Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) GSEA plots of transmembrane RTK-related gene sets enriched in PlexinD1-knockdown vs. control 22Rv1 cells, and PLXND1 -high vs. -low PCa patient samples in TCGA cohort. ( B ) Representative images of a RTK phosphorylation antibody array and quantification of p-ErbB3 levels in control and PlexinD1-knockdown C4-2B ENZR cells ( n = 2 technical replicates). The raw values from measurement of p-ErbB3 spot intensity after background subtraction are presented. ( C ) Western blot of p-ErbB3, p-ErbB2, p-cMet, p-ERK, p-AKT, and their total protein forms in control and PlexinD1-knockdown C4-2B ENZR and 22Rv1 cells. ( D ) qPCR of ErbB3 ( NRG1 and NRG2 ) and cMet ( HGF ) ligand mRNA levels in control and PlexinD1-knockdown C4-2B ENZR and 22Rv1 cells ( n = 3 biological replicates). ( E ) Representative PLA images and quantification of PlexinD1-ErbB3/cMet interaction by per-cell cytoplasmic fluorescence intensity in C4-2B ENZR cells treated with recombinant Sema3E/Sema3C proteins (200–500 ng/ml, 4 h) or SEMA3E / SEMA3C siRNA (10 μM, 48 h). PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 μm. ( F ) Co-IP assays of PlexinD1-ErbB3/cMet interaction in 22Rv1 cells. IgG was used in the IP step as negative control. Five percent of input was blotted as positive control. ( G ) Cell proliferation assays of 22Rv1 cells pre-treated with an ErbB3 neutralizing antibody (100 ng/ml, 24 h) or SGX-523 (5 µM, 24 h) and then subjected to PLXND1 siRNA addition followed by a 5-day observation period, and control and PlexinD1-overexpressing LNCaP cells upon treatment with an ErbB3 neutralizing antibody or SGX-523 during a 5-day observation period. Data represent the fold changes of cell proliferation on day 5 relative to siRNA treatment day (day 1) for 22Rv1 cells or treatment day (day 1) for LNCaP cells ( n = 3 biological replicates), with fold changes in non-treated groups of control cells set as 1 for normalization of other groups. ( H ) qPCR of stemness, basal, and NE markers as indicated in 22Rv1 cells receiving an ErbB3 neutralizing antibody (100 ng/ml, 24 h), SGX-523 (5 µM, 24 h), or transient transfection of ErbB3/cMet expression plasmids followed by PLXND1 siRNA treatment for another 48 h ( n = 3 biological replicates). ( I ) qPCR of stemness, basal, NE, and EMT markers as indicated in control and PlexinD1-overexpressing LNCaP cells upon treatment with 100 ng/ml ErbB3 neutralizing antibody or 5 µM SGX-523 for 24 h ( n = 3 biological replicates). Data information: In ( B , D , E , G , H , I ), data are presented as mean ± SEM. In ( A ), P values were determined by permutation test. In ( B , D ), P values were determined by unpaired two-tailed Student’s t -test. In ( E ), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. In ( G , H , I ), P values were determined by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human BRN2 siRNA , Santa Cruz Biotechnology , sc-29837.

