Journal: Nature communications

Article Title: Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

doi: 10.1038/s41467-022-34610-0

Figure Lengend Snippet: Fig. 3 | Structural basis of PELP1-PELP1 dimerization. a Schematic representation of PELP1’s Rix1 domain with localization of LxxLL and PxxP motifs. Specific motifs involved in PELP1 dimerization, or residing close to dimer interfaces, are noted below the schematic. b Bottom view of segmented cryo-EM density showing two symmetric PELP1 dimerization interfaces between LM81-LM12-LM22 (superscript denotes specific protomer). c Model zoom of LM81-LM12-LM22 dimer interface exhibiting a hydrophobic environment produced by mainly leucine residues from LxxLL motifs. d Top view of cryo-EM density showing a single symmetric PELP1 dimerization interface between LM11 and α-helix 22 of each PELP1 protomer. e Model zoom of LM111+2 - α221+2 interface showing contributing leucine residues to another hydrophobic interface environment.

Article Snippet: Membranes were incubated overnight at 4 °C with ERα antibody (1/500, Millipore #06-935), PELP1 antibody (1/5000, Bethyl Labs #A300-180A-M) or WDR18 antibody (1/125, Sigma #HPA050200) and imaged using Azur Biosystem c600 imager.

Techniques: Cryo-EM Sample Prep, Produced

Journal: Nature communications

Article Title: Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

doi: 10.1038/s41467-022-34610-0

Figure Lengend Snippet: Fig. 5 | PELP1’s solvent-exposed LxxLL motifs are incompatible with steroid receptor binding. a Schematic representation of PELP1’s Rix1 domain with locali- zation of LxxLL and PxxP motifs. Motifs illustrated in model views are noted below the schematic (solvent motif labels colored blue). b Model view of a single PELP1 Rix1 domain protomer with solvent-exposed LxxLL motifs colored magenta. c, d, e Individual zooms of each solvent LxxLL motif with residue positions dis- played. Solvent face is illustrated in each panel with dashed line for spatial orien- tation of the motifs. The invariant leucine residues required for AF-2 binding of SRs are buried away from the solvent face, rendering them inaccessible for SR binding.

Article Snippet: Membranes were incubated overnight at 4 °C with ERα antibody (1/500, Millipore #06-935), PELP1 antibody (1/5000, Bethyl Labs #A300-180A-M) or WDR18 antibody (1/125, Sigma #HPA050200) and imaged using Azur Biosystem c600 imager.

Techniques: Solvent, Binding Assay, Residue

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Immunohistochemical staining of PELP1 in TNBC. Positive immunostaining of PELP1 mainly distributed in nuclei of tumor cells, no cytoplasmic staining was found ( a , b ). Low grade lymph node stage TNBC showed weak PELP1 nuclear expression ( a ), High grade lymph node stage TNBC showed strong PELP1 nuclear expression ( b ). PELP1 nuclear staining was absent in negative control ( c ). Bar = 50 μm.

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Immunohistochemical staining, Staining, Immunostaining, Expressing, Negative Control

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Correlation between PELP1 protein expression and clinicopathological variables in patients with TNBC

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Clinicopathological variables and outcomes of patients with TNBC. Kaplan–Meier survival curve showed that TNBC patients with positive lymph node metastasis had significantly reduced DFS ( a1 ) and OS ( a2 ); TNBC patients in stage III and IV also demonstrated significantly reduced DFS ( b1 ) and OS ( b2 ); PELP1 was not associated with DFS or OS in TNBC patients when observed independently, although patients in the high PELP1 group demonstrated a trend of reduced DFS ( c1 ) and OS ( c2 ), compared with those in the low PELP1 group.

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques:

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Univariate analysis of DFS and OS according to clinicopathological variables

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques:

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: PELP1 protein expression and patients’ outcome in subgroups of TNBC. Kaplan–Meier survival curve showed that, in the tumor size ≤ 2 cm subgroup, patients with high PELP1 expression had significantly shorter DFS ( a1 ); in the high Ki-67 LI subgroups, patients with high PELP1 expression have significantly shorter DFS ( b1 ) and OS ( b2 ).

