Journal: Development (Cambridge, England)

Article Title: Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome

doi: 10.1242/dev.204571

Figure Lengend Snippet: Targeted insertion of an ALFA tag using optimized knock-in parameters. (A) Sequence and flanking target insertion sites for foxc1a , prox1a and rasa1a . Left and right homology arms are indicated by magenta and blue, respectively. Black lines denote the Cas9 spacer sequence (line above or below sequence denotes positive or negative strand, respectively) and arrowheads mark the DSB sites. Start or stop codons are underlined. Restriction enzyme sites used to estimate nuclease activity are indicated by boxes and labeled. (B) Camera lucida of a zebrafish embryo at 31 hpf. Reproduced with permission from . (C-E) Whole-mount immunostained embryos imaged by confocal microscopy; lateral views, anterior to the left, dorsal is up. (C) Wild-type embryos immunostained with polyclonal Prox1 antibody. Arrows denote fluorescence in neural tube (nt), lateral line primordium (lat) and muscle pioneers (mp). (D,E) Immunostaining of alfa-prox1a um529 heterozygous (D) and wild-type sibling (E) embryos with a nanobody against ALFA. Scale bar: 80 µm.

Article Snippet: The following antibodies and dilutions were used: sdAb/ALFA-HRP (1:10,000; N1505-HRP, RRID:AB_3075989, NanoTag Biotechnologies), anti-Prox1 polyclonal rabbit antibody (1:1000; 11-002, RRID:AB_10013720, AngioBio) in blocking buffer (PBSTw containing 0.1% Triton X-100/2% DMSO/5% bovine serum albumin/1% goat serum).

Techniques: Knock-In, Sequencing, Activity Assay, Labeling, Confocal Microscopy, Fluorescence, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: PROX1 is an early driver of lineage plasticity in prostate cancer

doi: 10.1172/JCI187490

Figure Lengend Snippet: ( A ) Differentially upregulated genes ranked by adjusted P ( P adj) value in prostate cancer patient tumors that converted to DNPC after enzalutamide treatment are shown from the Westbrook et al. 2022 cohort . PROX1 is the top-ranked gene. ( B – D ) PROX1 mRNA levels were quantified by RNA-Seq in the indicated molecular subtypes of prostate cancer patient tumors from 3 different cohorts: Labrecque et al. 2019 ( n = 98) ( B ), WCDT ( n = 210) ( , ) ( C ), and Beltran et al. 2016 ( n = 49) ( D ). Molecular subtypes ARPC (AR + NE – ), amphicrine (AR + NE + ), AR activity–low (AR low NE – ), DNPC (AR – NE – ), and NEPC (AR – NE + ) are indicated with the sample sizes of each group. Data are reported as the mean ± SD. P values were calculated by unpaired 2-sample Wilcoxon’s test with Benjamini-Hochberg correction for multiple comparison ( B and C ) and unpaired 2-sample Wilcoxon’s test ( D ). * P < 0.05; *** P < 0.001; **** P < 0.0001. ( E and F ) Kaplan-Meier curves represent overall survival probability for patients in the WCDT ( , ) ( E ) or Abida et al. 2019 ( F ) cohort stratified by quantiles of PROX1 expression. Q1 represents the lowest-quartile group, and Q4 represents the highest-quartile group. The log-rank test was used to determine significance.

Article Snippet: The following antibodies were used in sequence at the specified dilutions: PROX1 (1:50; Cell Signaling Technology catalog 14963), PSA (1:50; Cell Signaling Technology, 2475T), INSM1 (1:50; Santa Cruz Biotechnology, sc-271408), and AR (1:100; Cell Signaling Technology, 5153T).

Techniques: RNA Sequencing, Activity Assay, Comparison, Expressing

Journal: The Journal of Clinical Investigation

Article Title: PROX1 is an early driver of lineage plasticity in prostate cancer

doi: 10.1172/JCI187490

Figure Lengend Snippet: ( A and B ) PROX1 mRNA expression was measured in the indicated prostate cancer models using reverse transcriptase quantitative PCR (RT-qPCR). β-Actin served as endogenous housekeeping control. Data are reported as the mean ± SD ( n = 3) ( A ). PROX1 protein expression was measured in the indicated prostate cancer models using Western blotting. AR and PSA served as markers of ARPC. INSM1 served as marker for NEPC. β-Actin served as loading control ( B ). ARPC models are marked in blue, DNPC model in purple, and NEPC models in red text. ( C ) PROX1 mRNA levels were quantified by RNA-Seq in prostate cancer patient-derived xenografts (PDXs) ( n = 114) of the indicated molecular subtypes with their sample sizes (GEO series GSE199596 ). Data are reported as the mean ± SD. Statistical significance was calculated by unpaired 2-sample Wilcoxon’s test with Benjamini-Hochberg correction for multiple comparison. * P < 0.05; **** P < 0.0001. ( D ) PROX1 expression in prostate cancer PDXs was determined by IHC, and representative images with their molecular subtype are shown. Scale bars: 100 μm. ( E ) Expression levels of indicated mRNAs were quantified by RNA-Seq in LTL331 PDXs at different time points during progression from LTL331 (PreCx) to LTL331R (Relapsed). Log 2 transcripts per million (TPM) values are indicated in the heatmap. ( F ) LTL331 progression model tumors were stained by IHC with indicated antibodies before castration (PreCx/LT331), 12 weeks after castration (Cx12wks), or after relapse (Relapsed/LT331R), and representative images are shown. Scale bar: 100 μm.