Techniques: Knockdown, Control, Phospho-proteomics, Ab Array, Western Blot, Fluorescence, Recombinant, Incubation, Co-Immunoprecipitation Assay, Negative Control, Positive Control, Transfection, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) IPA upstream regulator analysis of RNA-seq data from PlexinD1-knockdown vs. control C4-2B ENZR cells, with significantly downregulated hits shown (absolute value of z-score of ≥2, p < 0.05). ( B ) STRING analysis of Gli1 linked to multiple stemness, NE, and EMT marker and/or driver genes. ( C ) Western blot of Gli1 in control and PlexinD1-manipulated PCa cells. ( D ) Determination of Gli-Luc reporter activity in control and PlexinD1-manipulated PCa cells ( n = 3 biological replicates). ( E ) qPCR of Gli1 target genes in control and PlexinD1-knockdown C4-2B ENZR cells ( n = 3 biologial replicates). ( F ) qPCR of Hh ligands in control and PlexinD1-knockdown C4-2B ENZR and 22Rv1 cells ( n = 3 biological replicates). ( G ) Determination of Gli-Luc reporter activity in 22Rv1 cells receiving an ErbB3 neutralizing antibody (100 ng/ml, 24 h), SGX-523 (5 µM, 24 h), or transient transfection of ErbB3/cMet expression plasmids followed by PLXND1 siRNA treatment for another 48 h ( n = 3 biological replicates), and in control and PlexinD1-overexpressing LNCaP cells upon treatment with 100 ng/ml ErbB3 neutralizing antibody or 5 µM SGX-523 for 24 h ( n = 3 biological replicates). ( H ) Representative images of Gli1 (red color) and α-tublin (a cytoplasm marker, green color) co-IF images and quantification of Gli1+ cells in the nuclei of control and PlexinD1-manipulated 22Rv1 and LNCaP cells under the conditions as in ( G ) ( n = 3 biological replicates). Scale bars: 10 µm. ( I ) Western blot of Gli1 in nuclear and cytoplasmic fractions of control and PlexinD1-manipulated 22Rv1 and LNCaP cells under the conditions as in ( G ). ( J ) Cell proliferation assays of control and PlexinD1-overexpressing LNCaP cells upon treatment with 10 µM cyclopamine or 5 µM GANT61 for 5 days ( n = 3 biological replicates). ( K ) Cell proliferation assays of C4-2B ENZR and 22Rv1 cells upon treatment with 10 µM cyclopamine or 5 µM GANT61 for 5 days ( n = 3 biological replicates). ( L ) qPCR of stemness, NE and EMT markers in control and PlexinD1-overexpressing LNCaP cells under treatment with GANT61 (5 µM, 24 h) ( n = 3 biological replicates). ( M ) GSEA plots of two Hh signaling-related gene sets enriched in PLXND1 -high vs. -low PCa patient samples from TCGA cohort. ( N ) Chi-square analysis of distribution of select Gli1 target gene mRNA expression (L, low; H, high) in PLXND1 -low and -high PCa patient samples from TCGA cohort. Data information: In ( D – H , J – L ), data are presented as mean ± SEM. In ( A ), P values were determined by Fisher’s exact test. In ( D – F , K ), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test [for C4-2B ENZR and 22Rv1 in ( D )] and unpaired two-tailed Student’s t -test [for LNCaP in ( D )]. In ( G , H , J , L ), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. In ( M ), P values were determined by permutation test. In ( N ), P values were determined by chi-square test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human BRN2 siRNA , Santa Cruz Biotechnology , sc-29837.

Techniques: RNA Sequencing, Knockdown, Control, Marker, Western Blot, Activity Assay, Transfection, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human BRN2 siRNA , Santa Cruz Biotechnology , sc-29837.

Techniques: Recombinant, Sequencing, Control, Transfection, Membrane, Reverse Transcription, Software, Microarray, Subcloning, shRNA, Cell Viability Assay, Viability Assay, Reporter Assay, In Situ, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Extraction, Bicinchoninic Acid Protein Assay, Magnetic Beads, Gel Extraction, Purification, Ligation, Mutagenesis, Chromatin Immunoprecipitation

Journal: Cell reports

Article Title: BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis

doi: 10.1016/j.celrep.2022.111826

Figure Lengend Snippet: (A) Schematic of the drug screen design. (B) Percentages of dead PI-positive cells in cells treated for 24h with indicated drugs in the presence or absence of BCLi navitoclax. Median values from 6 quantified images are shown per each condition. N=6. (C) Representative microscopy images from the library screen shown in B. Numbers in the top left corners indicate the percentages of apoptotic cells. Scale bar 275μm. (D) Identification of drug screen hits. Drugs that, combined with BCLi, induced at least double the percentage of cell death induced by BCLi alone (indicated by the punctate line) were designated as “BCLi-enhanced.”, and all others as “Unaffected.” Statistics using 2-way ANOVA with Sidak’s multiple comparison test. (E) Pairwise comparison of the percentages of senescent cells with and without BCLi addition. Statistical analysis using paired t-test. (F) Comparison of the percentages of senescent cells induced by BCLi-enhanced and unaffected drugs with and without BCLi. Statistics as in D. (G) Drug categories enriched in BCLi-enhanced and unaffected drug groups. (H) Representative images of crystal violet-stained melanoma cells treated with 1μM navitoclax (BCLi), 1μM alisertib (AURKAi), or both drugs combined for 24 h. Scale bar 200 μm. (I) Cell count from the experiment in H. Data is shown as percentages of cells relative to vehicle control. Statistical comparisons using one-way ANOVA with Dunnett’s post-test. N=3. (J) Representative images of crystal violet-stained A375 cells after 24h treatment with vehicle, 1μM navitoclax (BCLi), 1μM paclitaxel, or both drugs combined for 24 h. Scale bar 50μm. Cell quantification relative to vehicle-treated cells is shown on right. Statistical comparisons using one-way ANOVA with Tukey’s test. N=9 random fields from 3 individual wells (3 images/well). (K) Representative images of crystal violet-stained A375 cells after 24h treatment with 1μM navitoclax (BCL-2/xLi), 1μM venetoclax (BCL-2i), 1μM A-1155463 (BCL-xLi), 1μM A-1210477 (MCL-1i) in the presence or absence of 1μM alisertib (AURKAi). Scale bar 150μm. (L) Relative cell numbers from the experiment in K. Data are presented in percentages relative to untreated cells (Vehicle/-) with statistical analysis using 2-way ANOVA with Sidak’s post-test. Experiment was repeated three times with consistent results. (M) Left: representative images of crystal violet-stained Hs294T cells transfected with AURKA or non-targeting siRNA and treated with 1μM navitoclax (BCL-2/xLi) for 24h. Scale bar 275μm. Experiment was performed with three biological replicates. Middle: quantification from 6 random fields per condition and one-way ANOVA statistical analysis with Sidak’s post-test. Right: efficiency of target knockdown determined by western. (N) Same as M, except BCL-xL-targeting siRNA was used and cells were treated with 1μM alisertib (AURKAi) for 24h. All panels: Ns P > 0.05, * P≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. When applicable, p-values were adjusted for multiple comparisons. See also Fig. S1.

