Journal: bioRxiv

Article Title: Nutrient-dependent mTORC1 signaling in coral-algal symbiosis

doi: 10.1101/723312

Figure Lengend Snippet: A) Maximum Likelihood phylogeny (PhyML) of MITF-family proteins across vertebrates and invertebrates. Only bootstrap values (ML percentage) ≥ 50 are shown. We used upstream regulatory factors, which are sister to the MITF-like TFs within the bHLH (basic helix-loop-helix) TFs, as an outgroup. Species names are italicized. Triangles represent collapsed branches. The position of Aiptasia is highlighted in light red. For raw sequences, trimmed alignments, and tree information, see File S3. For alignment of Aiptasia MITF-like and Homo sapiens TFEB, see . B) Overview of gene expression of Aiptasia homologs of CLEAR network genes involved in autophagy and lysosomes. Presence of E-box motifs in the promoter region of a gene is marked with “Y”. Asterisks “*” indicate differentially expressed genes (log2-fold change > 2; false-discovery-rate < 0.01). C) Representative Western blots of phosphorylated 4-EBP1 (Thr37/46) (p4-EBP1) comparing aposymbiotic and symbiotic polyps with (feeding 3 times per week, for > 3 weeks) and without food (no feeding for ≥ 3 weeks). For representative images of lipids in polyp macerates see . D) Representative images of LAMP1-immunofluorescence analysis of Aiptasia -LAMP1 (see also ) in aposymbiotic and symbiotic larvae 6 dpf. Colors in merge are nuclei in blue (Hoechst 33258), LAMP1 in green and LAMP1 detected with Alexa488-anti-rabbit IgG, and symbiont autofluorescence in red; scale bars represent 25 μm for overviews and 5 μm for inset. E) Representative images of mTOR-immunofluorescence analysis occurring in ~50% of symbiotic larvae, 6 dpf. Colors in merge are nuclei in blue (Hoechst 33258), mTOR in green and symbiont autofluorescence in red; scale bars represent 25 μm for whole larva images (upper panels) and 5 μm for close up (lower panels). F) Protein sequence alignment of transmembrane domain 8 of SLC38A9 homologs. Key conserved phenylalanine (F) and tyrosine (Y) residues within the CARC and CRAC motifs boxed in red. G) Model of mTORC1 signaling to coordinate nutrient input by symbionts with host physiology in cnidarian endosymbiosis.

Article Snippet: The top was incubated with α-tubulin antibody (1:3000; T9026, Sigma-Aldrich Co., LLC), the lower half incubated with the p4-EBP1 primary antibody (1:1000; 2855T, Cell Signaling Technology) overnight at 4°C.

Techniques: Expressing, Western Blot, Immunofluorescence, Sequencing

Journal: Nature communications

Article Title: Human stem cells alter the invasive properties of somatic cells via paracrine activation of mTORC1.

doi: 10.1038/s41467-017-00661-x

Figure Lengend Snippet: Fig. 2 Human stem cells trigger the invasion of various somatic cell types. a Outline of the invasion experiments in b–i. The table summarises the analysed target cells and their mean invasive capacity. *Invasion data are given as the percentage of invasive cells evaluated via gradient-based transwell invasion assay (n ≥3; mean ± s.d.). N/A, not applicable. b–g Transwell invasion assay of indicated target cells upon co-culture with stem cells. Data are presented as the fold change of the corresponding medium control or the absolute number of invasive cells per membrane (n ≥3; mean ± s.d.). h Calcein-staining of invasive cardiac fibroblasts upon AFSC co-culture representative of data in c. Scale bar, left panels, 800 μm; right panels, 100 μm. i Calcein-staining of invasive hepatocytes upon AFSC co-culture representative of data in f. Scale bar, left panels, 100 μm; right panels, 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, P > 0.05 (not significant) by unpaired, two-tailed Student’s t-test analysis. n refers to biological replicates

Article Snippet: Somatic cells and embryonal carcinoma cell lines are the following: CCD1079Sk, primary foreskin fibroblasts (ATCC, CRL-2097, p4); HCF, primary cardiac fibroblasts isolated from the ventricle of an adult heart (Promocell, C-12375, p2); HCH, primary chondrocytes isolated from normal human articular cartilage from the femoral head (Promocell, C-12710, p2); Hep G2, hepatocellular carcinoma cells (ATCC, HB-8065); HFF-1, primary foreskin fibroblasts (ATCC, SCRC-1041, p13); hNHeps, primary hepatocytes isolated from non-transplantable donor tissue (Lonza, CC-2591); HT-1080, fibrosarcoma cells (ATCC, CCL-121, p18); IMR-90, primary lung fibroblasts (ATCC, CCL-186, p10); MCF7, mammary carcinoma cells (ATCC, HTB-22, p146); MEF, immortalised mouse embryonic fibroblasts (provided by David J. Kwiatkowski, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA); NCCIT, embryonal carcinoma cells (ATCC, CRL-2073) and NTERA-2 cl.

