Journal: Cell reports

Article Title: MCK2-mediated MCMV infection of macrophages and virus dissemination to the salivary gland depends on MHC class I molecules.

doi: 10.1016/j.celrep.2023.112597

Figure Lengend Snippet: Figure 2. Neuropilin 1 (Nrp1) and CX3CR1 do not mediate MCK2-dependent MCMV infection of macrophages (A) Representative flow cytometry histograms plots of Nrp1 levels on NIH/3T3 fibroblasts or RAW 264.7 monocyte/macrophage cells without nucleofection or nucleofected with CRISPR-Cas9 ribonucleoparticles targeting Nrp1 (Nrp1 RNPs). (B) Quantification of mCherry signal at 20 hpi with indicated MCMV strains at MOI of 1 from cells treated with control (Ctrl.) RNPs or Nrp1 RNPs. (C) Representative flow cytometry histograms plots of CX3CR1 levels on NIH/3T3 fibroblasts or RAW 264.7 monocyte/macrophage cells that were nucleofected with Ctrl. or CX3CR1 RNPs. (D) Quantification of mCherry signal at 20 hpi with MCMV-3DR at MOI of 1 from cells treated with Ctrl. RNPs or CX3CR1 RNPs. (E) Quantification of mCherry signal at 20 hpi with MCMV-3D or -3DR at MOI of 1 from alveolar macrophages collected by bronchoalveolar lavage from wild-type (WT) or Cx3cr1/ mice. (B and D) Data are from 3–4 independent experiments. One dot equals a mean of the triplicates from one experiment, line at mean value per group. (E) Data are from 2 experiments with 5–6 animals per group (dots), and line represents mean value per group. (C–E) Statistical analysis: one-way ANOVA test followed by Sidak’s multiple comparison test; ns, not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Liberase Roche Cat#: 11988409001 Trypsin Biochrom AG Cat#: L2103-20 Critical commercial assays SF Cell Line 4D-NucleofectorTM X Kit L Lonza Inc. Cat#: V4XC-2012 SG Cell Line 4D-NucleofectorTM X Kit L Lonza Inc. Cat#: V4XC-3024 QIAamp DNA Mini Kit Qiagen Cat#: 51304 QIAquick PCR Purification Kit Qiagen Cat#: 28104 Mouse Brie CRISPR knockout lentiviral prep Addgene Cat#: 73633-LV Experimental models: Cell lines RAW 264.7 monocytes/macrophages ATCC ATCC TIB-71 NIH/3T3 fibroblasts ATCC ATCC CRL-1658 Primary mouse embryonal fibroblasts (MEF) from BALB/c mice In house N/A Hoxb8-immortalized Cas9-progenitor cells Hammerschmidt et al.43 N/A RMA T cell leukemia cells In house N/A RMA-S T cell leukemia cells In house N/A Experimental models: Organisms/strains Mouse: C57BL/6N Charles River Strain Code: 027 Mouse: BALB/c Charles River Strain Code: 028 Mouse: C3H Charles River Strain Code: 025 Mouse: B6.129P2-B2mtm1Unc/J mice The Jackson Laboratory Strain #:002070; RRID:IMSR_JAX:002070 Mouse: B6.129P2(Cg)-Cx3cr1tm1Litt/J The Jackson Laboratory Strain #:005582; RRID:IMSR_JAX:005582 Oligonucleotides For a list of used crRNAs please see Table S1.

Techniques: Infection, Cytometry, CRISPR, Control, Comparison

Journal: Frontiers in Oncology

Article Title: PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation

doi: 10.3389/fonc.2022.911466

Figure Lengend Snippet: PTEN positively regulates FBP1 in human PCa cell lines and murine prostate tumors. (A, B) WB (A) and RT-PCR (B) were performed in 22Rv1 and DU145 cells infected with lentivirus expressing control or PTEN shRNAs for 48 h. β-Tubulin was used as a WB loading control. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (C, D) WB (C) and RT-PCR (D) were performed in PC-3 and C4-2 cells transfected with pcDNA3.1 or FBP1 expression plasmid for 24 h. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (E, F) WB (E) and RT-PCR (F) were performed in MEFs generated from Pten p/p conditional mice infected with or without infected with lentivirus expressing CMV-driven Cre. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (G) Photos of IHC of Fbp1 and Pten protein in the FFPE prostate tissues of Cre-negative mice (left column) and prostate tumors in Cre-positive mice (right column) at 5 months of age. (H) LNCaP cells were treated with LY294002 at the indicated concentrations for 24 h and then analyzed by WB.

