Journal: Molecular cell

Article Title: The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2024.04.007

Figure Lengend Snippet: Figure 4. Exosome function controls the dynamics of the MYCN/MAX/MXD network (A) Immunoblot of IMR-32 cells stably expressing an shRNA targeting EXOSC10 treated for 72 h with Dox or EtOH as control. ACTB was loading control. Blot is representative of n = 3 independent replicates. (B) Read distribution of MYCN ChIP-Rx, MAX, MNT, and SIN3A cleavage under targets & release using nuclease (CUT&RUN) at the CDK5RAP3 and PHB genes. Cells treated as in (A) (n = 3 for MYCN ChIP-Rx; n = 2 for all C&Rs).

Article Snippet: 3 mg of each, MAX (Proteintech), MNT (Thermo Fisher Scientific), or SIN3A (Novus Biologicals) antibody were added per sample and incubated with shaking (800 rpm) at 4 C overnight.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of the MAP kinase and NF-κB signaling pathways in mediating ECFP/Ang II-induced NHE3 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that in wild-type mPCT cells, ECFP/Ang II stimulated NHE3 expression significantly, and the response was attenuated by the MEK1/MEK2 kinase inhibitor U0126 and the NF-κB activation inhibitor Ro 106–9920, respectively. However, the MEK inhibitor PD 980659 and the p38 MAP kinase inhibitor SB202196 failed to attenuate the effect of ECFP/Ang II on NHE3 expression. Panel ( B ) shows that in Agtr1a -/- mPCT cells, ECFP/Ang II failed to stimulate NHE3 expression, and the inhibitors of the MAP kinases and NF-κB signaling pathways had no significant effects on NHE3 expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced Na + /HCO 3 - cotransporter expression in wild-type mPCT cells. Panel ( A ) shows that ECFP/Ang II significantly increased Na + /HCO 3 - expression, and the response was attenuated by losartan but not by PD123319, suggesting a dominant role of AT 1 receptors in mPCT cells. Panel ( B ) shows that the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659 attenuated the effects of ECFP/Ang II on expression, but the p38 MAP kinase inhibitor SB202196 had no effect on Na + /HCO 3 - expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that ECFP/Ang II increased NF-κB, p65 expression in wild-type mPCT cells, and the response was attenuated by both losartan and PD123319, supporting an important role of AT 1 and AT 2 receptors in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. Panel ( B ) shows that ECFP/Ang II alone had no significant effect on NF-κB, p65 expression in Agtr1a -/- mPCT cells, but both losartan and PD123319 potentiated this response. Panel ( C ) shows that in wild-type mPCT cells, the effect of ECFP/Ang II on NF-κB, p65 expression was attenuated by the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659, respectively. However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. ** p < 0.01 vs. control WT or Agtr1a -/- mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II, or Agtr1a -/- mPCT cells transfected with ECFP/ANG II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: PLoS Pathogens

Article Title: Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

doi: 10.1371/journal.ppat.1006668

Figure Lengend Snippet: (A) MCPyV early region showing nucleotide positions for LT start (5387), ST stop (4827), LT stop (2503), and LT splice donor (5154) and acceptor (4722) and approximate positions of mutations that result in truncated LT found in MCC. LT and ST share an N-terminal J domain. The ST unique domain contains the LSD and Zn fingers. LT splices from J domain to a second exon containing the LXCXE or RB1 binding motif. Antibody Ab3 binds LT only and Ab5 binds both LT and ST. (B) Identification of co-precipitating proteins by MudPIT with antibodies Ab3 (LT, blue), Ab5 (LT/ST, red), EP400 (green) and MAX (black). See for details. (C) MKL-1 lysates were immunoprecipitated (IP) with indicated antibodies (top) followed by immunoblotting with indicated antibodies (left). Asterisks indicate non-specific bands in IgG control immunoprecipitation lane. (D) MKL-1 lysates (Input) were separated in a Superose 6 column and fractions (#) were blotted with antibodies indicated on left. Protein size markers in kDa indicated at top and right. (E) Three MYCL isoforms (i1, i2, i3) are indicated (see also ). Immunogen of MYCL antibody contained MYCL-i1 residues 16–139. (F) Fractions #5, 13 and 21 from Fig 1D were immunoprecipitated with MAX antibody and blotted.

