Journal: bioRxiv

Article Title: MYC-associated factor MAX is an essential regulator of the clock core network

doi: 10.1101/771329

Figure Lengend Snippet: MAX-mediated circadian gene expression also operates in non-cancerous cells. ( A-B ) Expression of MAX , PER1 , PER2 , CRY1 , and CRY2 genes upon MAX silencing in foreskin fibroblast BJ-5ta and epithelial MCF10A cell lines. Relative expression was determined by qRT-PCR using GAPDH for normalization. Values of control cells were set to 1. Shown as mean + SEM, n ≥ 3. *P < 0.05. **P < 0.01 and ***P < 0.001, two-way ANOVA with Bonferroni post hoc test, silencing versus control. ( C ) Recruitment of MAX on the promoter of PER1 , PER2 , CRY1 and CRY2 in MCF10A cells. Enrichment of MAX was evaluated by quantitative PCR of immunoprecipitated DNA compared with input DNA (% of input). Immunoglobulin G (IgG) was used as a negative control. Shown as mean + SEM, n =3. *P < 0.05. **P < 0.01 and ***P < 0.001, unpaired two-tailed T-test. ( D ) Real-time bioluminescence oscillatory pattern in MAX-silenced and control MCF10A cells expressing a firefly luciferase reporter controlled by the circadian-responsive BMAL1 promoter. Shown as baseline-subtracted luminescence data fitted to a sine wave.

Article Snippet: Human epithelial MCF10A cell line (obtained from ATCC) were maintained in a 1:1 mixture of DMEM and Ham’s F12 (Sigma, catalog#51651C) media supplemented with 20 ng/ml human Epidermal Growth Factor (EGF), (Sigma, catalog#E9644), 2mM L-glutamine, 5% horse serum (Sigma, catalog#H1270), 10 μg/ml human recombinant insulin (Sigma, catalog#I9278), 0.5 mg/ml hydrocortisone (Sigma, catalog#H6909), 100 ng/ml cholera toxin (Sigma, catalog#C8052) and 1X penicillin/streptomycin solution.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Control, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Two Tailed Test, Luciferase

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet:

Article Snippet: Human: MDAMB468 , ATCC/CRUKCI Biorepository , Cat# HTB-26; RRID: CVCL_0419.

Techniques: Recombinant, In Vivo, Protease Inhibitor, Plasmid Preparation, Purification, Sample Prep, Sequencing, RNA Sequencing Assay, shRNA, Software

Journal: Oncogene

Article Title: Epithelial Splicing Regulatory Protein 1 is a Favorable Prognostic Factor in Pancreatic Cancer that Attenuates Pancreatic Metastases

doi: 10.1038/onc.2013.392

Figure Lengend Snippet: A: ESRP1 was strongly expressed in the nuclei of cancer cells in PDAC sections, especially in well-to-moderately differentiated adenocarcinoma forming ductal structure (arrows), while it was weakly expressed in poorly-differentiated adenocarcinoma, which are characterized by trabecular or solid structure, but not ductal structure. In serial tissue sections, high ESRP1 expression was associated with stronger immunostaining for FGFR-2 IIIb (white arrows), whereas FGFR-2 IIIc immunoreactivity was increased in tumors with low ESRP1 levels (arrowheads). Upper panel, H&E staining; bar; 100 µm. B and C: ESRP1 and FGFR-2 IIIb indices in well/moderate differentiated (well/mod) lesions were significantly higher than in poorly-differentiated (poor) lesions (** P <0.01). D: The FGFR-2 IIIc index was greater in poor lesions (* P <0.05).