Article Snippet: The following antibodies were used in sequence at the specified dilutions: PROX1 (1:50; Cell Signaling Technology catalog 14963), PSA (1:50; Cell Signaling Technology, 2475T), INSM1 (1:50; Santa Cruz Biotechnology, sc-271408), and AR (1:100; Cell Signaling Technology, 5153T).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Western Blot, Marker, RNA Sequencing, Derivative Assay, Comparison, Staining

Journal: The Journal of Clinical Investigation

Article Title: PROX1 is an early driver of lineage plasticity in prostate cancer

doi: 10.1172/JCI187490

Figure Lengend Snippet: ( A ) PROX1 promoter methylation in prostate cancer patient tumors was extracted from the Zhao et al. 2020 dataset ( n = 100) , and 5-methylcytosine (5-mC) score is shown. Data are reported as the mean ± SD. PROX1 promoter is significantly hypermethylated in NEPC (AR – NE + ) tumors as indicated by P values calculated by unpaired 2-sample Wilcoxon’s test with Benjamini-Hochberg correction for multiple comparison. ** P < 0.01. ( B ) Scatterplots and linear fitted lines of PROX1 promoter DNA methylation versus log 2 PROX1 expression in samples from the WCDT dataset ( , ). Spearman’s correlation coefficient (ρ) and P values are shown. ( C ) Genome tracks from whole-genome bisulfite sequencing analysis of indicated PDX samples indicate hypermethylation of PROX1 promoter region (highlighted in yellow) in adenocarcinoma PDXs (blue) and hypomethylation in NEPC PDXs (red). ( D ) Methylation-specific PCR (MSPCR) was used to amplify a region of the PROX1 promoter from prostate cancer PDXs and cell models. Methylated (M) and unmethylated (U) specific bands are shown for the indicated samples, which are color-coded: ARPC by blue, amphicrine by orange, AR activity–low by green, DNPC by purple, and NEPC by red. ( E ) The indicated cell lines were treated with 400 nM dAza (decitabine) daily for 5 days. RT-qPCR was performed to quantify PROX1 expression with β-actin used as an endogenous control. Data are reported as the mean ± SD ( n = 3). Statistical significance was calculated with a Student’s t test with Welch’s correction. * P < 0.05. ( F ) MSPCR was performed using DNA extracted from cells treated in E . Ratio of unmethylated (U) to methylated (M) products from densitometry analysis is shown below the respective bands.

Article Snippet: The following antibodies were used in sequence at the specified dilutions: PROX1 (1:50; Cell Signaling Technology catalog 14963), PSA (1:50; Cell Signaling Technology, 2475T), INSM1 (1:50; Santa Cruz Biotechnology, sc-271408), and AR (1:100; Cell Signaling Technology, 5153T).

Techniques: Methylation, Comparison, DNA Methylation Assay, Expressing, Methylation Sequencing, Activity Assay, Quantitative RT-PCR, Control

Journal: The Journal of Clinical Investigation

Article Title: PROX1 is an early driver of lineage plasticity in prostate cancer

doi: 10.1172/JCI187490

Figure Lengend Snippet: ( A – C ) Scatterplots and linear fitted lines of log 2 TPM expression of AR versus PROX1 in the indicated molecular subtypes of prostate cancer samples from Labrecque et al. 2019 ( n = 98) ( A ), WCDT ( n = 210) ( , ) ( B ), and Beltran et al. 2016 ( n = 49) datasets ( C ). Pearson’s correlation coefficient ( R ) and P values are shown. ( D ) Feature plots of PROX1 , INSM1 , AR , and KLK3 expression extracted from scRNA-Seq meta-atlas published in Cheng et al. 2024 . Populations of NEPC (green ovals), KRT7 + DNPC (purple ovals), and progenitor-like DNPC (blue circles) are marked according to the original publication. ( E ) Violin plot showing the expression level of PROX1 across different cell populations in the scRNA-Seq meta-atlas of human prostate cancer published in Cheng et al. 2024 . PROX1 is only highly expressed in NEPC and progenitor-like DNPC populations. ( F ) Expression levels of indicated proteins were measured by Western blots in V16D and LNCaP cells transfected with empty vector (EV) or PROX1 overexpression vector after 72 hours. LASCPC-01 serves as a positive control for PROX1 and INSM1. β-Actin served as loading control.

