Journal: Nature Communications

Article Title: Primary cilia and SHH signaling impairments in human and mouse models of Parkinson’s disease

doi: 10.1038/s41467-022-32229-9

Figure Lengend Snippet: a Overlap of genome wide predicted GLI3 target genes using the MatInspector (Genomatix) with all DEGs from NSC1a. b Normalized gene expression levels of the SHH receptors PTCH1 , PTCH2 , the SHH transcription factors GLI1 , GLI2 , GLI3 and other signaling targets FOXA2, GBP1, SHOX2 analyzed on mRNA level by RT-qPCR in hNPCs. c Quantification of full length GLI3 (GLI3-FL) and GLI3 transcriptional repressor (GLI3-R) in the cytoplasmic (cf) or nuclear (nf) fraction of protein extracts from hNPCs and ( d ) DAns analyzed by Western blot. Cf and nf GLI3 levels were normalized to levels of ACTB or H1-0, respectively. All experiments were performed in triplicates, n = 5 Ctrl and 7 sPD clones. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value. Each dot represents one patient. P -values were determined by two-sided t -test b ( PTCH1 p = 0.9180; PTCH2 p = 0.9333; GLI1 p = 0.0508; GLI2 p = 0.0402; GLI3 p = 0.0030; GBP1 p = 0.0318; SHOX2 p = 0.0986), c (cf GLI3-FL p = 0.0156; nf GLI3-FL p = 0.0009; GLI3-R p = 0.0001), d (cf GLI3-FL p = 0.8308; nf GLI3-FL p = 0.2297; GLI3-R p = 0.0048; nf GLI3-FL/R ratio p = 0.9541); two-sided Mann-Whitney-U test b ( FOXA2 p = 0.0303), c (nf GLI3-FL/R ratio p = 0.9001); one-sided Fisher’s Exact Test a ( p = 3.31 × 10 − 13 ). # p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: For RT-qPCR 25 ng (278 ng/μl) cDNA was quantitatively amplified on a QuantStudio 7 Flex (Thermo Fisher Scientific) using TaqMan universal PCR MM no Ung (Thermo Fisher Scientific, 4324018) and gene specific TaqMan primers (Thermo Fisher Scientific, 4331182): ACTB (Hs99999903_m1); HPRT1 (Hs99999909_m1); GAPDH (Hs99999905_m1); GLI1 (Hs00171790_m1); GLI2 (Hs01119974_m1); GLI3 (Hs00609233_m1); FOXA2 (Hs05036278_s1); PITX3 (Hs01013935_g1); BBS5 (Hs00537098_m1); GET4 (Hs00944514_m1); LINGO2 (Hs01102041_s1); SLC1A2 (Hs01102423_m1); SRCAP (Hs00198472_m1); PTCH1 (Hs00181117_m1); PTCH2 (Hs00184804_m1); SHOX2 (Hs00243203_m1); GBP1 (Hs00977005_m1).

Techniques: Genome Wide, Gene Expression, Quantitative RT-PCR, Western Blot, Clone Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Primary cilia and SHH signaling impairments in human and mouse models of Parkinson’s disease