Article Snippet: The following antibodies were used: Ab5 and Ab3 [ , ]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Control

Journal: PLoS Pathogens

Article Title: Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

doi: 10.1371/journal.ppat.1006668

Figure Lengend Snippet: (A) MKL-1 cells transduced with lentiviral scramble shRNA (shScr) or shRNA specific for LT and ST (shPanT) or ST only (shST) for 1 day followed by selection in puromycin (1 μg/ml) for additional 3 days were lysed (Input) and immunoprecipitated for MAX, EP400 or non-specific IgG and blotted. (B) Lysates from HCT116 (lanes 1 and 2) or UISO (lanes 3, 4 and 5) cells stably expressing MCPyV ST (lanes 2 and 5) or a C-terminal epitope tagged ST (lane 4) were immunoblotted (Input) or immunoprecipitated with antibodies to MAX or non-specific IgG. See also . (C) MCPyV ST residues 70–112 is shown with corresponding substitution mutations. See also . Residues in the LT stabilization domain (LSD) are indicated [ , ]. (D) HCT116 cells stably expressing wild type (WT) MCPyV ST or indicated mutant constructs. Lysates were immunoprecipitated with Ab5 (ST) or MAX antibodies and blotted. Red dashed lines are shown to indicate 11 lanes in ST-Input lanes. Identical panel is also shown in for input.

Article Snippet: The following antibodies were used: Ab5 and Ab3 [ , ]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).

Techniques: Transduction, shRNA, Selection, Immunoprecipitation, Stable Transfection, Expressing, Mutagenesis, Construct

Journal: PLoS Pathogens

Article Title: Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

doi: 10.1371/journal.ppat.1006668

Figure Lengend Snippet: (A) CRISPR-Cas9 screen of MKL-1 cells was analyzed in the MAGeCK-VISPR pipeline. Cumulative distribution function of p-values plotted based on 18,493 human genes. EP400 complex components and MYCL were identified in CRISPR screen negative selection with p-values < 0.05 were indicated. See also and . (B) Lysates from virus-positive MCC cell lines MKL-1, WaGa, MS-1, PeTa, BroLi and MKL-2, virus-negative MCC cell line UISO, and additional lines were immunoblotted. ST-CT are UISO cells stably expressing C-terminal epitope tagged ST. (C) Lysates from MKL-1 cell lines containing Dox-inducible shRNA (shMYCL) or miRNA (mirMYCL) specific for MYCL, prepared 2 days after addition of 0.3 μg/ml Dox (Input), were immunoprecipitated for MAX, Ab5, EP400 or non-specific IgG and blotted. (D) MKL-1 cells containing Dox-inducible HA tagged Omomyc before (-) or after (+) 5 days of Dox treatment. Dox (0.3 μg/ml) was added every two days. Lysates (Input) were immunoprecipitated with non-specific IgG, MAX, Ab5 and HA antibodies and blotted. (E) Viability of MKL-1 Dox-inducible cell lines described in C and D. 3,000 cells of each line were aliquoted in 96 well plate on day 0. Total days of Dox treatment is indicated on the X axis. Fresh medium or medium with 0.3 μg/ml Dox was supplemented every two days. At the end of time course (day 10), all samples were assessed for viability by CellTiter-Glo (Promega). Values were normalized to untreated samples of each inducible cell line. Three biological replicas were performed. Data are presented as mean (SD).

Article Snippet: The following antibodies were used: Ab5 and Ab3 [ , ]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).

