Journal: Science Advances

Article Title: A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia

doi: 10.1126/sciadv.aeb7238

Figure Lengend Snippet: ( A ) Illustration of F2R highlighting its activation, inhibition by vorapaxar, and signaling through G proteins. ( B ) Parental NIH-3T3 cells and cells transduced with F2r CRISPRa sgRNAs were serum starved for 24 hours, followed by immunofluorescence analysis of cilia (Arl13b) and centrioles [γ-tubulin (γ-tub)]. Where indicated, cells were treated with 6 μM vorapaxar during serum starvation. ( C ) Quantification of ciliation from (B). Bars show means of N = 3 independent replicates (N > 75 cells each). ( D ) Diagram of SARM1, including NADase TIR domain that cleaves NAD + to produce nicotinamide (NAM) and cADPR. ( E ) Analysis of ciliation in NIH-3T3 cell lines as in (A). Where indicated, cells were treated with 30 μM DSRM-3716 during serum starvation. ( F ) Quantification of ciliation from (E). Bars show means of N = 3 independent replicates ( N > 80 cells each). ( G ) Analysis of ciliation in NIH-3T3 cells expressing doxycycline (Dox)–inducible F2r (Tet-F2r-Flag). Cells were treated with Dox (1 μg/ml) and vorapaxar during serum starvation. Bars show means of N = 3 independent experiments ( N > 125 cells each). ( H ) Analysis of ciliation in NIH-3T3 Tet-F2r-Flag cells treated as indicated. Lines show means from N = 3 independent experiments ( N > 95 cells each). ( I ) Analysis of ciliation in serum-starved RPE1 cells after treatment with thrombin (10 U/ml). Thin lines show N = 3 replicates ( N > 100 cells each); thick lines show means. ( J ) Live-cell imaging of RPE1 cells with labeled cilia (Htr6-3xmNeonGreen) and centrioles (miRFP670-Centrin2). Time after thrombin addition is noted in hour:minute:second. See also fig. S3C. Scale bars, 5 μm. Error bars show standard deviations; asterisks denote significant (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001) differences in mean, as determined by two-sided t test.

Article Snippet: PB-Tet-F2r-3xFlag was cloned by Gibson assembly of a synthesized mouse F2r gene product (Twist Biosciences) into a piggyBac Tet-Cas9 plasmid with Neo resistance provided by I. Cheeseman (Massachusetts Institute of Technology).

Techniques: Activation Assay, Inhibition, Transduction, Immunofluorescence, Expressing, Live Cell Imaging, Labeling

Journal: Science Advances

Article Title: A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia

doi: 10.1126/sciadv.aeb7238

Figure Lengend Snippet: ( A ) Diagram of proposed cilia disassembly pathway. ( B ) Immunofluorescence analysis of ciliation in NIH-3T3 Tet-F2r-Flag cells treated with Dox (1 μg/ml), 30 μM DSRM-3716, and/or 10 μM dHNN during 24-hour serum starvation. Bars show means of N ≥ 3 independent experiments ( N > 100 cells each). ( C ) Analysis of ciliation in RPE1 cells. Cells were serum starved prior to addition of thrombin (10 U/ml), with or without vorapaxar or DSRM-3716. Bars show means of N = 3 independent experiments ( N > 140 cells each). ( D ) NIH-3T3 Tet-F2r-Flag cells were serum starved for 24 hours, followed by analysis of cilia (Arl13b) and centrioles (γ-tubulin). Cells were treated with Dox, 5 μM dantrolene, 5 μM BAPTA-AM, and/or 10 μM Y-27632 during serum starvation (dantrolene was added 24 hours prior to serum starvation). ( E ) Quantification of ciliation in NIH-3T3 Tet-F2r-Flag cells treated as in (D). Bars show means of N ≥ 3 independent experiments ( N > 85 cells each). ( F ) Left: Illustration of centrosome-targeted calcium sink based on PACT targeting domain and Pvalb calcium-binding domain. Right: Analysis of cilia, centrioles [marked by pericentrin (PCNT)], and PACT-Pvalb-mCherry in RPE1 cells following serum starvation and 24 hours thrombin treatment. Where indicated, cells were treated with Dox (20 ng/ml) during thrombin treatment and for 24 hours prior. ( G ) Quantification of ciliation in RPE1 cell lines with inducible transgenes for a centrosome-targeted calcium sink (PACT-Pvalb-mCherry), a nontargeted calcium sink (Pvalb-mCherry), or centrosome-targeted mCherry (PACT-mCherry). Bars show means of N ≥ 3 independent experiments ( N > 100 cells each). Scale bars, 5 μm. Error bars show standard deviations; asterisks denote significant (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant) differences in mean, as determined by two-sided t test.