Article Snippet: Silencer AURKA siRNA , Cell signaling , 8883S.

Techniques: Microscopy, Comparison, Staining, Cell Counting, Control, Transfection, Knockdown, Western Blot

Journal: Cell reports

Article Title: BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis

doi: 10.1016/j.celrep.2022.111826

Figure Lengend Snippet: (A) Western blot of p21 in A375 melanoma cells treated with 1μM navitoclax (BCL-2/xLi), 1μM venetoclax (BCL-2i), 1μM A-1155463 (BCL-xLi), 1μM A-1210477 (MCL-1i) in the presence or absence of 1μM alisertib (AURKAi) for 24hrs. Experiment was repeated three times with consistent results. (B) Real-time PCR of relative CDKN1A (p21) mRNA expression in A375 cells after 24hrs of treatment with vehicle, 1μM navitoclax (BCLi), 1μM alisertib (AURKAi), or combination of both drugs. The experiment was repeated three times with 2 replicates each. Statistical comparison using one-way ANOVA with Tukey’s post- test. (C) Western blot analysis of p21 degradation. A375 melanoma cells were treated as in B for 16hrs followed by the addition of Cycloheximide (150μg/ml) ± MG-132 (1μM). Cell lysates were collected at indicated time points (0.25h – 4h). P21 bands from 3 independent experiments were quantified using densitometry and normalized to corresponding actin bands and plotted on the graph below. The square root transformation was used to correct for heteroscedasticity. Statistical analysis using 2-way ANOVA with Tukey’s post-test. (D) Western blot in A375 cells treated as in B for 24hrs in the absence or presence of pan-caspase inhibitor Z-VAD-FMK (20 μg/ml). Three independent experiments were performed with consistent results. (E) Western blot analysis of p21 knockdown. (F) Representative images of Hs294T cells transfected with p21-targeting or non-targeting siRNA, treated with vehicle or 1μM alisertib (AURKAi) for 24hrs, and stained with Hoechst and PI. Scale bar 50μm. Experiment was performed with 3 biological replicates. Right: data quantification from 6 random fields (n=6) and statistics by 2-way ANOVA with Sidak’s post-test. (G) Western blot analysis of cleaved PARP in Hs294T cells treated as in F. (H) Cristal violet staining in Hs294T cells treated as in F. Statistical analysis as in F. (I) Representative images and quantification of crystal violet-stained A375 cells treated as in B for 24hrs in serum-containing (+ serum, control culture condition) and serum-free (- serum, cell cycle-arresting conditions) media. Scale bar 200μm. Statistical comparisons using one-way ANOVA with Sidak’s post-test. N=3 biological replicates. (J) Western blot analysis of cleaved caspase 3 in A375 cells treated as described in I. Experiment was repeated twice with consistent results. (K) Western blot of BAX protein expression in A375 cells treated with vehicle, 1μM navitoclax (BCLi), 1μM alisertib (AURKAi), or their combination for 16hrs. Three independent experiments were performed with consistent results. (L) Real-time PCR analysis of BAX mRNA in A375 cells treated as in B for 24hrs. Experiment was repeated two times with two replicates each. Statistical analysis using one-way ANOVA with Tukey’s post-test. N=4. (M) Representative images of crystal violet-stained A375 cells transfected with non-targeting and BAX-targeting siRNA. Scale bar 150μm. Two independent experiments were performed with consistent results. Right: quantified cell numbers and statistical comparison using one-way ANOVA with Sidak’s post-test. N=6. (N) Western blot for BAX siRNA efficiency. (O-P) Same as M and N, except BAK-specific siRNA was used. All panels: Ns - P > 0.05, * - P≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001. P-values were adjusted for multiple comparisons. See also Fig. S6.