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Univariate analysis of DFS and OS according to PELP1 protein expression in different subgroups

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Combining PELP1 status and Ki-67 LI as a prognostic biological marker. Kaplan–Meier survival curve showed that, combination of PELP1 status with Ki-67 status was significantly correlated with DFS ( a1 ) and OS ( a2 ) in patients with TNBC; patients with TNBC in PELP1/Ki-67 double high group had significantly reduced DFS ( b1 ) and OS ( b2 ) compared with others.

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Marker

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Multivariate analysis of DFS and OS according to clinical pathological variables

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: LAS1L associates with PELP1, TEX10, WDR18, RanBP5, NOL9, and SENP3. (A) LAS1L was immunoprecipitated (IP) with an anti-LAS1L–specific antibody from HEK 293T cell lysates. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate; *, presence of an unspecific band. (B) PELP1 was immunoprecipitated (IP) with an anti-PELP1 antibody from HEK 293T cell lysates. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate. (C) HEK 293T cells were transfected with nontargeting control (represented by the letter “C”) or LAS1L siRNA for 48 h. Cells were lysed and immunoprecipitated with an anti-PELP1 antibody. Proteins associating with PELP1 in the absence of LAS1L were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate; *, presence of an unspecific band; #, an IgG band.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Negative Control, Transfection, Control

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: Depletion of LAS1L-interacting proteins induces a p53-dependent G1 cell cycle arrest. (A) Cell cycle profiles of HCT116 cells transfected with control, LAS1L, PELP1, TEX10, NOL9, SENP3, and WDR18 siRNA. Seventy-two hours after transfection the cells were pulse-labeled with BrdU for 30 min, stained with propidium iodide, and analyzed by flow cytometry to determine the percentage of cells in G1 and S phase. Error bars indicate SD from triplicate experiments. (B) Western blot analysis of the siRNA-transfected cells from (A) with specific antibodies against p53, p21, and β-actin. (C) Total RNA was extracted from the siRNA-transfected cells from panel (A), and knockdowns were confirmed by qRT-PCR with gene-specific primers. Relative mRNA levels for each gene-specific primer were normalized to β-actin. Error bars indicate SD from triplicate experiments.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Transfection, Control, Labeling, Staining, Flow Cytometry, Western Blot, Quantitative RT-PCR

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: LAS1L-associated proteins localize to the nucleolus. Immunofluorescence analysis of U2OS cells with complex protein-specific antibodies. Cells were preextracted with 0.1% Triton, fixed, and immunostained with anti-LAS1L, PELP1, TEX10, NOL9, and WDR18 antibodies (green). Colocalization with SENP3 was confirmed using an anti-SENP3 antibody (red). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all three panels.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Immunofluorescence, Staining

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: LAS1L and NOL9 interact with the mammalian Rix1 complex on pre-60S ribosomal particles. Nuclear extracts from HCT116 cells were fractionated by centrifugation on a 10–30% sucrose gradient. Fractions were collected, and the optical density was measured at 260 nm (A 260 ). Based on the A 260 profile, fractions corresponding to free nuclear proteins (1, 2, and 3), pre-40S ribosomal particles (8, 9, and 10), and pre-60S ribosomal particles (12, 13, and 14) were then combined and immunoprecipitated (IP) with rabbit IgG (A) as a negative control or with PELP1 (B) or LAS1L (C) antibodies. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). WCL, whole-cell lysate; *, presence of an unspecific band; #, an IgG band.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Centrifugation, Immunoprecipitation, Negative Control, SDS Page, Western Blot

