pre-stained protein ladder for accurate sizing of protein bands. D, single blot was split into two halves ( green line ) to be incubated with either rabbit anti-IDH1 antibody (ab172964; 0.115 μg/ml) or the corresponding rabbit monoclonal IgG isotype control (ab172730; 0.166 μg/ml) to detect the endogenous IDH1 protein present in HeLa and HEK293T as well IDH1 recombinant protein. Both halves were incubated with HRP-conjugated goat anti-mouse IgG (H + L). E, single blot was split into two halves ( green line ) to be incubated with either mouse anti-c-Myc antibody (ab32; 1 μg/ml) or the corresponding mouse monoclonal IgG1 isotype control (ab18443; 1 μg/ml) to detect c-Myc protein tag present on the purified IDH1 recombinant protein but absent in HEK293T and HeLa whole-cell lysates. Both halves were incubated with HRP-conjugated goat anti-rabbit IgG (H + L). Blots were detected with WesternSure® PREMIUM chemiluminescent substrate (LI-COR 926–95000) and imaged on an Odyssey® Fc with the following resolution: 125 μm and exposure of 2 min. Lysate loaded per lane: 20 μg; protein ladder: WesternSure® pre-stained chemiluminescent protein ladder (LI-COR 926-980000); blocking buffer: intercept blocking buffer (TBS); intercept T20 (TBS) antibody diluent.

Article Snippet: Isocitrate dehydrogenase (IDH1) (NM_005896) human recombinant protein (OriGene no. TP310582), IDH1 (NM_005896) human overexpression lysate supplied with parental HEK293T lysate (OriGene no. LY401782; HEK293T LY500001; lot no. 0076CF), and IDH1 knockout cell lysate (supplied with parental HeLa control lysate) (Origene no. LC810112, LC810Hela; lot no. 1601) were mixed with either 2× protein loading buffer (PLB) (LICOR no. 928-40004) or 2× SDS buffer (OriGene) and denatured by boiling at 97 °C for 5 min.

Techniques: Biomarker Discovery, Purification, Recombinant, Western Blot, Blocking Assay, Staining, Incubation, Control

Journal: Nature

Article Title: Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

doi: 10.1038/nature13441

Figure Lengend Snippet: Figure 2 | Mutant IDH blocks hepatocyte differentiation by silencing HNF- 4a. a, Heat map of GSEA showing top-ranked gene sets distinguishing IDH1(R132C) or IDH2(R172K) from wild-type (WT) or empty vector (EV) control hepatoblasts (pairwise analysis; replicates for each condition; see Methods). NES, normalized enrichment score; NS, not significant. b, c, Hepatoblasts analysed by immunoblot (b) and qRT–PCR (c). d, e, Analysis of wild-type hepatoblasts expressing the indicated short hairpin (sh)RNAs (uncoated plates). CTL, control. d, Hepatocyte sphere formation. e, Proliferation of shRNA-expressing hepatoblast cells co-expressing EV or shRNA-resistant Hnf4a(1) complementary DNA. f–h, Control and R132C- expressing hepatoblasts co-expressing vector control (EV2) or HNF-4a, grown on uncoated plates. f, Hepatocyte sphere formation. g, Hepatocyte gene expression. h, Proliferation. Scale bars, 100mm (d), 250mm (f). *P , 0.05.

Article Snippet: Human wild-type IDH1 cDNA (NCBI RefSeq accession number NM_ 005896.3) was obtained from Origene and subcloned into pRetro-Puro using EcoRI and BamHI fragments.

Techniques: Mutagenesis, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Expressing, shRNA, Gene Expression

Journal: Genome Biology

Article Title: ATRX regulates glial identity and the tumor microenvironment in IDH-mutant glioma