Article Snippet: PB-Tet-F2r-3xFlag was cloned by Gibson assembly of a synthesized mouse F2r gene product (Twist Biosciences) into a piggyBac Tet-Cas9 plasmid with Neo resistance provided by I. Cheeseman (Massachusetts Institute of Technology).

Techniques: Immunofluorescence, Binding Assay

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: PAR1–4 mRNA expression in GBM specimen and its association with patients´ overall survival time. (A) Comparison of PAR1, PAR2, PAR3 and PAR4 mRNA expression in non-malignant brain (NMB, n=7) and all analyzed GBM samples (both primary and relapsed GBM, n=118). (B) Subdivision of GBM specimen in primary GBM (prGBM, n=78), first (1 st , n=33) and second (2 nd , n=7) relapses and comparison with NMB. (A+B) Gene expression was measured by qPCR. Each mRNA level of the target genes (PAR1-4) was normalized to the mean of GAPDH and β-actin using the 2 -ΔΔct method. Data are shown as scatter plots representing the median as horizontal bars. Mann Whitney U test, *p < 0.05 and ***p < 0.001 for (A) and OneWay ANOVA/Kruskal Wallis test with Dunn’s Multiple Comparison Test, *p < 0.05, **p < 0.01 and *** p < 0.001 for (B) . (C) Kaplan Meier survival analyses of PAR1–4 mRNA expression in GBM patients. Association of the relative mRNA expression of each single PAR receptor with the survival time of patients with primary GBM. The patients were divided into two subgroups depending on the median gene expression. No significant association was found.

Article Snippet: The following Gene Expression Assays on Demand from Applied Biosystems were used for qPCR: F2R FAM-Hs00169258_m1, F2RL1 FAM-Hs00608346_m1, F2RL2 FAM-Hs00765740_m1, F2RL3 FAM-Hs01006385_g1. qPCR was performed in a 7900 HT Fast Real-Time PCR system from Applied Biosystems using TaqManTM Gene Expression Master Mix.

Techniques: Expressing, Comparison, Gene Expression, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: Protein expression of PAR1–4 in GBM patients´ samples. (A) Representative immunoblots of PAR1–4 in GBM tissue in comparison to non-malignant brain samples (NMB). Detection of GAPDH was used as loading control for normalization of the respective PAR protein level to a housekeeping protein. (B) Relative protein expression of PAR1–4 in GBM and NMB after densitometric evaluation and normalization to GAPDH. Data are shown as scatter plots representing the median as horizontal bars. Mann Whitney U test, ***p < 0.001. (C) Kaplan Meier survival analyses of PAR1–4 protein expression in GBM patients. Association of the relative protein expression of each single PAR receptor with the survival time of patients with primary GBM. The patients were divided into two subgroups depending on the median protein expression. The protein content of the respective PARs was determined by western blotting and normalized to the housekeeping protein GAPDH. Log-rank (Mantel-Cox) Test, *p < 0.05.

Article Snippet: The following Gene Expression Assays on Demand from Applied Biosystems were used for qPCR: F2R FAM-Hs00169258_m1, F2RL1 FAM-Hs00608346_m1, F2RL2 FAM-Hs00765740_m1, F2RL3 FAM-Hs01006385_g1. qPCR was performed in a 7900 HT Fast Real-Time PCR system from Applied Biosystems using TaqManTM Gene Expression Master Mix.