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: LAS1L-associated proteins are required for proper processing of ITS-2. (A) Schematic representation of the primary 47S rRNA transcript and the two major processing pathways with rRNA intermediates, as indicated (adapted from Hadjiolova et al. , 1993 ). (B) Northern blot analysis of total RNA from HCT116 cells transfected with control, LAS1L, PELP1, TEX10, NOL9, SENP3, RanBP5, and WDR18 siRNA. Seventy-two hours after transfection, equal amounts of total RNA were hybridized with specific probes for ITS-1, ITS-2, 28S, and 18S rRNA intermediates (indicated on the right). The right panel shows longer exposure times for each probe. The positions of the specific probes used for Northern blot analysis are indicated on the schematic in (A). (C) The 32S/28S, 12S/28S, and 30S/28S ratios were determined by quantification of the relative band intensities of the 32S, 30S, and 28S rRNA intermediates in the lower exposures and the 12S rRNA intermediate in the longer exposure from (B). Intensities were normalized to the control siRNA-treated sample. (D) Knockdowns were confirmed by qRT-PCR with gene-specific primers. Relative mRNA levels for each gene-specific primer were normalized to β-actin. Error bars indicate SD from triplicate experiments.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Northern Blot, Transfection, Control, Quantitative RT-PCR

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: PELP1 requires active Pol I transcription for nucleolar localization. (A) U2OS cells were treated with either DMSO or 20 nM actinomycin D for 2 h. Cells were fixed and immunostained with an anti-PELP1 antibody (green) and an anti-UBF1 antibody (red). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of both panels. (B) Cells were treated with DMSO (−) or actinomycin D (+) as in (A), and lysates were immunoprecipitated (IP) with an anti-PELP1 antibody. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Staining, Immunoprecipitation, SDS Page, Western Blot, Negative Control

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: SENP3 is necessary for LAS1L and PELP1 nucleolar localization. (A) HCT116 cells were transfected with control or SENP3 siRNA for 72 h. Cells were fixed and immunostained with anti-LAS1L (green) and anti-SENP3 (red) antibodies. DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (B) HCT116 cells were transfected with control or SENP3 siRNA for 72 h. Cells were fixed and immunostained with anti-PELP1 (green) and anti-SENP3 (red) antibodies. DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (C) Cells were transfected with a Control (−) or SENP3 (+) siRNA for 72 h. Lysates were then immunoprecipitated with rabbit IgG (as negative control) or PELP1 antibody. Coprecipitating proteins were separated on SDS–PAGE and analyzed by Western blotting using specific antibodies (indicated on the left). WCL, whole-cell lysate; *, presence of an unspecific band; #, an IgG band.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Transfection, Control, Staining, Immunoprecipitation, Negative Control, SDS Page, Western Blot

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: NPM1 is required for LAS1L and PELP1 nucleolar localization. (A) HCT116 cells were transfected with control or NPM1 siRNA for 48 h. Subcellular localization of LAS1L was determined by immunofluorescence analysis using a LAS1L antibody (green). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (B) HCT116 cells were transfected with control or NPM1 siRNA for 48 h. Subcellular localization of PELP1 was determined by immunofluorescence analysis using a PELP1 antibody (green). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (C) Knockdowns of NPM1 for (A) and (B) were confirmed by Western blotting using specific antibodies (indicated on the left). An anti-CDK2 antibody was used as loading control. (D) Cells were transfected with a control (“C”) or NPM1 siRNA for 72 h. Lysates were then immunoprecipitated with rabbit IgG (as negative control) or PELP1 antibody. Coprecipitating proteins were separated on SDS–PAGE and analyzed by Western blotting using specific antibodies (indicated on the left). WCL, whole-cell lysate.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Transfection, Control, Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Negative Control, SDS Page

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.E11-06-0530

Figure Lengend Snippet: LAS1L and PELP1 are modified by SUMO in an SENP3-dependent manner. (A) HEK 293T cells were transfected with FLAG-LAS1L (+) plus either empty vector (−) or plasmids expressing 6xHis-tagged SUMO-1 or SUMO-3 (+). Forty-eight hours after transfection, SUMOylated proteins were pulled down from cell lysates using Ni-NTA agarose beads. Eluates were analyzed on SDS–PAGE and by Western blotting with the indicated antibodies. (B) HEK 293T cells were transfected with either control (−) or SENP3 (+) siRNA. The next day, cells were transfected with empty vector (−) or a 6xHis-tagged SUMO-3 (+) expressing plasmid. Forty-eight hours after transfection, pulldowns and protein analyses were performed as in (A). (C) HEK 293T cells were transfected with either control (−) or NPM1 (+) siRNA. The next day, cells were transfected with empty vector (−) or a plasmid expressing 6xHis-tagged SUMO-3 (+). Forty-eight hours after transfection, pulldowns and protein analyses were performed as in (A).