Article Snippet: Silencer AURKA siRNA , Cell signaling , 8883S.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Comparison, Transformation Assay, Knockdown, Transfection, Staining, Control

Journal: Cell reports

Article Title: BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis

doi: 10.1016/j.celrep.2022.111826

Figure Lengend Snippet: (A) RNAseq of tumors shown in Fig. 4A. The heat map of genes modulated by either combined AURKAi and BCLi treatment or corresponding individual treatments. N=3. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. (B) Results of the principal component analysis of gene expression data sin A. (C) Top 10 KEGG pathways enriched within the genes differentially expressed after combo (top panel) and alisertib (bottom panel) treatments. Log10-transformed Benjamini-adjusted p-values were plotted. (D) The heat map shows relative mRNA expression of genes within the KEGG p53 signaling pathway across. “Row max” corresponds to the highest expression value of a protein in this row across all tested samples. “Row min” is expression in the sample with lowest expression value. (E) Western blot analysis of p53 protein in melanoma cells treated with vehicle, 1μM navitoclax (BCLi), 1μM alisertib (AURKAi) or combination for 24 hrs. Three independent experiments were performed with consistent results. (F) Western blot shows the efficiency of p53 knockdown/knockout. (G) Representative images and quantificationof crystal violet-stained Hs294T melanoma cells transfected with p53 siRNA or non-targeting siRNA and treated as described in E. Scale bar 200μm. N=3. Statistical analysis using one-way ANOVA with Sidak’s post- test. Two independent experiments were performed with consistent results. (H) Same as G, except A375 cells were used. (I) Representative images and quantification of crystal violet-stained HCT-116 wild-type and p53-null cells treated as in E. Scale bar 200μm. Statistical analysis as in G. (J) Representative images and quantification of Sk-Mel-28 cells with mutated TP53 treated as in E. Scale bar 200μm. Statistical analysis using one-way ANOVA with Dunnett’s test. (All panels) Ns - P > 0.05, * - P≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001. P-values were adjusted for multiple comparisons.

Article Snippet: Silencer AURKA siRNA , Cell signaling , 8883S.

Techniques: Gene Expression, Transformation Assay, Expressing, Western Blot, Knockdown, Knock-Out, Staining, Transfection

Journal: Cell reports

Article Title: BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis

doi: 10.1016/j.celrep.2022.111826

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Silencer AURKA siRNA , Cell signaling , 8883S.

Techniques: Virus, Recombinant, Modification, Staining, Membrane, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Transfection, Saline, Protein Array, Control, Software

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) GSEA of multiple cellular event and plasticity related gene sets enriched in C4-2B ENZR vs. LNCaP cells. ( B ) Dot plot depicting the axon guidance pathway among KEGG pathways highly enriched in C4-2B ENZR vs. LNCaP cells. ( C ) Volcano plot of axon guidance regulator genes detected in C4-2B ENZR vs. LNCaP cells ( n = 2 biological replicates). ( D ) GSEA plots of two semaphorin/plexin signaling-related gene sets enriched in C4-2B ENZR vs. LNCaP cells. ( E ) Western blot of PlexinD1, Sema3E, Sema3C, and NE and AR signaling markers in LNCaP and C4-2B ENZR cells. ( F ) Representative PLA images and quantification of PlexinD1-Sema3E/Sema3C interaction by per-cell cytoplasmic fluorescence intensity in LNCaP and C4-2B ENZR cells. PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 µm. ( G ) Representative Western blotting images and quantification of PlexinD1, Sema3E, and Sema3C protein expression in a panel of cell lines as indicated, with the averaged individual protein levels after normalization to β-actin in RWPE-1 cells from three independent experiments set as 1. Note that unequal amounts of total cell lysates from different cell lines were loaded intentionally due to the large discrepancy in target protein levels among different cell lines. Data information: In ( F ), data are presented as mean ± SEM. In ( A , D ), P values were determined by permutation test. In ( C ), P values were determined by unpaired two-tailed Student’s t -test. In ( F ), P values were determined by one‐way ANOVA with Dunnett’s multiple comparisons test. ** P < 0.01. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3C siRNA , Santa Cruz Biotechnology , sc-44091.