Techniques: CRISPR, Selection, Virus, Stable Transfection, Expressing, shRNA, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

doi: 10.1371/journal.ppat.1006668

Figure Lengend Snippet: (A) MKL-1 cells containing three different Dox-inducible shRNA targeting EP400 (shEP400–1, shEP400-2, or shEP400-3) or shScramble (shScr) treated with Dox (0.3 μg/ml) every two days for five days. Lysates (Input) were immunoprecipitated with EP400 or control IgG antibodies and blotted for cells before (-) or after (+) 5 days of Dox treatment. (B) Same as 4A except lysates were immunoprecipitated with control IgG, MAX or Ab5 antibodies and blotted for cells after (+) 5 days of Dox treatment. (C) Cell viability assay of MCPyV positive MCC cell line MKL-1 containing Dox-inducible shRNA targeting EP400 (shEP400) or scramble (shScr). Dox added for indicated number of days. Three biological replicas were performed. Data are presented as mean (SD). (D) Lysates from UISO cells containing an inducible scramble shRNA (shScr) or 3 different shRNAs specific for EP400, prepared after 5 days Dox treatment were immunoblotted (Input) or immunoprecipitated with EP400 antibody or control IgG and blotted with indicated antibodies. (E) Cell viability assay of MCPyV negative MCC cell line UISO containing Dox-inducible shRNA targeting EP400 (shEP400) or scramble (shScr). Dox added for indicated number of days. Three biological replicas were performed; data are presented as mean (SD). (F) Lysates from parental Kelly cells or containing Dox inducible scramble shScr or shEP400-1 prepared after 5 days Dox treatment were immunoblotted (Input) or immunoprecipitated with MAX antibody or non-specific IgG and blotted with antibodies indicated. (G) Cell viability assay of Kelly cells containing Dox-inducible shRNA targeting EP400 (shEP400) or scramble (shScr). Three biological replicas were performed; data are presented as mean (SD).

Article Snippet: The following antibodies were used: Ab5 and Ab3 [ , ]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).

Techniques: shRNA, Immunoprecipitation, Control, Viability Assay

Journal: PLoS Pathogens

Article Title: Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

doi: 10.1371/journal.ppat.1006668

Figure Lengend Snippet: (A) HFK-hTERT cells were transduced with Dox-inducible OCT4, SOX2 and KLF4 (P) and stably expressed MYCL, 3M or 4M MCPyV ST. Cells were treated with Dox for 31 days and then were immunostained with fluorescent antibodies to TRA-1-60 or TRA-1-81. Light field images demonstrate flat iPSC colonies formed with 3M and MYCL but not from 4M. (B) Cells were stained with alkaline phosphatase one day after immunostaining (Fig 5A). (C) Number of iPSC colonies detected after 31 days. Three biological replicas were performed. Data are presented as mean (SD). (D) IMR90 cells stably expressing dominant negative p53 and hTERT (PH) were transduced with MYCL (PHL) or tumor derived MCPyV ER region containing truncated LT and wild type ST (PHE) and MYCL (PHEL) or 3M mutant ST (PH3) and 4M mutant ST (PH4). Lysates (Input) were prepared from indicated cells, immunoprecipitated with Ab5 or MAX antibodies followed by immunoblotting with the indicated antibodies. (E) Images of soft agar colonies from PHEL cells (4X or 20X magnification). (F) Anchorage independent growth of IMR90 cells indicated in D (10 5 cells) plated in soft agar and cultured for 4 weeks. Three biological replicas were performed. Data are presented as mean (SD).

Article Snippet: The following antibodies were used: Ab5 and Ab3 [ , ]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).