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Modification, Transfection, Plasmid Preparation, Expressing, SDS Page, Western Blot, Control

Journal: Cells

Article Title: Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

doi: 10.3390/cells6040042

Figure Lengend Snippet: PELP1 expression in human tissues of ACC and H295R cells. ( A ) Western blot analysis of PELP1 was performed on 50 μg of total protein extracted from normal human adrenal tissues (normal) and ACCs (C1–C6); ( B ) Western analysis of PELP1 was performed on 50 μg of total protein extracted from LNCaP and H295R cells. GAPDH was used as a loading control. Results are representative of three different experiments.

Article Snippet: The protein content was determined by the Bradford method and 500 μg of protein lysates were first precleared with 10 μL of protein A agarose beads (Santa Cruz) for 1 h. Then precleared cell lysates were incubated over night with primary anti-PELP1 antibody together with 20 μL of protein A agarose beads at 4 °C in HNTG buffer (20 m M HEPES pH 7.5, 150 m M NaCl, 0.1% Triton X-100, 10% glycerol, 0.1 m M Na 3 VO 4 ).

Techniques: Expressing, Western Blot, Control

Journal: Cells

Article Title: Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

doi: 10.3390/cells6040042

Figure Lengend Snippet: PELP1 is recruited to form a multiprotein complex in H295R cells after treatment with E2 and IGF-II. H295R cells were treated for 10 min with E2 (100 nM) or IGF-II (100 ng/mL). Total protein extract (500 μg) was immunoprecipitated with 1 μg of anti-PELP1 antibody. The samples were immunoblotted for IGF1R, ERα, and c-Src. Protein expression for each sample was normalized to PELP1 content. Results are representative of three independent experiments.

Article Snippet: The protein content was determined by the Bradford method and 500 μg of protein lysates were first precleared with 10 μL of protein A agarose beads (Santa Cruz) for 1 h. Then precleared cell lysates were incubated over night with primary anti-PELP1 antibody together with 20 μL of protein A agarose beads at 4 °C in HNTG buffer (20 m M HEPES pH 7.5, 150 m M NaCl, 0.1% Triton X-100, 10% glycerol, 0.1 m M Na 3 VO 4 ).

Techniques: Immunoprecipitation, Expressing

Journal: Cells

Article Title: Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

doi: 10.3390/cells6040042

Figure Lengend Snippet: PELP1 knockdown decreases ERK1/2 phosphorylation. ( A ) H295R cells were transfected with PELP1 siRNA (100 nM and 200 nM) or a non-targeting siRNA (control siRNA) for 24 h. Western blot analyses of PELP1 were performed on 50 μg of total protein; ( B ) H295R cells were transfected with control siRNA or PELP1 siRNA. After 24 h cells were treated for 10 min with E2 (100 nM) or IGF-II (100 ng/mL). Western blot analyses of PELP1 were performed on 10 μg of total protein. Results are representative of three independent experiments. GAPDH and ERK1/2 were used as a loading control; upper panel graph represents mean of pERK1/2 optical density (O.D.) from three independent experiments with similar results normalized to ERK1/2 content (* p < 0.001 compared to untreated control sample (basal) assumed as 100).

Article Snippet: The protein content was determined by the Bradford method and 500 μg of protein lysates were first precleared with 10 μL of protein A agarose beads (Santa Cruz) for 1 h. Then precleared cell lysates were incubated over night with primary anti-PELP1 antibody together with 20 μL of protein A agarose beads at 4 °C in HNTG buffer (20 m M HEPES pH 7.5, 150 m M NaCl, 0.1% Triton X-100, 10% glycerol, 0.1 m M Na 3 VO 4 ).