Techniques: Western Blot, Fluorescence, Incubation, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) qPCR of PLXND1 , SEMA3C, SEMA3E, KLK3 , and NCAM1 in LNCaP, LAPC4, and VCaP cells upon culture under CSS condition for 24 h followed by R1881 stimulation (10 nM, 24 h) ( n = 3 biological replicates). ( B , C ) Western blot of PlexinD1, AR, and PSA in LNCaP and LAPC4 cells upon treatment with 10 nM R1881 ( B ) or 10 µM ENZ ( C ) for the indicated times. ( D ) Genomic browser representation of AR binding at PLXND1 promoter encompassing three AREs, with the nucleotides identical to the canonical ARE highlighted in red, by interrogating AR ChIP-seq dataset GSE125245. ( E ) ChIP-qPCR of AR and H3K9ac occupancy at an ARE-centric PLXND1 promoter sequence as well as an AR-bound KLK3 promoter region upon R1881 stimulation (10 nM, 6 h) in LNCaP cells. Data represent the percent of input ( n = 3 technical replicates). ( F ) Schematic diagrams of WT and mutated forms of individual AREs in PLXND1 ARE-Luc constructs. ( G ) Determination of WT and mutated PLXND1 ARE-Luc activities upon R1881 stimulation (10 nM, 6 h) in LNCaP cells ( n = 3 biological replicates). ( H ) Pearson correlation analysis of PLXND1 mRNA expression with AR score in the indicated datasets from cBioPortal. ( I ) Pearson correlation analysis of mRNA co-expression between PLXND1 vs. different AR target genes in the indicated datasets from cBioPortal. Data information: In ( A , E , G ), data are presented as mean ± SEM. In ( A ), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. In ( E ), P values were determined by two-way ANOVA with Tukey’s multiple comparisons test. In ( G ), P values were determined by unpaired two-tailed Student’s t -test. In ( H ), P values were determined by Pearson’s correlation coefficient t -test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3C siRNA , Santa Cruz Biotechnology , sc-44091.

Techniques: Western Blot, Binding Assay, ChIP-sequencing, ChIP-qPCR, Sequencing, Construct, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) GSEA plots of transmembrane RTK-related gene sets enriched in PlexinD1-knockdown vs. control 22Rv1 cells, and PLXND1 -high vs. -low PCa patient samples in TCGA cohort. ( B ) Representative images of a RTK phosphorylation antibody array and quantification of p-ErbB3 levels in control and PlexinD1-knockdown C4-2B ENZR cells ( n = 2 technical replicates). The raw values from measurement of p-ErbB3 spot intensity after background subtraction are presented. ( C ) Western blot of p-ErbB3, p-ErbB2, p-cMet, p-ERK, p-AKT, and their total protein forms in control and PlexinD1-knockdown C4-2B ENZR and 22Rv1 cells. ( D ) qPCR of ErbB3 ( NRG1 and NRG2 ) and cMet ( HGF ) ligand mRNA levels in control and PlexinD1-knockdown C4-2B ENZR and 22Rv1 cells ( n = 3 biological replicates). ( E ) Representative PLA images and quantification of PlexinD1-ErbB3/cMet interaction by per-cell cytoplasmic fluorescence intensity in C4-2B ENZR cells treated with recombinant Sema3E/Sema3C proteins (200–500 ng/ml, 4 h) or SEMA3E / SEMA3C siRNA (10 μM, 48 h). PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 μm. ( F ) Co-IP assays of PlexinD1-ErbB3/cMet interaction in 22Rv1 cells. IgG was used in the IP step as negative control. Five percent of input was blotted as positive control. ( G ) Cell proliferation assays of 22Rv1 cells pre-treated with an ErbB3 neutralizing antibody (100 ng/ml, 24 h) or SGX-523 (5 µM, 24 h) and then subjected to PLXND1 siRNA addition followed by a 5-day observation period, and control and PlexinD1-overexpressing LNCaP cells upon treatment with an ErbB3 neutralizing antibody or SGX-523 during a 5-day observation period. Data represent the fold changes of cell proliferation on day 5 relative to siRNA treatment day (day 1) for 22Rv1 cells or treatment day (day 1) for LNCaP cells ( n = 3 biological replicates), with fold changes in non-treated groups of control cells set as 1 for normalization of other groups. ( H ) qPCR of stemness, basal, and NE markers as indicated in 22Rv1 cells receiving an ErbB3 neutralizing antibody (100 ng/ml, 24 h), SGX-523 (5 µM, 24 h), or transient transfection of ErbB3/cMet expression plasmids followed by PLXND1 siRNA treatment for another 48 h ( n = 3 biological replicates). ( I ) qPCR of stemness, basal, NE, and EMT markers as indicated in control and PlexinD1-overexpressing LNCaP cells upon treatment with 100 ng/ml ErbB3 neutralizing antibody or 5 µM SGX-523 for 24 h ( n = 3 biological replicates). Data information: In ( B , D , E , G , H , I ), data are presented as mean ± SEM. In ( A ), P values were determined by permutation test. In ( B , D ), P values were determined by unpaired two-tailed Student’s t -test. In ( E ), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. In ( G , H , I ), P values were determined by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3C siRNA , Santa Cruz Biotechnology , sc-44091.