Article Snippet: The following were purchased: anti-ESRP1, anti-lamin A+C, anti-Perilipin 3, and anit-IQGAP1 antibodies from Abcam plc (Cambridge, MA); anti-filamin A from Novus Biologicals (Colorado, USA); anti-14-3-3ε from Cell signaling technology (Boston, MA); anti-CD31 antibody from AbD Serotec (Kidlington, UK); anti-cytokeratin (CK) 19 antibody from Boehringer Mannheim GmbH (Mannheim, Germany); anti-insulin antibody from DAKO JAPAN (Tokyo, Japan); anti-HLA class I-A, B, C antibody from Hokudo Co., Ltd. (Tokyo, Japan); Histofine Simple Stain MAX PO (M) and (R) kits from Nichirei (Tokyo, Japan); FastPure RNA kit and pBApo-CMV Neo DNA vector from Takara Bio Inc. (Tokyo, Japan); TaqMan Gene Expression Assays for ESRP1 (Hs00214472_m1), CD44s (Hs01081473_m1), CD44v (Hs00153304_m1), E-cadherin (Hs01012953_m1), Vimentin (Hs00185584_m1), HSP 70 (Hs03044880_gH), VCP (Hs00997642_m1), ENO1 (Hs00361415_m1), IQGAP1 (Hs00896595_m1), 14-3-3ε (Hs00356749_g1) FLNA (Hs00924645_m1, Hs00926963_m1), PLIN3 (Hs00998416_m1), and 18S rRNA (Hs99999901_s1), and siRNAs targeting human ESRP1 (s29571, 29572) and negative control siRNA (Silencer select Negative control #2) from Life Technologies Corporation (Carlsbad, CA), We prepared 2 different types of TaqMan primers for filamin alpha: one is specific for a splicing variant and the other is for common site of native filamin alpha and its splicing isoforms.

Techniques: Expressing, Immunostaining, Staining

Journal: Oncogene

Article Title: Epithelial Splicing Regulatory Protein 1 is a Favorable Prognostic Factor in Pancreatic Cancer that Attenuates Pancreatic Metastases

doi: 10.1038/onc.2013.392

Figure Lengend Snippet: A: The overall survival rate of the group exhibiting high ESRP1 immunoreactivity was significantly higher than that of the low-immunoreactivity ESRP1 group ( P =0.0189). B: The disease-free survival rate of the high ESRP1 group tended to higher than that of the low ESRP1 group ( P =0.0767).

Article Snippet: The following were purchased: anti-ESRP1, anti-lamin A+C, anti-Perilipin 3, and anit-IQGAP1 antibodies from Abcam plc (Cambridge, MA); anti-filamin A from Novus Biologicals (Colorado, USA); anti-14-3-3ε from Cell signaling technology (Boston, MA); anti-CD31 antibody from AbD Serotec (Kidlington, UK); anti-cytokeratin (CK) 19 antibody from Boehringer Mannheim GmbH (Mannheim, Germany); anti-insulin antibody from DAKO JAPAN (Tokyo, Japan); anti-HLA class I-A, B, C antibody from Hokudo Co., Ltd. (Tokyo, Japan); Histofine Simple Stain MAX PO (M) and (R) kits from Nichirei (Tokyo, Japan); FastPure RNA kit and pBApo-CMV Neo DNA vector from Takara Bio Inc. (Tokyo, Japan); TaqMan Gene Expression Assays for ESRP1 (Hs00214472_m1), CD44s (Hs01081473_m1), CD44v (Hs00153304_m1), E-cadherin (Hs01012953_m1), Vimentin (Hs00185584_m1), HSP 70 (Hs03044880_gH), VCP (Hs00997642_m1), ENO1 (Hs00361415_m1), IQGAP1 (Hs00896595_m1), 14-3-3ε (Hs00356749_g1) FLNA (Hs00924645_m1, Hs00926963_m1), PLIN3 (Hs00998416_m1), and 18S rRNA (Hs99999901_s1), and siRNAs targeting human ESRP1 (s29571, 29572) and negative control siRNA (Silencer select Negative control #2) from Life Technologies Corporation (Carlsbad, CA), We prepared 2 different types of TaqMan primers for filamin alpha: one is specific for a splicing variant and the other is for common site of native filamin alpha and its splicing isoforms.