Techniques: Knockdown, Phospho-proteomics, Transfection, Control, Western Blot

Journal: Cells

Article Title: Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

doi: 10.3390/cells6040042

Figure Lengend Snippet: PELP1 knockdown decreases IGF1R expression in H295R cells. ( A ) H295R cells were transfected with PELP1 siRNA or a non-targeting siRNA (control siRNA) for 24 h. After transfection cells were treated for 24 h with E2 (100 nM). Western blot analyses of PELP1 and IGF1R were performed on 50 μg of total protein. Results are representative of three independent experiments. GAPDH was used as a loading control; ( B ) The graph represents mean of PELP1 and IGF1R optical densities (O.D.) from three independent experiments with similar results normalized to GAPDH content (* p < 0.001 compared to untreated control sample (Basal) assumed as 100).

Article Snippet: The protein content was determined by the Bradford method and 500 μg of protein lysates were first precleared with 10 μL of protein A agarose beads (Santa Cruz) for 1 h. Then precleared cell lysates were incubated over night with primary anti-PELP1 antibody together with 20 μL of protein A agarose beads at 4 °C in HNTG buffer (20 m M HEPES pH 7.5, 150 m M NaCl, 0.1% Triton X-100, 10% glycerol, 0.1 m M Na 3 VO 4 ).

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot

Journal: Cells

Article Title: Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

doi: 10.3390/cells6040042

Figure Lengend Snippet: PELP1 knockdown decreases Cyclin D1 expression in H295R cells. Cells were transfected with PELP1 siRNA or a non-targeting siRNA (control siRNA) for 24 h. After transfection cells were treated for 24 h with E2 (100 nM) and IGF-II (100 ng/mL). Western blot analyses of Cyclin D1 were performed on 50 μg of total protein. Results are representative of at least three independent experiments. GAPDH was used as a loading control; the right panel graph represents mean of Cyclin D1 optical density (O.D.) from three independent experiments with similar results normalized to GAPDH content (* p < 0.001 compared to untreated control sample (Basal) assumed as 100).

Article Snippet: The protein content was determined by the Bradford method and 500 μg of protein lysates were first precleared with 10 μL of protein A agarose beads (Santa Cruz) for 1 h. Then precleared cell lysates were incubated over night with primary anti-PELP1 antibody together with 20 μL of protein A agarose beads at 4 °C in HNTG buffer (20 m M HEPES pH 7.5, 150 m M NaCl, 0.1% Triton X-100, 10% glycerol, 0.1 m M Na 3 VO 4 ).

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot

Journal: Cells

Article Title: Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

doi: 10.3390/cells6040042

Figure Lengend Snippet: PELP1 knockdown reduces cell proliferation in H295R. ( A , B ), H295R cells were transfected with PELP1 siRNA or a non-targeting siRNA (control siRNA). Twenty-four hours after silencing, cells were left untreated (Basal) or treated for an additional 48 h with IGF-II (100 ng/mL) or E2 (100 nM). (* p < 0.0001 compared with Basal). At the end of the experiment, cells were incubated with MTT ( A ) or [3H] thymidine ( B ) as described in Materials and Methods.

Article Snippet: The protein content was determined by the Bradford method and 500 μg of protein lysates were first precleared with 10 μL of protein A agarose beads (Santa Cruz) for 1 h. Then precleared cell lysates were incubated over night with primary anti-PELP1 antibody together with 20 μL of protein A agarose beads at 4 °C in HNTG buffer (20 m M HEPES pH 7.5, 150 m M NaCl, 0.1% Triton X-100, 10% glycerol, 0.1 m M Na 3 VO 4 ).

Techniques: Knockdown, Transfection, Control, Incubation

Journal: Molecular cancer research : MCR

Article Title: Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

doi: 10.1158/1541-7786.MCR-17-0598

Figure Lengend Snippet: AIB1 interacts with cytoplasmic PELP1. (A) Western blot of TAP pull-down (left) and whole cell lysates (right) in hTERT mammary epithelial cells. (B) Western blot showing co-immunoprecipitation (left) and whole cell lysates (right) of PELP1 and AIB1 in MCF10A cells.

Article Snippet: Stable Cell Line Generation MCF-7 cells were transfected per manufacturer’s instructions with human specific PELP1 double nickase constructs (sc-405376-NIC-2; Santa Cruz Biotechnology) to knockdown endogenous PELP1 (h2 MCF-7).