doi: 10.1186/s13059-021-02535-4

Figure Lengend Snippet: Phenotypic differences between ATRX knockout and wildtype gliomas in an IDH-mutant background. A A schematic overview of the murine studies. B An enzymatic cleavage assay, indicating homozygous knockout in ATRX exon 9 (Top), a Western blot showing complete loss of ATRX protein (bottom). C Fluorometric 2-HG detection assay, comparing wildtype IDH1 and IDH1R132H overexpression in ATRX-KO SB28 cells, * t test p <0.05. D Representative BLI images of ATRX-KO and wildtype intercranial tumors. E BLI timeseries. F Percentages of tumor-infiltrating monocytic-lineage cells expressing the given markers in ATRX-KO vs. wildtype, in an IDH1R132H background. G (Top) Differential expression test of snRNA-seq data via MAST, comparing tumor-infiltrating monocytic-lineage cells between ATRX-KO and wildtype tumors in an IDH1R132H background. (Bottom) Differential expression test of snRNA-seq data via MAST, comparing neoplastic cells between ATRX-KO and ATRX-wildtype tumors in an IDH1R132H background. H Differential peaks from scATAC-seq data called between neoplastic cells from ATRX-KO and ATRX-wildtype tumors with an IDH1R132H background. Peaks are called via MACS at p <0.05 and represented as a heatmap (bottom) and moving average (top) of reads per 10 bp, in a 2 Kbp window around the transposase cut site. I ScATAC-seq motif enrichment in differential peaks between ATRX-KO and ATRX-wildtype neoplastic cells. Motif frequency relative to a genome-wide background was scored via HOMER. J ) Extracellular-matrix invasion assay, comparing ATRX-KO and wildtype cells with an IDH1R132H background

Article Snippet: SB28 ATRX-KO cells were transfected with plasmids expressing either IDH1R132H (ORIGENE, CAT#: RC400096) or wildtype IDH1 (Origene CAT#: RC210582) and selected via neomycin.

Techniques: Knock-Out, Mutagenesis, Cleavage Assay, Western Blot, Detection Assay, Over Expression, Expressing, Quantitative Proteomics, Genome Wide, Invasion Assay

Journal: Genome Biology

Article Title: ATRX regulates glial identity and the tumor microenvironment in IDH-mutant glioma

doi: 10.1186/s13059-021-02535-4

Figure Lengend Snippet: ATRX loss induces a global loss of CTCF, correlated gene dysregulation, and protects glioma cells from therapy-induced senescence. A Anti-CTCF single-cell CUT&Tag in SB28+IDH1R132H cells with ATRX KO or ATRX-wildtype scrambled control. Reads per 10 bp are shown as heatmaps (bottom) and summarized as averages (top) in a neighborhood of JASPAR CTCF motif sites. B CTCF motif frequencies in scCUT&Tag peaks, represented as standardized deviances from a data-driven null distribution via ChromVAR, for ATRX KO vs. wildtype cells (top), *** t test p < 1e−16. Numbers of, and overlap between, peaks in ATRX KO and wildtype datasets. C Browser shots of CTCF scCUT&Tag read pile-ups with significant peaks and genes annotated. D The percentages of NSC chromatin-loops affected, directly or indirectly, by CTCF loss upon ATRX KO (top). A Q-Q plot of the average ATRX-KO/WT cell-averaged log fold-changes in gene expression for each chromatin loop determined from NSC Hi-C data. Values on the y -axis are represented as percentages of a corresponding null distribution (Methods). The x -axis quantiles are provided by a Beta distribution fit to the inter-quartile range of y -axis percentiles. Loops containing specific genes are annotated. E Browser shots of a chromatin loop containing VEGFA and closeup of one boundary domain (left). A null distribution of average ATRX-KO/WT cell-averaged log fold-changes in gene expression measured via snRNA-seq in vivo in neoplastic cells, taken across all consecutive windows of four adjacent genes in chromosome 17. The loop containing VEGFA and the percentile of its average log fold-change are annotated (right-top). Violin plots of Vegfa expression in ATRX KO and WT neoplastic cells, in vivo, *MAST q < 0.05 (right-bottom). F As in E , except for a loop containing TNC. G Fluorometric assay for β-galactosidase activity in ATRX-KO and scrambled-control SB28 cells, both expressing IDH1R132H, post-treatment with one of Doxorubicin or CDKi. Error bars indicate standard error. * t test p < 0.05. B As in A , but for patient-derived ATRX/IDH1 double-mutant cells (SF10602) compared with an ATRX-wildtype, IDH1-mutant control (SF10417). Cells were treated with one of Temozolomide, Imatinib, Doxorubicin, or CDKi. C Visualization of β-galactosidase expression in ATRX-KO and scrambled-control SB28 cells, both expressing IDH1R132H, before and after treatment with CDKi. H Model of gene dysregulation due to CTCF disruption in chromatin-loop boundaries

Article Snippet: SB28 ATRX-KO cells were transfected with plasmids expressing either IDH1R132H (ORIGENE, CAT#: RC400096) or wildtype IDH1 (Origene CAT#: RC210582) and selected via neomycin.

Techniques: Control, Gene Expression, Hi-C, In Vivo, Expressing, Activity Assay, Derivative Assay, Mutagenesis, Disruption