Techniques: Transduction, Stable Transfection, Staining, Immunostaining, Expressing, Dominant Negative Mutation, Derivative Assay, Mutagenesis, Immunoprecipitation, Western Blot, Cell Culture

Journal: PLoS Pathogens

Article Title: Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

doi: 10.1371/journal.ppat.1006668

Figure Lengend Snippet: (A) Venn diagram of annotated genes corresponding to peaks identified by ChIP-seq with indicated antibodies. Two biological replicas of MAX and EP400 were performed and shared genes indicated. Shared genes identified with Ab5 and ST-HA are indicated. See also . (B) De novo DNA motif identification with indicated antibodies. (C) Distribution of peaks by Metagene analysis. (D) Heatmaps of H3K4me3, MAX, EP400 and ST (Ab5) ChIP peaks ranked by read density of H3K4me3 and scaled against the 75th percentile of genome-wide read density for each ChIP. (E) Meta-track analysis of ChIP-seq read density for MAX, EP400 and ST at all H3K4me3 peaks genome-wide. Regions are centered and ranked for H3K4me3 peaks over input.

Article Snippet: The following antibodies were used: Ab5 and Ab3 [ , ]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).

Techniques: ChIP-sequencing, Genome Wide

Journal: PLoS Pathogens

Article Title: Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

doi: 10.1371/journal.ppat.1006668

Figure Lengend Snippet: (A) Heatmap depicts average mean-centered and standard-deviation-scaled gene expression profiles for each of 62 clusters created by applying model-based clustering to the differentially expressed genes (DEG) in MKL-1 cells after depletion of EP400 or MYCL in comparison to shScr control. Model-based clusters (1–62) are labeled on the right-hand side and their gene members are listed in . Merged Clusters (CL1-4) are indicated on the left-hand side. (B) Diagram illustrating BETA Activating/Repressing Function Prediction of transcription factors by correlation of distance of peaks from corresponding TSS obtained in ChIP-seq of ST, MAX and EP400 with changes in gene expression by RNA-seq after Dox-induction with shRNA targeting EP400 or MYCL. (C) Venn diagram showing common direct target genes of MAX, EP400 and ST identified by BETA based on ChIP-seq of MAX, EP400, ST and RNA-seq of shEP400–2, -3 and MYCL shRNA (BETA3). (D) Heatmap depicts average mean-centered and standard-deviation-scaled gene expression profiles for each of 37 clusters created by applying model-based clustering to the 951 BETA3 target genes in MKL-1 cells after depletion of EP400 or MYCL in comparison to shScr control. Model-based clusters (1–37) are labeled on the right-hand side and their gene members are listed in . Merged Clusters (CL1-4) are indicated on the left-hand side. (E) MKL-1 cells containing Dox inducible shRNA for shScr, shMYCL or EP400 (shEP400-2, -3) were treated with dox for 5 days. Lysates were blotted with indicated antibodies. EP400 immunoprecipitations were blotted with EP400 antibody.

Article Snippet: The following antibodies were used: Ab5 and Ab3 [ , ]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).

Techniques: Standard Deviation, Gene Expression, Comparison, Control, Labeling, ChIP-sequencing, RNA Sequencing, shRNA