Techniques: Expressing, Western Blot, Comparison, Control, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: PAR1–4 mRNA and protein content in adherent and neurospheric LN-18 GBM cells. (A) Comparative PAR1–4 mRNA expression of both adherent (LN-18-adh, grey bars) and neurospheric (LN-18-NS, black bars) LN-18 cells of the passages one to four (Pass. 1-4) as well as of parental LN-18 cells (LN-18-par, white bars). Gene expression was measured by qPCR. Each mRNA level of the target genes (PAR1-4) was normalized to the mean of 18S rRNA, GAPDH and β-actin using the 2 -ΔΔct method, n=4. (B) Comparative PAR1–4 protein content of both adherent (LN-18-adh, grey bars) and neurospheric (LN-18-NS, black bars) LN-18 cells of passage three. The protein content of the respective PARs was determined by western blotting and normalized to the housekeeping protein GAPDH. Representative immunoblots are shown above the diagram, n=3-4. OneWay ANOVA with Bonferroni’s Multiple Comparison Test, *p < 0.05, **p < 0.01 and ***p < 0.001 LN-18-adh vs. LN-18-NS; #p < 0.05, ##p < 0.01 and ###p < 0.001 LN-18-adh vs. LN-18-NS.

Article Snippet: The following Gene Expression Assays on Demand from Applied Biosystems were used for qPCR: F2R FAM-Hs00169258_m1, F2RL1 FAM-Hs00608346_m1, F2RL2 FAM-Hs00765740_m1, F2RL3 FAM-Hs01006385_g1. qPCR was performed in a 7900 HT Fast Real-Time PCR system from Applied Biosystems using TaqManTM Gene Expression Master Mix.

Techniques: Expressing, Gene Expression, Western Blot, Comparison

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: Impact of PAR subtype specific inhibitors and agonists on viability of LN-18 GBM cells. Adherent and neurospheric LN-18 cells of passage 1 or 3 were treated for 48 or 72h with the respective compounds followed by measurement of cell viability using the Resazurin assay. (A+B) Treatment of adherent (A) and neurospheric (B) LN-18 cells with inhibitors of PAR1 (RWJ), PAR2 (FSLLRY) and PAR4 (tcY) (each 5 and 20 µM), n=3-4. (C+D) Incubation of adherent (C) and neurospheric (D) LN-18 cells with Thrombin (TB, 30 U/ml), Factor Xa (FXa, 30 nM) or PAR subtype specific agonists (AP1 to AP4, each 100 and 200 µM), n=3-4. OneWay ANOVA Dunnett’s Multiple Comparison Test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Con.

Article Snippet: The following Gene Expression Assays on Demand from Applied Biosystems were used for qPCR: F2R FAM-Hs00169258_m1, F2RL1 FAM-Hs00608346_m1, F2RL2 FAM-Hs00765740_m1, F2RL3 FAM-Hs01006385_g1. qPCR was performed in a 7900 HT Fast Real-Time PCR system from Applied Biosystems using TaqManTM Gene Expression Master Mix.

Techniques: Resazurin Assay, Incubation, Comparison

Journal: Frontiers in Oncology

Article Title: Significance of FXa and its receptor PAR2 for the growth of colon cancer cells in vitro and in vivo

doi: 10.3389/fonc.2025.1631350

Figure Lengend Snippet: Key figures of the animal study. Trial days. (A) Shown is the number of days the animals were subjected to the study, with a maximum of 21 days. PAR2-KO (black) mice stayed significantly longer in the trial than WT (white) mice. This finding is applicable to all three treatment groups. The animals were administered either 5 mg/kg or 50 mg/kg body weight Apixaban plus vehicle or vehicle only (control). Tumor size. (B) Additionally, PAR2-KO mice exhibited a significantly reduced tumor size when compared to their WT counterparts. Ratio of spleen weight to body weight. (C) Macroscopic discrepancies between the animal strains were observed in the ratio of spleen weight to body weight. Compared to WT (white) animals, PAR2-KO (black) animals showed a significantly higher spleen weight in relation to body weight. Apixaban concentration. (D) Apixaban plasma concentrations measured by UPLC-MS/MS in WT (white) and PAR2-KO mice (black) treated with 50 mg/kg or w/o (control) Apixaban. (E) Significantly higher Apixaban levels could also be detected in the tumors of treated animals. PCR analyzes. (F) Apixaban is metabolized via CYP3A4. The murine counterpart is CYP3A11. PCR analyses show that the treatment of the animals with Apixaban significantly increases the expression of CYP3A11 in the liver at RNA level in both, WT (white) and PAR2-KO mice (black). Further Specific RNA analyses of the removed spleens demonstrate elevated expression levels of the following genes in PAR2-KO mice: MMP2 (G) , MMP9 (H) , COX2 (I) , PAR1 (J) , EGFR (L) . PAR2 can only be detected in WT mice (K) . N = 3-6 (I-VI), Shown is the relative expression of the target normalized to Gapdh and relative to the control approaches. Mean ± SD of n independent experiments is presented for all data. One-way ANOVA, Dunnett posthoc test, p < 0.05 (*).