doi: 10.1038/s41467-022-32229-9

Figure Lengend Snippet: a Quantification of full length GLI3 (GLI3-FL) and GLI3 transcriptional repressor (GLI3-R) in the cytoplasmic (cf) or nuclear (nf) fraction of protein extracts from hNPCs upon cyclopamine (Cyc) inhibition (10 µM for 4 days). b SHH receptor PTCH1 , transcription factor GLI3 and the signaling targets FOXA2 , GBP1 , SHOX2 analyzed on mRNA level by RT-qPCR in hNPCs (DMSO ctrl and Cyc treated − 10 µM for 4 days). c (right) Density plot illustrating the distribution of PC length (in µm) in a hNPC population (DMSO ctrl and Cyc treated − 10 µM for 4 days). (left) Fraction of ciliated cells. PC length was measured in immunostainings of hNPCs positive for anti-NES and anti-ARL13B. Analyzed were n = 40 cilia per clone. d Cell proliferation rate of hNPCs (DMSO ctrl and Cyc treated − 10 µM for 4 days) determined by EdU incorporation after 2 h of EdU treatment. e Mitochondrial stress test performed in hNPC (DMSO ctrl and Cyc treated − 10 µM for 4 days) using a Seahorse XFe96 Extracellular Flux Analyzer. Injected were (A) Oligomycin (1 µg/ml), (B) FCCP (0.5 µM), (C) Rotenone (5 µM)/Antimycin A (2 µM). Measurement progression is shown with means ± standard error of the mean (SEM). Boxplots display means of serial measurements for basal and maximal respiration, proton leakage as well as the difference between basal respiration and proton leak for ATP linked respiration and the difference between basal and maximal respiration for spare capacity. All experiments were performed in triplicates, n = 5 Ctrl and 7 sPD clones. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value. Each dot represents one patient. P -values were determined by two-sided t -test a (Cyc cf p = 0.1688; nf GLI3-FL p = 0.9198; GLI3-R p = 0.1932; nf GLI3-FL/R ratio p = 0.0940), b (Cyc PTCH1 p = 0.5821, GLI3 p = 0.8361, GBP1 p = 0.3518, SHOX2 p = 0.3658), c (left - untreated p = 0.2691; Cyc p = 0.5563), d (untreated p = 0.9068; Cyc p = 0.2045), e (untreated - basal p = 0.0009; proton p = 0.0095; max p = 0.0046; ATPl p = 0.0008; spare p = 0.0449) (Cyc - basal p = 0.5347; proton p = 0.6068; max p = 0.6061; ATPl p = 0.5199; spare p = 0.7502); two-sided Mann–Whitney-U test b (Cyc FOXA2 p = 0.4762); two-sided Kolmogorov-Smirnov test (ks) + linear mixed effects model (lm) c (right - untreated ks p = 4.8 × 10 − 5 ; lm p = 0.009) (right - Cyc ks p = 0.86; lm p = 0.80). # p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: For RT-qPCR 25 ng (278 ng/μl) cDNA was quantitatively amplified on a QuantStudio 7 Flex (Thermo Fisher Scientific) using TaqMan universal PCR MM no Ung (Thermo Fisher Scientific, 4324018) and gene specific TaqMan primers (Thermo Fisher Scientific, 4331182): ACTB (Hs99999903_m1); HPRT1 (Hs99999909_m1); GAPDH (Hs99999905_m1); GLI1 (Hs00171790_m1); GLI2 (Hs01119974_m1); GLI3 (Hs00609233_m1); FOXA2 (Hs05036278_s1); PITX3 (Hs01013935_g1); BBS5 (Hs00537098_m1); GET4 (Hs00944514_m1); LINGO2 (Hs01102041_s1); SLC1A2 (Hs01102423_m1); SRCAP (Hs00198472_m1); PTCH1 (Hs00181117_m1); PTCH2 (Hs00184804_m1); SHOX2 (Hs00243203_m1); GBP1 (Hs00977005_m1).

Techniques: Inhibition, Quantitative RT-PCR, Injection, Clone Assay, MANN-WHITNEY

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 1. Analysis of Hh pathway activation in human MPM specimens and rat MPM model in tumor and stromal fractions. A, immunohistochemical detection of GLI1, DHH, PTCH1, and HHIP in tumor (T) and stroma (S) and box plot of semiquantitative expression levels (H-score) of GLI1 and DHH in 146 MPM specimens, and PTCH1 and HHIP in 8 MPM specimens (lines indicate medians). B, histology of rat MPM model revealed cellular areas of closely packed spindled tumor cells (T) with relatively large, irregular nuclei, adjacent to stroma (S) containing fibroblasts (, organized small spindle cells), blood vessels (X), and inflammatory cells (small round cells with dense blue chromatin; arrow). Of note is the presence of some fibroblasts and inflammatory cells also in the tumor area. Scatter plot shows semiquantitative expression levels of GLI1, DHH, PTCH1, and HHIP (H-score) of 6 tumors (lines indicate medians).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Activation Assay, Immunohistochemical staining, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 2. The comparison of Hh pathway activation in vitro and in vivo rat MPM model. A, expression levels (mRNA) of pathway components were monitored in IL45-luc cultured in 10% serum, primary cell culture in 3% O2 without serum compared with tumor from the rat model (in vivo). B, cell growth measurement by MTT (left) and colony formation assay (right) shows that IL45-luc and the parental line (IL45) cultured in 20% O2 with serum are identically sensitive to vismodegib (data are given in mean SD; , P < 0.05; , P < 0.001; , P < 0.0001). C and D, IL45 parental cells were exposed to increasing concentration of vismodegib and measured for the downregulation of hedgehog pathway target gene (Gli1 and Ptch1) in addition to Gli2 and Gli3. No significant suppression of all genes was observed upon the treatment course (24 and 48 hours).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Comparison, Activation Assay, In Vitro, In Vivo, Expressing, Cell Culture, Colony Assay, Concentration Assay