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Establishment of a prognostic hypoxic gene signature in osteosarcoma in the TARGET cohort. (A) The chromosomal locations of 197 hypoxic genes. (B) The relationships between the number of trees and error rates. (C) The rank of relative importance of the top five variables. (D) Univariate cox regression analysis of TES, SDC3, MXI1, CAVIN1, and EGFR in osteosarcoma. (E) Heat map visualizing the up- (red) and down-regulated (green) genes in high- and low-risk groups. (F) The distributions and proportions of survival status (alive: green; dead: red). (G) Kaplan-Meier curves of OS between high- and low-risk specimens. The differences were compared by log-rank tests.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Univariate cox regression analysis for prognosis-related hypoxic genes in the TARGET cohort.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: The relative importance of prognosis-related hypoxic genes.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Prognostic values of hypoxic genes from the gene signature in osteosarcoma. Kaplan-Meier curves for overall survival of osteosarcoma patients in the high and low (A) CAVIN1, (B) EGFR, (C) SCD3, (D) TES, and (E) MXI1 expression groups from the TARGET cohort. The survival differences were assessed via log-rank tests.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Verification of the expression of hypoxic genes from the gene signature. (A–E) RT-qPCR for detecting the expression of SCD3, EGFR, MXI1, CAVIN1, and TES in U2OS osteosarcoma cells and hFOB1.19 normal cells. (F, G) Western blotting for the expression of the above genes in U2OS osteosarcoma cells and hFOB1.19 normal cells. (H, I) Western blotting for the expression of the above genes in Saos-2 osteosarcoma cells and hFOB1.19 normal cells. (J–N) RT-qPCR for the expression of the above genes in tumors with Saos-2 osteosarcoma cells and hFOB1.19 normal cells. ** p < 0.01; *** p < 0.001; **** p < 0.0001. U2OS osteosarcoma cells compared with hFOB1.19 normal cells.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Silencing MXI1 attenuates proliferation, migration, and invasion of osteosarcoma cells. (A, B) RT-qPCR for verifying the expression of MXI1 in U2OS and Saos-2 cells following transfection with its specific siRNAs. (C, D) Colony formation for measuring the proliferative capacity of U2OS and Saos-2 cells after silencing MXI1 expression. (E–H) Transwell assays for detecting the (E, F) migration and (G, H) invasion of U2OS and Saos-2 cells with MXI1 knockdown. ** p < 0.01; **** p < 0.0001.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, Knockdown

Journal: Science Advances

Article Title: A MYC inhibitor selectively alters the MYC and MAX cistromes and modulates the epigenomic landscape to regulate target gene expression

doi: 10.1126/sciadv.abh3635

Figure Lengend Snippet: ( A ) Peak overlap analysis of MYC and MAX consensus peaks ( n = 4). ( B ) Percentage of differential MAX binding peaks lost in response to MYCi975 compared to MAX consensus peaks ( n = 4). ( C ) Total number of differentially bound MAX peaks following differential binding analysis ( n = 4). ( D ) Peak overlap analysis of differential MYC- and MAX-bound sites (MYCi975 sensitive) in MYCi975-treated 22Rv1 cells. ( E ) Heatmap representation of MYC, MAX, MGA, MNT, and MXD1 at MYCi975-sensitive sites demonstrating no significant change in MAX occupancy (6812 peaks in total). ( F ) Log 2 (normalized tag counts) for MYC, MAX, MGA, MNT, and MXD1 at MAX-retained peaks in 22Rv1 cells. The middle dashed line represents the sample median, and the upper and lower dotted lines represent the upper and lower quartiles, respectively. ( G ) Differential gene expression analysis of MAX-retained peaks annotated to promoters (±2 kb from the TSS). ( H ) Gene ontology analysis of promoter-bound MAX-retained peaks. ( I ) Gene browser tracks of AURKB showing loss of MYC (green), retention of MAX (blue), an increase in MXD1 (red), and loss of AURKB m RNA (black) in 48-hour MYCi975-treated cells. MGA (gray) and MNT (yellow) are also displayed.

Article Snippet: Antibodies used in this study were CTCF (Active Motif, catalog no. 61932), c-MYC (Abcam, ab56-9E11), c-MYC (Abcam, ab32072- Y69 ), WDR5 (Proteintech, catalog no. 15544-1-AP), FOXA1 (Bethyl Laboratories, A305-249A), H3K27ac (Active Motif, catalog no. 39685), AR (Abcam, ab108341), MAX (Proteintech, catalog no. 10426-1-AP), full-length PARP (Cell Signaling Technologies, 46D11, catalog no. 9532T), cleaved PARP (Cell Signaling Technologies, D64E10, catalog no. 5625T), MNT (Bethyl Laboratories, A303-627A), MXD1 (Proteintech, catalog no. 17888-1-AP), MGA (Bethyl Laboratories, A302-865A), FOXM1 (Diagenode, catalog no. C15410232), histone H3 (Abcam, ab10799), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich, G9545).

Techniques: Binding Assay, Gene Expression