Article Snippet: The utilized Assays on demand [Applied Biosystems, Cat. #4331182] were used: Mm00433160_m1, Mm00438851_m1, Mm00439498_m1, Mm00445021_m1, Mm004488841_g1, Mm00468695_s1, Mm00473016_m1, Mm00476227_m1, Mm00486079_m1, Mm02619656_s1, Mm02620181_s1, Mm02620565_s1, Mm03039020_m1, Mm99999915_g1, Mm03928990_g1, Mm00731567_m1.

Techniques: Control, Concentration Assay, Clinical Proteomics, Tandem Mass Spectroscopy, Expressing

Journal: Frontiers in Immunology

Article Title: Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis

doi: 10.3389/fimmu.2024.1398120

Figure Lengend Snippet: GzmK stimulates human keratinocyte proliferation via PAR-1. (A) Western blot immunodetection of PAR-1 and GAPDH of HaCaTs transfected with or without F2R (PAR-1) or Control siRNA (10 nM or 20 nM or 40 nM, 72 h). (B) Western blot immunodetection of PAR-1 and GAPDH of HaCaTs transfected with or without F2R (PAR-1) or Control siRNA (40 nM, 120 h). (C) HaCaTs transfected with or without F2R (PAR-1) or Control siRNA (40 nM, 120 h) and stimulated with or without GzmK (100 nM, 48 h). Proliferation was detected by Ki67 immunocytochemistry, Ki67 = red, Hoechst nuclear stain = blue. n ≥ 3 per group (C) . Scale bars represent 20 µm (C) , with representative images shown.

Article Snippet: Two siRNA targeting the F2R gene (PAR-1) and a negative control siRNA with no complementary target sequences were used (Qiagen).

Techniques: Western Blot, Immunodetection, Transfection, Control, Immunocytochemistry, Staining

Journal: Frontiers in Immunology

Article Title: Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis

doi: 10.3389/fimmu.2024.1398120

Figure Lengend Snippet: GzmK activation of PAR-1 phosphorylates p38 and p44/42 MAPK in human keratinocytes; GzmK induces STAT3 (Ser727) phosphorylation in a PAR-1/p44/42 MAPK-dependent manner in human keratinocytes. (A) Western blot immunodetection of p-p38 MAPK, total p38 MAPK, and GAPDH of HaCaTs transfected with or without F2R (PAR-1) or Control siRNA and stimulated with or without GzmK (100 nM, 15 min). (B) Western blot immunodetection of p-STAT3 (Ser727), p-STAT3 (Tyr705), total STAT3, p-p44/42 MAPK, total p44/42 MAPK, and GAPDH of HaCaTs transfected with or without F2R (PAR-1) or Control siRNA and stimulated with or without GzmK (100 nM, 15 min). (C) Western blot immunodetection of p-STAT3 (Ser727), p-STAT3 (Tyr705), total STAT3, p-p44/42 MAPK, and GAPDH of HaCaTs pre-treated with U0126 (10 µM, 45 min) followed by stimulation with or without GzmK (100 nM, 15 min).

Article Snippet: Two siRNA targeting the F2R gene (PAR-1) and a negative control siRNA with no complementary target sequences were used (Qiagen).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Immunodetection, Transfection, Control