Techniques: Western Blot, Immunoprecipitation

Journal: Molecular cancer research : MCR

Article Title: Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

doi: 10.1158/1541-7786.MCR-17-0598

Figure Lengend Snippet: Generation of PELP1 models in SR+ breast cancer cells. (A) mRNA levels of PELP1 and AIB1 in a panel of luminal breast cancer lines. Graphed data represents the mean ± SD (n = 3). (B) Western blot showing knockdown of endogenous PELP1 in MCF-7 cells (h2 MCF-7) using a double nickase approach. (C) Western blot showing overexpression of PELP1 in LXSN (vector control), WT PELP1, and cyto PELP1 in h2 MCF-7 PELP1 cells. (D) Western blot showing PELP1 expression in cellular fractionation (CE, cytoplasmic extract; NE, nuclear extract) from h2 MCF-7 PELP1 cells. p65 and HDAC2 (loading controls). (E) Immunofluorescence in h2 MCF-7 PELP1 cells. DAPI; blue. Western blot showing co-immunoprecipitation (left) and whole cell lysates (right) of (F) PELP1 and AIB1, (H) PELP1 and ER, and (I) ER and AIB1 in h2 MCF-7 PELP1 cells (under basal conditions). (G) Western blot showing co-immunoprecipitation from cytoplasmic extract (left) and cytoplasmic extract (right) of PELP1 and AIB1 (under basal conditions).

Article Snippet: Stable Cell Line Generation MCF-7 cells were transfected per manufacturer’s instructions with human specific PELP1 double nickase constructs (sc-405376-NIC-2; Santa Cruz Biotechnology) to knockdown endogenous PELP1 (h2 MCF-7).

Techniques: Western Blot, Over Expression, Plasmid Preparation, Expressing, Cell Fractionation, Immunofluorescence, Immunoprecipitation

Journal: Molecular cancer research : MCR

Article Title: Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

doi: 10.1158/1541-7786.MCR-17-0598

Figure Lengend Snippet: AIB1 activation (pThr24) levels are altered in the presence of cytoplasmic PELP1. Cellular fractionation in mammary epithelial cells (A) MCF10A and breast cancer cells (B) T47D CO and (C) MCF-7. Western blots show phosphorylated (pAIB1; Thr24) and total AIB1.

Article Snippet: Stable Cell Line Generation MCF-7 cells were transfected per manufacturer’s instructions with human specific PELP1 double nickase constructs (sc-405376-NIC-2; Santa Cruz Biotechnology) to knockdown endogenous PELP1 (h2 MCF-7).

Techniques: Activation Assay, Cell Fractionation, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

doi: 10.1158/1541-7786.MCR-17-0598

Figure Lengend Snippet: Cytoplasmic PELP1-induced gene expression in h2 MCF-7 PELP1 models. (A) Supervised heat map of differentially expressed genes in h2 MCF-7 PELP cells (LXSN, WT PELP1, and cyto PELP1). Cells were treated with vehicle (EtOH) or E2 (1 nM) for 6 h. Genes were hierarchically clustered using average clustering method and Pearson correlation as distance in R ComplexHeatmap package (PMID: 27207943). The columns are grouped by experimental condition and biological replicates for display purposes. (B) qRT-PCR validation of select genes from RNA-Seq analysis in h2 MCF-7 PELP1 cells. Graphed data represents the mean ± SD (n = 3).

Article Snippet: Stable Cell Line Generation MCF-7 cells were transfected per manufacturer’s instructions with human specific PELP1 double nickase constructs (sc-405376-NIC-2; Santa Cruz Biotechnology) to knockdown endogenous PELP1 (h2 MCF-7).