Article Snippet: The following antibodies were used in sequence at the specified dilutions: PROX1 (1:50; Cell Signaling Technology catalog 14963), PSA (1:50; Cell Signaling Technology, 2475T), INSM1 (1:50; Santa Cruz Biotechnology, sc-271408), and AR (1:100; Cell Signaling Technology, 5153T).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Over Expression, Positive Control, Control

Journal: The Journal of Clinical Investigation

Article Title: PROX1 is an early driver of lineage plasticity in prostate cancer

doi: 10.1172/JCI187490

Figure Lengend Snippet: ( A ) PROX1 knockdown with 2 different doxycycline-inducible (Dox-inducible) shRNAs versus non-targeted control (NC) shRNA was performed in NCI-H660 and LASCPC-01 cells. Cell viability was measured by CCK-8 assays at the indicated time points. Data are reported as the mean ± SD ( n = 6 for NCI-H660, n = 4 for LASCPC-01). PROX1 knockdown efficiency for each shRNA was confirmed by Western blotting. β-Actin served as loading control. For statistical analysis, shNC +Dox was compared with each shRNA +Dox by Student’s t test with Welch’s correction. **** P < 0.0001. ( B ) Apoptosis and EdU assays were performed using cells with Dox (1 μg/mL) treatment for 8 days. Data are reported as the mean ± SD ( n = 3). For statistical analysis, 1-way ANOVA with Dunnett’s multiple-comparison test was performed. * P < 0.05; ** P < 0.01; **** P < 0.0001. ( C ) RT-qPCR was used to measure expression of the indicated NEPC markers ( CHGA and SYP ) or PROX1 in NCI-H660 cells. β-Actin served as housekeeping control. Data are reported as the mean ± SD ( n = 3). For statistical analysis, unpaired t test with Holm-Šidák method for multiple comparison was performed. ** P < 0.01; **** P < 0.0001. ( D ) Western blots were used to measure expression of the indicated NEPC markers (CHGA and SYP) or PROX1 in NCI-H660 cells. β-Actin served as loading control. ( E ) Gene set enrichment analysis was performed on RNA-Seq samples ( n = 2) from Dox-inducible shPROX1 or shNC NCI-H660 cells harvested after 8 days of Dox (1 μg/mL) induction.

Article Snippet: The following antibodies were used in sequence at the specified dilutions: PROX1 (1:50; Cell Signaling Technology catalog 14963), PSA (1:50; Cell Signaling Technology, 2475T), INSM1 (1:50; Santa Cruz Biotechnology, sc-271408), and AR (1:100; Cell Signaling Technology, 5153T).

Techniques: Knockdown, Control, shRNA, CCK-8 Assay, Western Blot, Comparison, Quantitative RT-PCR, Expressing, RNA Sequencing

Journal: The Journal of Clinical Investigation

Article Title: PROX1 is an early driver of lineage plasticity in prostate cancer

doi: 10.1172/JCI187490

Figure Lengend Snippet: ( A ) A PROX1 antibody was used to pull down PROX1 in LASCPC-01 cells, NCI-H660 cells, and an LTL331R PDX tumor. IgG was used as a negative control. Western blots were used to measure the indicated proteins. Input samples were included as endogenous control. ( B ) The indicated cell lines were treated with fimepinostat (left) and romidepsin (right), and cell viability was measured by CellTiter-Glo assays. IC 50 values are shown. Data are reported as the mean ± SD ( n = 4). ( C ) The indicated cell lines were treated with 5 nM fimepinostat, 2 nM romidepsin, or 500 nM entinostat for 48 hours. The indicated proteins were measured by Western blot. Histone H3 serves as loading control. ( D ) Overlapping pathways that change with PROX1 knockdown from or treatment with the HDAC inhibitor fimepinostat (Fime) or romidepsin (Romi) in NCI-H660 cells using RNA-Seq data from Zhang et al. 2023 are presented as a bubble plot. ( E ) NSG mice were implanted with DNPC BCaP-1 PDXs and treated with vehicle, fimepinostat (75 mg/kg orally, 5 times per week), or romidepsin (1.5 mg/kg intraperitoneally, 2 times per week). Tumor volume (top) and mouse body weight (bottom) are shown. Data are reported as the mean ± SEM ( n = 9 for vehicle and romidepsin, n = 6 for fimepinostat). Statistical significance was calculated using 2-way ANOVA with Dunnett’s multiple-comparison test. * P < 0.05. ( F ) Left: Protein was extracted from 4 endpoint PDX tumors from vehicle-, fimepinostat-, or romidepsin-treated mice and probed with the indicated antibodies by Western blot. Right: Quantification of PROX1 protein from the Western blot is presented as a bar plot. Data are reported as the mean ± SEM ( n = 4). Statistical significance was calculated by 1-way ANOVA with Dunnett’s multiple-comparison test. ** P < 0.01.

Article Snippet: The following antibodies were used in sequence at the specified dilutions: PROX1 (1:50; Cell Signaling Technology catalog 14963), PSA (1:50; Cell Signaling Technology, 2475T), INSM1 (1:50; Santa Cruz Biotechnology, sc-271408), and AR (1:100; Cell Signaling Technology, 5153T).

Techniques: Negative Control, Western Blot, Control, Knockdown, RNA Sequencing, Comparison