Techniques:

Journal: Oncogene

Article Title: Epithelial Splicing Regulatory Protein 1 is a Favorable Prognostic Factor in Pancreatic Cancer that Attenuates Pancreatic Metastases

doi: 10.1038/onc.2013.392

Figure Lengend Snippet: A: Quantitative PCR. PANC-1 cells that were transiently transfected with an ESRP1-expressing vector (PANC-ESRP1) exhibited higher ESRP1 mRNA levels than empty vector-transfected cells (PANC-EV) and wild-type (PANC-1) cells (** P <0.01). PANC-ESRP1 expressed higher levels of FGFR-2 IIIb mRNA than PANC-EV and PANC-1 cells, while FGFR-2 IIIc expression was not altered. B: Western blots. ESRP1 protein was increased in the nuclear fraction of PANC-ESRP1 cell (PANC-ESRP1 nu) as compared to the nuclei of PANC-1 and PANC-EV cells (PANC-1 nu and PANC-EV nu). Lamin A + C, which is a marker of nuclear protein, was not detected in cytoplasm (cyt), and was abundant in the nuclear fraction (nu). β-actin immnoblotting was used to assess protein loading. rb ESRP1 and rb ESRP2 represent recombinant ESRP1 and ESRP2, respectively. C: In phase-contrast images, there were no characteristic morphological changes in PANC-1, PANC-EV and PANC-ESRP1 cells (upper panel). Immunofluorescence images of ESRP1 (green) showed significant increases of ESRP1 protein in PANC-ESRP1 cells as compared with PANC-1 and PANC-EV cells (lower panel). Upper panel: bar: 100µm, inset of lower panel: bar: 50 µm.

Article Snippet: The following were purchased: anti-ESRP1, anti-lamin A+C, anti-Perilipin 3, and anit-IQGAP1 antibodies from Abcam plc (Cambridge, MA); anti-filamin A from Novus Biologicals (Colorado, USA); anti-14-3-3ε from Cell signaling technology (Boston, MA); anti-CD31 antibody from AbD Serotec (Kidlington, UK); anti-cytokeratin (CK) 19 antibody from Boehringer Mannheim GmbH (Mannheim, Germany); anti-insulin antibody from DAKO JAPAN (Tokyo, Japan); anti-HLA class I-A, B, C antibody from Hokudo Co., Ltd. (Tokyo, Japan); Histofine Simple Stain MAX PO (M) and (R) kits from Nichirei (Tokyo, Japan); FastPure RNA kit and pBApo-CMV Neo DNA vector from Takara Bio Inc. (Tokyo, Japan); TaqMan Gene Expression Assays for ESRP1 (Hs00214472_m1), CD44s (Hs01081473_m1), CD44v (Hs00153304_m1), E-cadherin (Hs01012953_m1), Vimentin (Hs00185584_m1), HSP 70 (Hs03044880_gH), VCP (Hs00997642_m1), ENO1 (Hs00361415_m1), IQGAP1 (Hs00896595_m1), 14-3-3ε (Hs00356749_g1) FLNA (Hs00924645_m1, Hs00926963_m1), PLIN3 (Hs00998416_m1), and 18S rRNA (Hs99999901_s1), and siRNAs targeting human ESRP1 (s29571, 29572) and negative control siRNA (Silencer select Negative control #2) from Life Technologies Corporation (Carlsbad, CA), We prepared 2 different types of TaqMan primers for filamin alpha: one is specific for a splicing variant and the other is for common site of native filamin alpha and its splicing isoforms.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation, Western Blot, Marker, Recombinant, Immunofluorescence

Journal: Oncogene

Article Title: Epithelial Splicing Regulatory Protein 1 is a Favorable Prognostic Factor in Pancreatic Cancer that Attenuates Pancreatic Metastases

doi: 10.1038/onc.2013.392

Figure Lengend Snippet: A: Cell proliferation. Cell proliferation was similar in PANC-1, PANC-EV and PANC-ESRP1 cells at all tested time points. B: Single cell movement. PANC-ESRP1 cells movement was significantly decreased as compared with PANC-1 and PANC-EV cells (** P <0.01). bar: 500 µm. C and D: Both migration (C) and invasion (D) were markedly decreased in PANC-ESRP1 cells as compared with PANC-EV cells (** P <0.01). bar: 200µm.