Article Snippet: Pten Loxp/Loxp conditioned knockout mice (129/Balb/c) were originally generated in the laboratory of Dr. Hong Wu at the University of California, Los Angeles, CA , and purchased from Jackson Laboratory (004597).

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, Control, Transfection, Plasmid Preparation, Generated

Journal: Frontiers in Oncology

Article Title: PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation

doi: 10.3389/fonc.2022.911466

Figure Lengend Snippet: Loss of PTEN promotes FBP1 protein ubiquitination and degradation (A, B) WB and quantification of WB bands were carried out in 22Rv1 (A) and DU145 cells (B) infected with shControl or PTEN shRNAs for 48 h and followed by treatment with 50 μg/ml cycloheximide (CHX) for specific intervals of time. At every time point, the level of FBP1 protein was normalized to the level of β-Tubulin (a WB loading control) first and then to the value at the 0-h time point. (C, D) WB (C) and RT-PCR (D) were performed in 22Rv1 cells infected with lentivirus expressing control or PTEN shRNAs for 48 h and further treated with MG132 at 20 μM for 12 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (E, F) WB (E) and RT-PCR (F) were conducted in DU145 cells infected with lentivirus expressing control or PTEN shRNAs for 48 h and exposed with MG132 at 20 μM for another 12 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (G) WB evaluation was carried out using whole-cell lysate and co-IP samples obtained in 293T cells transfected with the indicated constructs and further treatment with 20 μM of MG132 for 12 more hours.

Article Snippet: Pten Loxp/Loxp conditioned knockout mice (129/Balb/c) were originally generated in the laboratory of Dr. Hong Wu at the University of California, Los Angeles, CA , and purchased from Jackson Laboratory (004597).

Techniques: Ubiquitin Proteomics, Infection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Co-Immunoprecipitation Assay, Transfection, Construct

Journal: Frontiers in Oncology

Article Title: PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation

doi: 10.3389/fonc.2022.911466

Figure Lengend Snippet: SKP2 mediates FBP1 protein ubiquitination and degradation induced by PTEN loss (A, B) WB (A) and quantitative RT-PCR (B) analyses were carried out in 22Rv1 and DU145 cells infected with lentivirus expressing control or PTEN-specific shRNAs for 48 hours. β-Tubulin was used as a WB loading control. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. *P < 0.05, **P<0.01, ***P<0.001. (C, D) WB (C) and quantitative RT-PCR (D) analyses were performed in 22Rv1 and DU145 cells infected with lentivirus expressing control or SKP2-specific shRNAs for 48 hours. β-Tubulin was used as a WB loading control. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (E, F) WB (E) and quantitative RT-PCR (F) were conducted using samples obtained from 22Rv1 and DU145 cells transfected with pcDNA3.1 or HA-SKP2 expression plasmid for 24 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (G, H) WB and quantification of WB bands were carried out in 22Rv1 (G) and DU145 cells (H) infected with shControl or SKP2-specific shRNAs for 48 hours and treated with 50 μg/ml cycloheximide (CHX) for different periods of time. At each time point, the intensity of FBP1 WB band was normalized to the intensity of β-Tubulin (a WB loading control) first and then to the value at the 0-h time point. (I, J) Western blot (I) and quantitative RT-PCR (J) analyses were conducted in 22Rv1 and DU145 cells infected with lentivirus expressing the indicated shRNAs for 48 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant.