Techniques: Expressing, Quantitative RT-PCR, RNA Sequencing Assay

Journal: Molecular cancer research : MCR

Article Title: Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

doi: 10.1158/1541-7786.MCR-17-0598

Figure Lengend Snippet: Cytoplasmic PELP1 promotes tumorsphere formation in breast cancer cells. (A,B) Soft agar colony formation in h2 MCF-7 PELP1 cells (LXSN, WT PELP1, or cyto PELP1). Cells were grown in vehicle (EtOH) or E2 (1 nM). Soft agar data were analyzed in terms of (A) total number of colonies and (B) average colony size. (C) Primary and (D) secondary tumorspheres in h2 MCF-7 PELP1 cells (LXSN, WT PELP1, or cyto PELP1). Cells were treated with vehicle (EtOH) or E2 (1 nM). (E) ALDH activity in primary tumorspheres formed from h2 MCF-7 PELP1 cells was assessed using flow cytometry. Graphed data represents the mean ± SD (n = 3). ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Stable Cell Line Generation MCF-7 cells were transfected per manufacturer’s instructions with human specific PELP1 double nickase constructs (sc-405376-NIC-2; Santa Cruz Biotechnology) to knockdown endogenous PELP1 (h2 MCF-7).

Techniques: Activity Assay, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

doi: 10.1158/1541-7786.MCR-17-0598

Figure Lengend Snippet: AIB1 knockdown affects cancer cell biology phenotypes in PELP1 models. (A) mRNA and (B) Western blot showing AIB1 knockdown in h2 MCF-7 PELP1 models. (C) mRNA levels of genes (e.g. SERPINE1, AMIGO2, NELL2) identified from our RNA-Seq analysis in AIB1 knockdown h2 MCF-7 PELP1 models. (D) Primary and (E) secondary tumorspheres in AIB1 knockdown h2 MCF-7 PELP1 models. Cells were treated with vehicle (EtOH) or E2 (1 nM). (F) Western blot showing co-immunoprecipitation (top) and whole cell lysates (bottom) of PELP1 and AIB1 in h2 MCF-7L PELP1 cells treated with vehicle (DMSO) or SI-2 (100 nM). (G) Primary tumorspheres in h2 MCF-7L PELP1 cells overexpressing LXSN, WT PELP1, or cyto PELP1 treated with vehicle (DMSO) or SI-2 (100 nM). Graphed data represents the mean ± SD (n = 3). ** p < 0.01, **** p < 0.0001.

Article Snippet: Stable Cell Line Generation MCF-7 cells were transfected per manufacturer’s instructions with human specific PELP1 double nickase constructs (sc-405376-NIC-2; Santa Cruz Biotechnology) to knockdown endogenous PELP1 (h2 MCF-7).

Techniques: Western Blot, RNA Sequencing Assay, Immunoprecipitation

Journal: Molecular cancer research : MCR

Article Title: Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

doi: 10.1158/1541-7786.MCR-17-0598

Figure Lengend Snippet: PELP1 knockdown prolongs survival time in mice transplanted with MMTV-AIB1 tumor cells. (A) Western blot showing knockdown of endogenous PELP1 in J110 mouse tumor cells (PELP1-low) using a double nickase approach. (B) Primary tumorspheres in J110 (parental or PELP1-low) cells. (C) Primary tumorspheres in J110 parental cells treated with vehicle (DMSO) or SI-2 (100 nM). Graphed data represents the mean ± SD (n = 3). * p < 0.05, ** p < 0.01 (Student t test). (D) Kaplan-Meier survival curve. J110 cells (parental or PELP1-low) were transplanted into recipient FVB mice (10 mice/group). Data represent two independent trials. **** p < 0.0001. (E) Model for cytoplasmic PELP1 signaling in SR+ breast cancer models. 1) PELP1/ER/PR/AIB1 complexes regulate E2-induced genes that promote proliferation and therapy endocrine therapy resistance (14). 2) Cytoplasmic PELP1 promotes AIB1 phosphorylation independent of E2 and ER, which results in regulation of genes, via yet to be identified transcription factors (TFs) that promote breast cancer stem cell (BCSC) survival and/or expansion.

Article Snippet: Stable Cell Line Generation MCF-7 cells were transfected per manufacturer’s instructions with human specific PELP1 double nickase constructs (sc-405376-NIC-2; Santa Cruz Biotechnology) to knockdown endogenous PELP1 (h2 MCF-7).

Techniques: Western Blot