Article Snippet: The following were purchased: anti-ESRP1, anti-lamin A+C, anti-Perilipin 3, and anit-IQGAP1 antibodies from Abcam plc (Cambridge, MA); anti-filamin A from Novus Biologicals (Colorado, USA); anti-14-3-3ε from Cell signaling technology (Boston, MA); anti-CD31 antibody from AbD Serotec (Kidlington, UK); anti-cytokeratin (CK) 19 antibody from Boehringer Mannheim GmbH (Mannheim, Germany); anti-insulin antibody from DAKO JAPAN (Tokyo, Japan); anti-HLA class I-A, B, C antibody from Hokudo Co., Ltd. (Tokyo, Japan); Histofine Simple Stain MAX PO (M) and (R) kits from Nichirei (Tokyo, Japan); FastPure RNA kit and pBApo-CMV Neo DNA vector from Takara Bio Inc. (Tokyo, Japan); TaqMan Gene Expression Assays for ESRP1 (Hs00214472_m1), CD44s (Hs01081473_m1), CD44v (Hs00153304_m1), E-cadherin (Hs01012953_m1), Vimentin (Hs00185584_m1), HSP 70 (Hs03044880_gH), VCP (Hs00997642_m1), ENO1 (Hs00361415_m1), IQGAP1 (Hs00896595_m1), 14-3-3ε (Hs00356749_g1) FLNA (Hs00924645_m1, Hs00926963_m1), PLIN3 (Hs00998416_m1), and 18S rRNA (Hs99999901_s1), and siRNAs targeting human ESRP1 (s29571, 29572) and negative control siRNA (Silencer select Negative control #2) from Life Technologies Corporation (Carlsbad, CA), We prepared 2 different types of TaqMan primers for filamin alpha: one is specific for a splicing variant and the other is for common site of native filamin alpha and its splicing isoforms.

Techniques: Migration

Journal: Oncogene

Article Title: Epithelial Splicing Regulatory Protein 1 is a Favorable Prognostic Factor in Pancreatic Cancer that Attenuates Pancreatic Metastases

doi: 10.1038/onc.2013.392

Figure Lengend Snippet: A: Cell proliferation. ESRP1 siRNA-transfected KLM-1 cells were transfected with siRNA targeting ESRP1 (siESRP1-A and siESRP1-B) and cell proliferation was monitored using the WST-8 cell counting assay. There was a significant increase in cell proliferation in both siESRP1-A and siESRP1-B cells as compared with negative control siRNA-transfected cells (siNeg, * P <0.05). B and C: Migration and invasion. Both migration and invasion were markedly increased in siESRP1-A and siESRP1-B cells as compared with siNeg cells (** P <0.01). bar: 200µm.

Article Snippet: The following were purchased: anti-ESRP1, anti-lamin A+C, anti-Perilipin 3, and anit-IQGAP1 antibodies from Abcam plc (Cambridge, MA); anti-filamin A from Novus Biologicals (Colorado, USA); anti-14-3-3ε from Cell signaling technology (Boston, MA); anti-CD31 antibody from AbD Serotec (Kidlington, UK); anti-cytokeratin (CK) 19 antibody from Boehringer Mannheim GmbH (Mannheim, Germany); anti-insulin antibody from DAKO JAPAN (Tokyo, Japan); anti-HLA class I-A, B, C antibody from Hokudo Co., Ltd. (Tokyo, Japan); Histofine Simple Stain MAX PO (M) and (R) kits from Nichirei (Tokyo, Japan); FastPure RNA kit and pBApo-CMV Neo DNA vector from Takara Bio Inc. (Tokyo, Japan); TaqMan Gene Expression Assays for ESRP1 (Hs00214472_m1), CD44s (Hs01081473_m1), CD44v (Hs00153304_m1), E-cadherin (Hs01012953_m1), Vimentin (Hs00185584_m1), HSP 70 (Hs03044880_gH), VCP (Hs00997642_m1), ENO1 (Hs00361415_m1), IQGAP1 (Hs00896595_m1), 14-3-3ε (Hs00356749_g1) FLNA (Hs00924645_m1, Hs00926963_m1), PLIN3 (Hs00998416_m1), and 18S rRNA (Hs99999901_s1), and siRNAs targeting human ESRP1 (s29571, 29572) and negative control siRNA (Silencer select Negative control #2) from Life Technologies Corporation (Carlsbad, CA), We prepared 2 different types of TaqMan primers for filamin alpha: one is specific for a splicing variant and the other is for common site of native filamin alpha and its splicing isoforms.

