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: Journal of Mammalian Ova Research

Article Title: The Expression and Roles of Semaphorin Type 3C in Granulosa Cells during The Luteinization Process

doi: 10.1274/jmor.31.31

Figure Lengend Snippet: Fig. 2. The expression and roles of Sema3C in cultured granu- losa cells. (A) The induction of Sema3c mRNA in cultured JUDQXORVD FHOOV ZLWK IRUVNROLQ30$ )RU 30$ RU $5(*15* $1 & JUDQXORVDFHOOVZHUHFXOWXUHG without any agonists. (B) The effects of Sema3c siRNA on the expression of Sema3c mRNA, *, P<0.05. (C) The cell migration of granulosa cells was induced by granu- ORVDFHOOVHFUHWHG6HPD&E\$5(*DQG15*$1 $5(*DQG15*

Article Snippet: After 8 h culture, the cells were washed, then cultured for 4 h in fresh, serum-free medium containing forskolin (For) (10 μM) and PMA (20 nM) which mimics LH stimulation of cAMP and diacylglycerol production, or EGF like factors (100 ng/ml of AREG and 10 ng/ml of NRG1), and harvested for RNA analysis [4, 8]. siRNA treatment procedure of cultured mouse granulosa cells Sema3c pre-designed siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Cell Culture, Migration

Journal: Journal of Mammalian Ova Research

Article Title: The Expression and Roles of Semaphorin Type 3C in Granulosa Cells during The Luteinization Process

doi: 10.1274/jmor.31.31

Figure Lengend Snippet: Fig. 4. The expression of genes involved in progesterone pro- duction, Cyp11a1, Hsd3b, Star and the secretion level in Sema3c siRNA-transfected granulosa cells cultured for KUZLWK$5(*DQG15* P<0.05.

Article Snippet: After 8 h culture, the cells were washed, then cultured for 4 h in fresh, serum-free medium containing forskolin (For) (10 μM) and PMA (20 nM) which mimics LH stimulation of cAMP and diacylglycerol production, or EGF like factors (100 ng/ml of AREG and 10 ng/ml of NRG1), and harvested for RNA analysis [4, 8]. siRNA treatment procedure of cultured mouse granulosa cells Sema3c pre-designed siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Transfection, Cell Culture

Journal: Journal of Mammalian Ova Research

Article Title: The Expression and Roles of Semaphorin Type 3C in Granulosa Cells during The Luteinization Process

doi: 10.1274/jmor.31.31

Figure Lengend Snippet: Fig. 3. The effects of Sema3c siRNA on the morphology (A) and the surface area (B) of JUDQXORVDFHOOVFXOWXUHGZLWK$5(*DQG15* P<0.05. (C) The activation of FAK pathway in Sema3c siRNA-transfected granulosa cells.

Article Snippet: After 8 h culture, the cells were washed, then cultured for 4 h in fresh, serum-free medium containing forskolin (For) (10 μM) and PMA (20 nM) which mimics LH stimulation of cAMP and diacylglycerol production, or EGF like factors (100 ng/ml of AREG and 10 ng/ml of NRG1), and harvested for RNA analysis [4, 8]. siRNA treatment procedure of cultured mouse granulosa cells Sema3c pre-designed siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Activation Assay, Transfection

Journal: Journal of Mammalian Ova Research

Article Title: The Expression and Roles of Semaphorin Type 3C in Granulosa Cells during The Luteinization Process

doi: 10.1274/jmor.31.31

Figure Lengend Snippet: Fig. 1. The expression of Sema3 family members in the ovary during the ovulation process in eCG- and hCG-treated mice. (A) The temporal changes of Sema3b and Sema3c mRNA expression in granulosa cells col- lected from ovaries of mice injected with eCG+hCG. For reference, the 0 hr value is set as 1, DQGWKHGDWDDUHSUHVHQWHGDVIROGLQFUHDVHV9DOXHVDUHPHDQí6(0RIWKUHHUHSOLFDWHV 6LJQL¿FDQWGLIIHUHQFHVZHUHREVHUYHGDVFRPSDUHGZLWKWKRVHLQJUDQXORVDFHOOVUHFRYHUHGIURP eCG primed mice (hCG 0hr), *, P<0.05. (B) The expression of Sema3c receptors, Plexina1(a1), Plexina2(a2) and Nrp1LQJUDQXORVDFHOORIH&*SULPHGPLFH0 ESPDUNHU3 SRVLWLYHFRQWURO P51$SXUL¿HGIURPDGXOWPRXVHEUDLQ* JUDQXORVDFHOOVIURPH&*SULPHGPLFHRYDU\ & 7KHNLQHWLFFKDQJHVRI6HPD&SURWHLQOHYHOVLQRYDU\RUJUDQXORVDFHOOVRIH&*K&*LQMHFWHG mice. (D) The localization of Sema3C in preovulatory (eCG) or periovulatory (hCG8hr) fol- licles. Green is Sema3C, Blue is nuclei (DAPI staining).