Techniques: Knockdown, Control, Phospho-proteomics, Ab Array, Western Blot, Fluorescence, Recombinant, Incubation, Co-Immunoprecipitation Assay, Negative Control, Positive Control, Transfection, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) Graphic depicting D1SP as a recombinant PlexinD1 decoy protein. ( B ) Western blot of D1SP in whole cell lysate (WCL) and conditioned medium (CM) of D1SP-expressing CHO-K1 cells using a hIgGFc-specific antibody. ( C ) Flow cytometric analysis of binding of IgG-PE antibody conjugated D1SP at various doses in 22Rv1 cells ( n = 3 biological replicates). ( D ) Representative PLA images and quantification of PlexinD1-Sema3E/Sema3C interaction by per-cell cytoplasmic fluorescence intensity in 22Rv1 cells treated with D1SP (1 µM, 2 h) or PBS as a vehicle. PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 µm. ( E ) Cell viability assays of C4-2B ENZR and 22Rv1 cells stimulated with 200 ng/µl recombinant Sema3E protein and then subjected to treatment with 1 µM D1SP for 7 days ( n = 3 biological replicates). ( F ) Transwell-based cell migration and invasion assays of C4-2B ENZR and 22Rv1 cells under 1 µM D1SP treatment ( n = 3 biological replicates). Scale bars: 400 µm. ( G ) Representative brightfield and fluorescence microscopic images and quantification of LuCaP 147CR, 49 and 173.1 PCa PDX-derived live organoids after incubation with 1 µM D1SP or PBS as a vehicle for 10 days ( n = 3 biological replicates). Scale bars: 100 µm. ( H – J ) Tumor growth curves ( H ), endpoint tumor weights ( I ), and anatomic tumor images ( J ) of s.c. 22Rv1 xenografts grown in male nude mice ( n = 6 tumors) and receiving intratumoral injection of D1SP (30 µg/tumor, 2–3 times per week) and saline on the right and left flanks of mice, respectively. ( K , L ) Representative images ( K ) and quantification ( L ) of IHC staining of Ki-67, p-ErbB3, p-ErbB2, and p-cMet ( n = 6 tumors) in control and D1SP-treated 22Rv1 tumors. Scale bars: 100 μm. ( M ) qPCR of select Gli1 target genes in control and D1SP-treated 22Rv1 tumors ( n = 6 tumors). Data information: In ( C – H ), data are presented as mean ± SEM. In ( C , D ), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. In ( E ), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. In ( F – H ), P values were determined by unpaired two-tailed Student’s t -test. In ( I , L , M ), P values were determined by paired two-tailed Student’s t -test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3C siRNA , Santa Cruz Biotechnology , sc-44091.

Techniques: Recombinant, Western Blot, Expressing, Binding Assay, Fluorescence, Incubation, Migration, Derivative Assay, Injection, Saline, Immunohistochemistry, Control, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human SEMA3C siRNA , Santa Cruz Biotechnology , sc-44091.

Techniques: Recombinant, Sequencing, Control, Transfection, Membrane, Reverse Transcription, Software, Microarray, Subcloning, shRNA, Cell Viability Assay, Viability Assay, Reporter Assay, In Situ, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Extraction, Bicinchoninic Acid Protein Assay, Magnetic Beads, Gel Extraction, Purification, Ligation, Mutagenesis, Chromatin Immunoprecipitation

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) GSEA of multiple cellular event and plasticity related gene sets enriched in C4-2B ENZR vs. LNCaP cells. ( B ) Dot plot depicting the axon guidance pathway among KEGG pathways highly enriched in C4-2B ENZR vs. LNCaP cells. ( C ) Volcano plot of axon guidance regulator genes detected in C4-2B ENZR vs. LNCaP cells ( n = 2 biological replicates). ( D ) GSEA plots of two semaphorin/plexin signaling-related gene sets enriched in C4-2B ENZR vs. LNCaP cells. ( E ) Western blot of PlexinD1, Sema3E, Sema3C, and NE and AR signaling markers in LNCaP and C4-2B ENZR cells. ( F ) Representative PLA images and quantification of PlexinD1-Sema3E/Sema3C interaction by per-cell cytoplasmic fluorescence intensity in LNCaP and C4-2B ENZR cells. PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 µm. ( G ) Representative Western blotting images and quantification of PlexinD1, Sema3E, and Sema3C protein expression in a panel of cell lines as indicated, with the averaged individual protein levels after normalization to β-actin in RWPE-1 cells from three independent experiments set as 1. Note that unequal amounts of total cell lysates from different cell lines were loaded intentionally due to the large discrepancy in target protein levels among different cell lines. Data information: In ( F ), data are presented as mean ± SEM. In ( A , D ), P values were determined by permutation test. In ( C ), P values were determined by unpaired two-tailed Student’s t -test. In ( F ), P values were determined by one‐way ANOVA with Dunnett’s multiple comparisons test. ** P < 0.01. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3E siRNA , Santa Cruz Biotechnology , sc-61520.