Techniques: Transfection, Cell Counting, Negative Control, Migration

Journal: Oncogene

Article Title: Epithelial Splicing Regulatory Protein 1 is a Favorable Prognostic Factor in Pancreatic Cancer that Attenuates Pancreatic Metastases

doi: 10.1038/onc.2013.392

Figure Lengend Snippet: Identification of Differentially Expressed Proteins in Transiently ESRP1-transfected PANC-1 Cells Proteomic analysis revealed that ESRP1-transfected PANC-1 cells transiently transfected to express high ESRP1 levels exhibited decreased expression of vimentin, 14-3-3ε, heat shock protein 70, and IQGAP1, and increased expression of valosin containing protein, α-enolase, filamin-α and perilipin 3

Article Snippet: The following were purchased: anti-ESRP1, anti-lamin A+C, anti-Perilipin 3, and anit-IQGAP1 antibodies from Abcam plc (Cambridge, MA); anti-filamin A from Novus Biologicals (Colorado, USA); anti-14-3-3ε from Cell signaling technology (Boston, MA); anti-CD31 antibody from AbD Serotec (Kidlington, UK); anti-cytokeratin (CK) 19 antibody from Boehringer Mannheim GmbH (Mannheim, Germany); anti-insulin antibody from DAKO JAPAN (Tokyo, Japan); anti-HLA class I-A, B, C antibody from Hokudo Co., Ltd. (Tokyo, Japan); Histofine Simple Stain MAX PO (M) and (R) kits from Nichirei (Tokyo, Japan); FastPure RNA kit and pBApo-CMV Neo DNA vector from Takara Bio Inc. (Tokyo, Japan); TaqMan Gene Expression Assays for ESRP1 (Hs00214472_m1), CD44s (Hs01081473_m1), CD44v (Hs00153304_m1), E-cadherin (Hs01012953_m1), Vimentin (Hs00185584_m1), HSP 70 (Hs03044880_gH), VCP (Hs00997642_m1), ENO1 (Hs00361415_m1), IQGAP1 (Hs00896595_m1), 14-3-3ε (Hs00356749_g1) FLNA (Hs00924645_m1, Hs00926963_m1), PLIN3 (Hs00998416_m1), and 18S rRNA (Hs99999901_s1), and siRNAs targeting human ESRP1 (s29571, 29572) and negative control siRNA (Silencer select Negative control #2) from Life Technologies Corporation (Carlsbad, CA), We prepared 2 different types of TaqMan primers for filamin alpha: one is specific for a splicing variant and the other is for common site of native filamin alpha and its splicing isoforms.

Techniques: Transfection, Expressing

Journal: Oncogene

Article Title: Epithelial Splicing Regulatory Protein 1 is a Favorable Prognostic Factor in Pancreatic Cancer that Attenuates Pancreatic Metastases

doi: 10.1038/onc.2013.392

Figure Lengend Snippet: A: Tumor growth. Pancreatic tumors derived from PANC-EV cells were larger (left panel) and heavier (right panel) than tumors derived from ESRP1-transfected cells, but these differences were not statistically significant (n=3 in each group). Bar: 10 mm. B: Hepatic metastases. ESRP1-2 and ESRP1-5 cells exhibited fewer hepatic metastases by comparison with PANC-EV2 and 9 cells. Consequently, the percentage of human HLA class I positive cancer areas/total liver areas was markedly decreased in ESRP1-2 and ESRP1-5 tumors (** P <0.01). C: Pulmonary metastases. ESRP1-2 and ESRP1-5 cells also exhibited a markedly decreased incidence of pulmonary metastases, as evidenced by the lower percentage of human HLA class I positive cancer areas/total liver areas in ESRP1-2 and ESRP1-5 derived tumors (** P <0.01). bars: 10 mm and 0.1 mm in B; 5 mm and 0.2 mm in C.