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 4. Efficient downregulation of Hh signaling and reduction of tumor cell growth following the treatment with vismodegib. A, mRNA levels of Hh target genes, Gli1 is strongly reduce in skin and tumor of vismodegib-treated rats. Expression level of other GLI1 target genes, Ptch1 and Hhip, isalso reduced in treated tumor compared with control. Dhh, Gli2, and Gli3 expression remains unchanged. B, immunohistochemical staining of GLI1 and HHIP showing reduced expression (intensity and frequency) in treated compared with control. More GLI1- and HHIP-negative stromal cells are present in the treated rat (see circles: T, tumor; S, stroma). C, quantitative analysis histo(H)-score of GLI1, HHIP, PTCH1, and DHH staining showing pronounced GLI1 and HHIP downregulation in stromal fractions. D, proliferation (Ki-67–positive), mitotic (phospho-histone H3–positive) indices are significantly reduced in the treated group. No change in necrosis and apoptosis level (TUNEL-positive nuclei) is observed between groups. (C, control, n ¼ 6; T, treated, n ¼ 6, data are given in mean SD; , P < 0.05; , P < 0.001).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Expressing, Control, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 5. Vismodegib dose dependently suppresses growth of mesothelioma cells but could not suppress Hh pathway in vitro. A, primary cells isolated from four different rat tumors cultured in 3% O2 without serum and IL45-luc cell line cultured 20% O2 were exposed to increasing concentration of vismodegib and measured for viability. No difference in terms of sensitivity to vismodegib was observed (data are given in mean SD). B, two lines of primary cell (isolated from control rats) were treated with vismodegib for 24 hours and analyzed for the downregulation of Gli1. No significant suppression of Gli1 was observed. C, IC50 of vismodegib determined in four cell lines after 72-hour treatment (MTT assay). D, the expression levels of Hh pathway components (Dhh, Smo, Ptch1, Gli1) of the four cell lines showing no correlation with their sensitivity to vismodegib.

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: In Vitro, Isolation, Cell Culture, Concentration Assay, Control, MTT Assay, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 6. Daily treatment with vismodegib causes downregulation of previously described fibroblast Hh-responsive genes. A, mRNA levels of canonical Hh target gene such as CyclinD1, Abcg2 remained unchanged in vismodegib-treated group compared with control. Previously described fibroblast Hh-responsive genes, i.e., Fn1, Vegfa, and Lif mRNA levels were reduced in vismodegib-treated group. (C, control, n ¼ 6; T, treated, n ¼ 6; data are given in mean SD; , P < 0.05; , P < 0.001). B, mouse embryonic fibroblast NIH3T3 cells were treated with supernatant collected from primary culture of rat MPM cells. Treatment with SMO agonist (SAG) was employed as a positive control. Gli1, Ptch1, and Fn1 expression levels were analyzed after 72-hour incubation at 37C 3% O2 (data represent mean of triplicates SD).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Control, Positive Control, Expressing, Incubation

Journal: Experimental & Molecular Medicine

Article Title: miR-30c regulates proliferation, apoptosis and differentiation via the Shh signaling pathway in P19 cells

doi: 10.1038/emm.2016.57

Figure Lengend Snippet: TaqMan assay IDs of genes used in the TaqMan qPCR

Article Snippet: Ptch , Mm00436026_m1.

Techniques: TaqMan Assay

Journal: Experimental & Molecular Medicine

Article Title: miR-30c regulates proliferation, apoptosis and differentiation via the Shh signaling pathway in P19 cells

doi: 10.1038/emm.2016.57

Figure Lengend Snippet: Effects of miR-30c on the Shh signaling pathway. ( a , b ) Identification of Gli2 as a potential target gene of miR-30c. ( a ) Dual luciferase reporter assay of miR-30c with a Gli2 3′ UTR reporter. The data are presented as the mean±s.d. of three experiments (* P <0.05 comparing miR-30c overexpression with control cells; # P <0.05 comparing miR-30c knockdown with control cells). ( b ) Western blotting of Gli2 protein levels in stable miR-30c overexpression or knockdown cell lines. ( c ) Immunostaining of Gli2 in stable miR-30c overexpression or knockdown cell lines. DAPI was used to stain the nuclei. Scale bar, 20 μm. ( d , e ) Ptch1 protein ( d ) and mRNA ( e ) levels were determined by western blotting or qRT-PCR, respectively, in miR-30c overexpression or knockdown cell line after treatment with DMSO for the indicated days. (* P <0.05 comparing Ptch1 mRNA levels in the cells on day 4 with those on day 0; # P <0.05 comparing Ptch1 mRNA levels in the cells on day 10 with those of day 0).

Article Snippet: Ptch , Mm00436026_m1.

Techniques: Luciferase, Reporter Assay, Over Expression, Western Blot, Immunostaining, Staining, Quantitative RT-PCR