Techniques: Western Blot, Fluorescence, Incubation, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) qPCR of PLXND1 , SEMA3C, SEMA3E, KLK3 , and NCAM1 in LNCaP, LAPC4, and VCaP cells upon culture under CSS condition for 24 h followed by R1881 stimulation (10 nM, 24 h) ( n = 3 biological replicates). ( B , C ) Western blot of PlexinD1, AR, and PSA in LNCaP and LAPC4 cells upon treatment with 10 nM R1881 ( B ) or 10 µM ENZ ( C ) for the indicated times. ( D ) Genomic browser representation of AR binding at PLXND1 promoter encompassing three AREs, with the nucleotides identical to the canonical ARE highlighted in red, by interrogating AR ChIP-seq dataset GSE125245. ( E ) ChIP-qPCR of AR and H3K9ac occupancy at an ARE-centric PLXND1 promoter sequence as well as an AR-bound KLK3 promoter region upon R1881 stimulation (10 nM, 6 h) in LNCaP cells. Data represent the percent of input ( n = 3 technical replicates). ( F ) Schematic diagrams of WT and mutated forms of individual AREs in PLXND1 ARE-Luc constructs. ( G ) Determination of WT and mutated PLXND1 ARE-Luc activities upon R1881 stimulation (10 nM, 6 h) in LNCaP cells ( n = 3 biological replicates). ( H ) Pearson correlation analysis of PLXND1 mRNA expression with AR score in the indicated datasets from cBioPortal. ( I ) Pearson correlation analysis of mRNA co-expression between PLXND1 vs. different AR target genes in the indicated datasets from cBioPortal. Data information: In ( A , E , G ), data are presented as mean ± SEM. In ( A ), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. In ( E ), P values were determined by two-way ANOVA with Tukey’s multiple comparisons test. In ( G ), P values were determined by unpaired two-tailed Student’s t -test. In ( H ), P values were determined by Pearson’s correlation coefficient t -test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3E siRNA , Santa Cruz Biotechnology , sc-61520.

Techniques: Western Blot, Binding Assay, ChIP-sequencing, ChIP-qPCR, Sequencing, Construct, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) GSEA plots of transmembrane RTK-related gene sets enriched in PlexinD1-knockdown vs. control 22Rv1 cells, and PLXND1 -high vs. -low PCa patient samples in TCGA cohort. ( B ) Representative images of a RTK phosphorylation antibody array and quantification of p-ErbB3 levels in control and PlexinD1-knockdown C4-2B ENZR cells ( n = 2 technical replicates). The raw values from measurement of p-ErbB3 spot intensity after background subtraction are presented. ( C ) Western blot of p-ErbB3, p-ErbB2, p-cMet, p-ERK, p-AKT, and their total protein forms in control and PlexinD1-knockdown C4-2B ENZR and 22Rv1 cells. ( D ) qPCR of ErbB3 ( NRG1 and NRG2 ) and cMet ( HGF ) ligand mRNA levels in control and PlexinD1-knockdown C4-2B ENZR and 22Rv1 cells ( n = 3 biological replicates). ( E ) Representative PLA images and quantification of PlexinD1-ErbB3/cMet interaction by per-cell cytoplasmic fluorescence intensity in C4-2B ENZR cells treated with recombinant Sema3E/Sema3C proteins (200–500 ng/ml, 4 h) or SEMA3E / SEMA3C siRNA (10 μM, 48 h). PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 μm. ( F ) Co-IP assays of PlexinD1-ErbB3/cMet interaction in 22Rv1 cells. IgG was used in the IP step as negative control. Five percent of input was blotted as positive control. ( G ) Cell proliferation assays of 22Rv1 cells pre-treated with an ErbB3 neutralizing antibody (100 ng/ml, 24 h) or SGX-523 (5 µM, 24 h) and then subjected to PLXND1 siRNA addition followed by a 5-day observation period, and control and PlexinD1-overexpressing LNCaP cells upon treatment with an ErbB3 neutralizing antibody or SGX-523 during a 5-day observation period. Data represent the fold changes of cell proliferation on day 5 relative to siRNA treatment day (day 1) for 22Rv1 cells or treatment day (day 1) for LNCaP cells ( n = 3 biological replicates), with fold changes in non-treated groups of control cells set as 1 for normalization of other groups. ( H ) qPCR of stemness, basal, and NE markers as indicated in 22Rv1 cells receiving an ErbB3 neutralizing antibody (100 ng/ml, 24 h), SGX-523 (5 µM, 24 h), or transient transfection of ErbB3/cMet expression plasmids followed by PLXND1 siRNA treatment for another 48 h ( n = 3 biological replicates). ( I ) qPCR of stemness, basal, NE, and EMT markers as indicated in control and PlexinD1-overexpressing LNCaP cells upon treatment with 100 ng/ml ErbB3 neutralizing antibody or 5 µM SGX-523 for 24 h ( n = 3 biological replicates). Data information: In ( B , D , E , G , H , I ), data are presented as mean ± SEM. In ( A ), P values were determined by permutation test. In ( B , D ), P values were determined by unpaired two-tailed Student’s t -test. In ( E ), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. In ( G , H , I ), P values were determined by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3E siRNA , Santa Cruz Biotechnology , sc-61520.