Article Snippet: The following were purchased: anti-ESRP1, anti-lamin A+C, anti-Perilipin 3, and anit-IQGAP1 antibodies from Abcam plc (Cambridge, MA); anti-filamin A from Novus Biologicals (Colorado, USA); anti-14-3-3ε from Cell signaling technology (Boston, MA); anti-CD31 antibody from AbD Serotec (Kidlington, UK); anti-cytokeratin (CK) 19 antibody from Boehringer Mannheim GmbH (Mannheim, Germany); anti-insulin antibody from DAKO JAPAN (Tokyo, Japan); anti-HLA class I-A, B, C antibody from Hokudo Co., Ltd. (Tokyo, Japan); Histofine Simple Stain MAX PO (M) and (R) kits from Nichirei (Tokyo, Japan); FastPure RNA kit and pBApo-CMV Neo DNA vector from Takara Bio Inc. (Tokyo, Japan); TaqMan Gene Expression Assays for ESRP1 (Hs00214472_m1), CD44s (Hs01081473_m1), CD44v (Hs00153304_m1), E-cadherin (Hs01012953_m1), Vimentin (Hs00185584_m1), HSP 70 (Hs03044880_gH), VCP (Hs00997642_m1), ENO1 (Hs00361415_m1), IQGAP1 (Hs00896595_m1), 14-3-3ε (Hs00356749_g1) FLNA (Hs00924645_m1, Hs00926963_m1), PLIN3 (Hs00998416_m1), and 18S rRNA (Hs99999901_s1), and siRNAs targeting human ESRP1 (s29571, 29572) and negative control siRNA (Silencer select Negative control #2) from Life Technologies Corporation (Carlsbad, CA), We prepared 2 different types of TaqMan primers for filamin alpha: one is specific for a splicing variant and the other is for common site of native filamin alpha and its splicing isoforms.

Techniques: Derivative Assay, Transfection, Comparison

Journal: iScience

Article Title: Epithelial uptake leads to fungal killing in vivo and is aberrant in COPD-derived epithelial cells

doi: 10.1016/j.isci.2024.109939

Figure Lengend Snippet: Single-cell comparison of uptake and intracellular killing by primary human AECs indicate that COPD-derived AECs are aberrant in their interaction with fungal spores (A) Percentage of AEC i when surveying 8,000 commercially derived cells infected with A1160 +/tdT ( Af tdT ) (MOI 0.5) by IFC. (B) Number of Af spores internalizzed when surveying 8,000 AEC in (A) by IFC. (C) Percentage of AEC i on 8,000 locally sourced cells surveyed by IFC. (D and E) (D) Uptake index by and (E) percentage of intracellular killing within commercially derived AEC i as quantified by live-cell microfluidic imaging. With the exception of (C), AEC tested were purchased from Lonza. For (C), cells were obtained from locally sourced lung resections from two healthy donors (D1 and D2) and two COPD sufferers (D3 and D4) via collaboration with the MFT ManARTS Biobank. For (A)–(D), infections of epithelial monolayers with Af were carried out for 6 h, while in (E) infections were carried out for 20 h. All experiments were conducted in biological duplicates and technical triplicates (ordinary 1-way ANOVA with Holm-Sidak’s multiple comparisons for A–C and unpaired t test for D and E). ∗∗∗∗ p ≤ 0.0001, ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, and ∗ p ≤ 0.05. Data are represented as mean ± SD.

Article Snippet: The epithelial cell line used in this study was the human pulmonary carcinoma epithelial cell line A549 (American type culture collection, CCL-185).

Techniques: Comparison, Derivative Assay, Infection, Imaging

Journal: iScience

Article Title: Epithelial uptake leads to fungal killing in vivo and is aberrant in COPD-derived epithelial cells

doi: 10.1016/j.isci.2024.109939

Figure Lengend Snippet:

Article Snippet: The epithelial cell line used in this study was the human pulmonary carcinoma epithelial cell line A549 (American type culture collection, CCL-185).

Techniques: Purification, Recombinant, Cloning, Southern Blot, Plasmid Preparation, Software, Saline, Red Blood Cell Lysis, Imaging, Flow Cytometry, Microscopy