Article Snippet: After 8 h culture, the cells were washed, then cultured for 4 h in fresh, serum-free medium containing forskolin (For) (10 μM) and PMA (20 nM) which mimics LH stimulation of cAMP and diacylglycerol production, or EGF like factors (100 ng/ml of AREG and 10 ng/ml of NRG1), and harvested for RNA analysis [4, 8]. siRNA treatment procedure of cultured mouse granulosa cells Sema3c pre-designed siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Injection, Staining

Journal: Journal of Mammalian Ova Research

Article Title: The Expression and Roles of Semaphorin Type 3C in Granulosa Cells during The Luteinization Process

doi: 10.1274/jmor.31.31

Figure Lengend Snippet: Fig. 2. The expression and roles of Sema3C in cultured granu- losa cells. (A) The induction of Sema3c mRNA in cultured JUDQXORVD FHOOV ZLWK IRUVNROLQ30$ )RU 30$ RU $5(*15* $1 & JUDQXORVDFHOOVZHUHFXOWXUHG without any agonists. (B) The effects of Sema3c siRNA on the expression of Sema3c mRNA, *, P<0.05. (C) The cell migration of granulosa cells was induced by granu- ORVDFHOOVHFUHWHG6HPD&E\$5(*DQG15*$1 $5(*DQG15*

Article Snippet: After 8 h culture, the cells were washed, then cultured for 4 h in fresh, serum-free medium containing forskolin (For) (10 μM) and PMA (20 nM) which mimics LH stimulation of cAMP and diacylglycerol production, or EGF like factors (100 ng/ml of AREG and 10 ng/ml of NRG1), and harvested for RNA analysis [4, 8]. siRNA treatment procedure of cultured mouse granulosa cells Sema3c pre-designed siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Cell Culture, Migration

Journal: Journal of Mammalian Ova Research

Article Title: The Expression and Roles of Semaphorin Type 3C in Granulosa Cells during The Luteinization Process

doi: 10.1274/jmor.31.31

Figure Lengend Snippet: Fig. 4. The expression of genes involved in progesterone pro- duction, Cyp11a1, Hsd3b, Star and the secretion level in Sema3c siRNA-transfected granulosa cells cultured for KUZLWK$5(*DQG15* P<0.05.

Article Snippet: After 8 h culture, the cells were washed, then cultured for 4 h in fresh, serum-free medium containing forskolin (For) (10 μM) and PMA (20 nM) which mimics LH stimulation of cAMP and diacylglycerol production, or EGF like factors (100 ng/ml of AREG and 10 ng/ml of NRG1), and harvested for RNA analysis [4, 8]. siRNA treatment procedure of cultured mouse granulosa cells Sema3c pre-designed siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Transfection, Cell Culture

Journal: Journal of Mammalian Ova Research

Article Title: The Expression and Roles of Semaphorin Type 3C in Granulosa Cells during The Luteinization Process

doi: 10.1274/jmor.31.31

Figure Lengend Snippet: Fig. 3. The effects of Sema3c siRNA on the morphology (A) and the surface area (B) of JUDQXORVDFHOOVFXOWXUHGZLWK$5(*DQG15* P<0.05. (C) The activation of FAK pathway in Sema3c siRNA-transfected granulosa cells.

Article Snippet: After 8 h culture, the cells were washed, then cultured for 4 h in fresh, serum-free medium containing forskolin (For) (10 μM) and PMA (20 nM) which mimics LH stimulation of cAMP and diacylglycerol production, or EGF like factors (100 ng/ml of AREG and 10 ng/ml of NRG1), and harvested for RNA analysis [4, 8]. siRNA treatment procedure of cultured mouse granulosa cells Sema3c pre-designed siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Activation Assay, Transfection