Techniques: Knockdown, Control, Phospho-proteomics, Ab Array, Western Blot, Fluorescence, Recombinant, Incubation, Co-Immunoprecipitation Assay, Negative Control, Positive Control, Transfection, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: ( A ) Graphic depicting D1SP as a recombinant PlexinD1 decoy protein. ( B ) Western blot of D1SP in whole cell lysate (WCL) and conditioned medium (CM) of D1SP-expressing CHO-K1 cells using a hIgGFc-specific antibody. ( C ) Flow cytometric analysis of binding of IgG-PE antibody conjugated D1SP at various doses in 22Rv1 cells ( n = 3 biological replicates). ( D ) Representative PLA images and quantification of PlexinD1-Sema3E/Sema3C interaction by per-cell cytoplasmic fluorescence intensity in 22Rv1 cells treated with D1SP (1 µM, 2 h) or PBS as a vehicle. PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 µm. ( E ) Cell viability assays of C4-2B ENZR and 22Rv1 cells stimulated with 200 ng/µl recombinant Sema3E protein and then subjected to treatment with 1 µM D1SP for 7 days ( n = 3 biological replicates). ( F ) Transwell-based cell migration and invasion assays of C4-2B ENZR and 22Rv1 cells under 1 µM D1SP treatment ( n = 3 biological replicates). Scale bars: 400 µm. ( G ) Representative brightfield and fluorescence microscopic images and quantification of LuCaP 147CR, 49 and 173.1 PCa PDX-derived live organoids after incubation with 1 µM D1SP or PBS as a vehicle for 10 days ( n = 3 biological replicates). Scale bars: 100 µm. ( H – J ) Tumor growth curves ( H ), endpoint tumor weights ( I ), and anatomic tumor images ( J ) of s.c. 22Rv1 xenografts grown in male nude mice ( n = 6 tumors) and receiving intratumoral injection of D1SP (30 µg/tumor, 2–3 times per week) and saline on the right and left flanks of mice, respectively. ( K , L ) Representative images ( K ) and quantification ( L ) of IHC staining of Ki-67, p-ErbB3, p-ErbB2, and p-cMet ( n = 6 tumors) in control and D1SP-treated 22Rv1 tumors. Scale bars: 100 μm. ( M ) qPCR of select Gli1 target genes in control and D1SP-treated 22Rv1 tumors ( n = 6 tumors). Data information: In ( C – H ), data are presented as mean ± SEM. In ( C , D ), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. In ( E ), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. In ( F – H ), P values were determined by unpaired two-tailed Student’s t -test. In ( I , L , M ), P values were determined by paired two-tailed Student’s t -test. * P < 0.05, ** P < 0.01; ns, not significant. Exact P values are listed in Appendix Table . .

Article Snippet: Human SEMA3E siRNA , Santa Cruz Biotechnology , sc-61520.

Techniques: Recombinant, Western Blot, Expressing, Binding Assay, Fluorescence, Incubation, Migration, Derivative Assay, Injection, Saline, Immunohistochemistry, Control, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human SEMA3E siRNA , Santa Cruz Biotechnology , sc-61520.

Techniques: Recombinant, Sequencing, Control, Transfection, Membrane, Reverse Transcription, Software, Microarray, Subcloning, shRNA, Cell Viability Assay, Viability Assay, Reporter Assay, In Situ, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Extraction, Bicinchoninic Acid Protein Assay, Magnetic Beads, Gel Extraction, Purification, Ligation, Mutagenesis, Chromatin Immunoprecipitation