Techniques: Transwell Invasion Assay, Co-Culture Assay, Control, Membrane, Staining, Two Tailed Test

Journal: Nature communications

Article Title: Human stem cells alter the invasive properties of somatic cells via paracrine activation of mTORC1.

doi: 10.1038/s41467-017-00661-x

Figure Lengend Snippet: Fig. 5 Stem cell-secreted IGFs activate the IGF-I receptor to promote target cell invasion. a Immunoblot for the detection of mTOR signalling proteins in serum-deprived IMR-90 cells stimulated with IGF-I or IGF-II. b Transwell invasion assay of Rapamycin-treated IMR-90 fibroblasts upon IGF stimulation (n ≥3; mean ± s.d.). c Immunoblots for the detection of mTOR signalling proteins in IGF-I receptor-depleted IMR-90 cells under steady state conditions (left panel) or upon IGF-I stimulation (right panel). d Transwell invasion assay of IGF-I receptor-depleted IMR-90 fibroblasts upon co-culture with stem cells (n ≥6; mean ± s.d.). e Immunoblot of tissue lysates from Matrigel- or ESC-injected mice for the detection of phosphorylated IGF-I receptor. The absence of human cells from non-teratoma tissues and equal loading was verified by detection of human topoisomerase IIβ and Ponceau-S staining. f Transwell invasion assay of IMR-90 fibroblasts co-cultured with IGF-I- or IGF-II-depleted stem cells (n ≥3; mean ± s.d.). Knockdown efficiency was evaluated via immunostaining of endogenous IGF-I and IGF-II. IGF detection in stem cells was further verified by co-analysing somatic (HT-1080, Hep G2) control cell lines. Scale bar, 50 μm. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, P > 0.05 (not significant) by unpaired, two-tailed Student’s t-test analysis. n refers to biological replicates

Article Snippet: Somatic cells and embryonal carcinoma cell lines are the following: CCD1079Sk, primary foreskin fibroblasts (ATCC, CRL-2097, p4); HCF, primary cardiac fibroblasts isolated from the ventricle of an adult heart (Promocell, C-12375, p2); HCH, primary chondrocytes isolated from normal human articular cartilage from the femoral head (Promocell, C-12710, p2); Hep G2, hepatocellular carcinoma cells (ATCC, HB-8065); HFF-1, primary foreskin fibroblasts (ATCC, SCRC-1041, p13); hNHeps, primary hepatocytes isolated from non-transplantable donor tissue (Lonza, CC-2591); HT-1080, fibrosarcoma cells (ATCC, CCL-121, p18); IMR-90, primary lung fibroblasts (ATCC, CCL-186, p10); MCF7, mammary carcinoma cells (ATCC, HTB-22, p146); MEF, immortalised mouse embryonic fibroblasts (provided by David J. Kwiatkowski, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA); NCCIT, embryonal carcinoma cells (ATCC, CRL-2073) and NTERA-2 cl.

Techniques: Western Blot, Transwell Invasion Assay, Co-Culture Assay, Injection, Staining, Cell Culture, Knockdown, Immunostaining, Control, Two Tailed Test

Journal: Nature communications

Article Title: Human stem cells alter the invasive properties of somatic cells via paracrine activation of mTORC1.

doi: 10.1038/s41467-017-00661-x

Figure Lengend Snippet: Fig. 7 Stem cell-induced invasion is mediated by the mTORC1-dependent activation of MMPs. a Gelatin zymography of conditioned medium for the analysis of secreted MMP2 in Rapamycin-treated IMR-90 cells co-cultured with stem cells. Results of independent experiments were densitometrically analysed (n ≥6; mean ± s.d.). The gel pictures were colour-inverted. b Transwell invasion assay of IMR-90 fibroblasts treated with Marimastat and co-cultured with stem cells (n ≥3; mean ± s.d.). c Transwell invasion assay of MMP2- or MMP14-depleted IMR-90 fibroblasts upon co-culture with stem cells (n ≥3; mean ± s.d.). Knockdown efficiency was assessed via gelatin zymography and immunoblotting. d Gelatin zymography of conditioned medium for the analysis of secreted MMP2 in MMP2- or MMP14-depleted IMR-90 fibroblasts upon co-culture with iPSCs. Data were densitometrically evaluated (OD). The bar indicates vertical cropping. e Reverse gelatin zymography of conditioned medium for the analysis of secreted TIMPs in IMR-90 fibroblasts co-cultured with stem cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired, two-tailed Student’s t-test analysis. n refers to biological replicates

Article Snippet: Somatic cells and embryonal carcinoma cell lines are the following: CCD1079Sk, primary foreskin fibroblasts (ATCC, CRL-2097, p4); HCF, primary cardiac fibroblasts isolated from the ventricle of an adult heart (Promocell, C-12375, p2); HCH, primary chondrocytes isolated from normal human articular cartilage from the femoral head (Promocell, C-12710, p2); Hep G2, hepatocellular carcinoma cells (ATCC, HB-8065); HFF-1, primary foreskin fibroblasts (ATCC, SCRC-1041, p13); hNHeps, primary hepatocytes isolated from non-transplantable donor tissue (Lonza, CC-2591); HT-1080, fibrosarcoma cells (ATCC, CCL-121, p18); IMR-90, primary lung fibroblasts (ATCC, CCL-186, p10); MCF7, mammary carcinoma cells (ATCC, HTB-22, p146); MEF, immortalised mouse embryonic fibroblasts (provided by David J. Kwiatkowski, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA); NCCIT, embryonal carcinoma cells (ATCC, CRL-2073) and NTERA-2 cl.

Techniques: Activation Assay, Zymography, Cell Culture, Transwell Invasion Assay, Co-Culture Assay, Knockdown, Western Blot, Two Tailed Test