Article Snippet: Pten Loxp/Loxp conditioned knockout mice (129/Balb/c) were originally generated in the laboratory of Dr. Hong Wu at the University of California, Los Angeles, CA , and purchased from Jackson Laboratory (004597).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Infection, Expressing, Control, Transfection, Plasmid Preparation, Western Blot

Journal: Frontiers in Oncology

Article Title: PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation

doi: 10.3389/fonc.2022.911466

Figure Lengend Snippet: CDK inhibitor blocks FBP1 degradation induced by PTEN loss (A) WB was carried out using the whole-cell lysate and co-IP samples obtained from 293T cells transfected with the indicated constructs. β-Tubulin was used as a WB loading control. (B, C) Western blot (B) and quantitative RT-PCR (C) analysis in 22Rv1 and DU145 cells infected with lentivirus expressing control or PTEN-specific shRNAs for 48 hours and treated with Roscovitine for 24 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (D, E) WB (D) and quantitative RT-PCR (E) were carried out in MEFs generated from Pten p/p conditional mice infected with or without lentivirus expression CMV-driven Cre and treated with Roscovitine for 24 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (F, G) WB and quantification of WB bands were performed in 22Rv1 (F) and DU145 cells (G) treated with Roscovitine for 48 hours followed by treatment with 50 μg/ml cycloheximide (CHX) for different periods of time. At each time point, the intensity of FBP1 was normalized to the intensity of β-Tubulin (a WB loading control) first and then to the value at the 0-h time point.

Article Snippet: Pten Loxp/Loxp conditioned knockout mice (129/Balb/c) were originally generated in the laboratory of Dr. Hong Wu at the University of California, Los Angeles, CA , and purchased from Jackson Laboratory (004597).

Techniques: Co-Immunoprecipitation Assay, Transfection, Construct, Control, Western Blot, Quantitative RT-PCR, Infection, Expressing, Generated

Journal: Frontiers in Oncology

Article Title: PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation

doi: 10.3389/fonc.2022.911466

Figure Lengend Snippet: Serine 271 phosphorylation is important for FBP1 degradation induced by PTEN loss (A) Amino acid sequence alignment among CDK phosphorylation consensus motif, those identified in known CDK substrates, and the putative CDK phosphorylation site (serine 271) on FBP1. (B) WB analysis in the whole cell lysate and co-IP samples in 293T cells transfected with indicated plasmids or infected with indicated lentivirus for 48 hours. (C, D) WB (C) and quantitative RT-PCR (D) analyses were performed in 22Rv1 and DU145 cells infected with lentivirus expressing control or PTEN-specific shRNAs and/or transfected with plasmid for Flag-tagged FBP1 WT or S271A for 48 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (E) WB analysis was carried out using the whole cell lysate and co-IP samples in 293T cells transfected with indicated plasmids or infected with indicated lentivirus for 48 hours.

Article Snippet: Pten Loxp/Loxp conditioned knockout mice (129/Balb/c) were originally generated in the laboratory of Dr. Hong Wu at the University of California, Los Angeles, CA , and purchased from Jackson Laboratory (004597).

Techniques: Phospho-proteomics, Sequencing, Co-Immunoprecipitation Assay, Transfection, Infection, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation

doi: 10.3389/fonc.2022.911466

Figure Lengend Snippet: A hypothetical model Left: Stabilization of FBP1 protein prevents over-activation of the Warburg effect in normal cells. Right: Aberrant activation of the PI3K/AKT pathway due to deregulation of signaling such as loss of PTEN leads to CDK-dependent phosphorylation and ubiquitination and degradation of FBP1 mediated by SKP2 E3 ubiquitin ligase, thereby leading to abnormal activation of the Warburg effect and cell growth of cancer such as PCa. P letter with circle represents protein phosphorylation.

Article Snippet: Pten Loxp/Loxp conditioned knockout mice (129/Balb/c) were originally generated in the laboratory of Dr. Hong Wu at the University of California, Los Angeles, CA , and purchased from Jackson Laboratory (004597).

Techniques: Activation Assay, Phospho-proteomics, Ubiquitin Proteomics