Journal: Chemical senses

Article Title: Green spotted puffers detect a nontoxic TTX analog odor using crypt olfactory sensory neurons.

doi: 10.1093/chemse/bjac011

Figure Lengend Snippet: Fig. 8. 5,6,11-TrideoxyTTX activates crypt OSNs. Reconstructed confocal microscopy images of 5,6,11-trideoxyTTX-administered OE displaying pS6-immunopositive (A), S100-immunopositive (B), and merged (pS6: magenta, S100: green; C). Double immunopositive crypt cell-like OSNs (arrows) had apical invagination, and their nuclei (the compartment missing cytoplasmic labeling of pS6/S100) were located in the lowest third of the soma. pS6/S100 double labeling was also observed in the elongated cells beneath the crypt cell-like OSNs (arrowheads). No pS6- immunopositive OSNs were observed in the vehicle (ACSF)-treated OE (D). Four fish examined for each condition showed similar results. For color figure refer online version. Scale bar: 10 µm.

Article Snippet: For double immunohistochemistry, sections were successively incubated with (i) a mixture of primary antibodies, anti-pS6 mouse monoclonal antibody (Ser235, Ser236 anti-pS6, 62016S, Cell Signaling Technology, Danvers, MA; 1:5,000) diluted with anti-S100 rabbit serum solution (Envision FLEX-S-100, Agilent Technologies, Santa Clara, CA) that was used in zebrafish crypt OSNs as validated in Germanà et al. (2004) for 2 days at 4°C; (ii) a mixture of secondary antibodies, Alexa Fluor 555-conjugated donkey anti-mouse IgG (A-31570, Thermo Fisher Scientific; 1:500) and biotinylated goat anti-rabbit IgG (BA1000, VECTOR Laboratories, Burlingame, CA; 1:500) for 1 day at 4°C; and (iii) an Alexa Fluor 488-conjugated streptavidin (S11223, Thermo Fisher Scientific; 1:500) for 2 h at 28°C.

Techniques: Confocal Microscopy, Labeling

Journal: Chemical senses

Article Title: Green spotted puffers detect a nontoxic TTX analog odor using crypt olfactory sensory neurons.

doi: 10.1093/chemse/bjac011

Figure Lengend Snippet: Fig. 7. pS6 immunohistochemistry of OE cross-sections exposed to 5,6,11-trideoxyTTX (10−6 M; A), l-Arg (10−6 M; B), vehicle (ACSF; C), or TTX (10−6 M; D). Scale bar: 10 µm. Six fish examined for each odorant showed similar results. Lower magnification confocal microscopy image of pS6- immunopositive OSNs in OE cross section exposed to 5,6,11-trideoxyTTX (E). Arrows: 5,6,11-trideoxyTTX-activated OSNs. Scale bar: 50 µm.

Article Snippet: For double immunohistochemistry, sections were successively incubated with (i) a mixture of primary antibodies, anti-pS6 mouse monoclonal antibody (Ser235, Ser236 anti-pS6, 62016S, Cell Signaling Technology, Danvers, MA; 1:5,000) diluted with anti-S100 rabbit serum solution (Envision FLEX-S-100, Agilent Technologies, Santa Clara, CA) that was used in zebrafish crypt OSNs as validated in Germanà et al. (2004) for 2 days at 4°C; (ii) a mixture of secondary antibodies, Alexa Fluor 555-conjugated donkey anti-mouse IgG (A-31570, Thermo Fisher Scientific; 1:500) and biotinylated goat anti-rabbit IgG (BA1000, VECTOR Laboratories, Burlingame, CA; 1:500) for 1 day at 4°C; and (iii) an Alexa Fluor 488-conjugated streptavidin (S11223, Thermo Fisher Scientific; 1:500) for 2 h at 28°C.

Techniques: Immunohistochemistry, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: IGF1-Stimulated Posttraumatic Hippocampal Remodeling Is Not Dependent on mTOR

doi: 10.3389/fcell.2021.663456

Figure Lengend Snippet: IGF1 potentiates mTOR activity in the dentate gyrus induced by moderate brain injury. (A) Representative microphotographs from confocal imaging of immunoreactivity of phospho-S6 Ribosomal Protein (pS6, white) in wildtype (WT) and insulin-like growth factor 1 transgenic (IGFtg) mice following sham injury and 24 and 72 h after moderate controlled cortical impact (CCI) injury. DAPI staining is shown as blue. Scale bar represents 100 μm. (B) Confocal images illustrate activation of S6 predominantly in neurons at 72 h after moderate CCI. Astrocytes (GFAP, green) did not colabel with pS6 (white) and most were not proliferating (PCNA, red). Scale bar represents 100 μm. (C) The area of pS6 immunostaining was significantly increased in injured IGFtg mice when compared to either injured WT mice or Sham controls in the granule cell layer (GCL) at 24 h and in the hilar layer (HL) at 72 h. In the molecular layer (ML), injury resulted in a small, transient increase in pS6 area at 24 h, which subsided by 72 h post-injury. pS6 + area is presented as a percent of the region of interest area. Data are presented as quartile box plots with min-max. One-way ANOVA, followed by post hoc tests: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 compared to Sham; # p < 0.05 compared to WT CCI. Group sizes: Sham ( n = 12; 7 WT, 5 IGFtg), 24 h CCI ( n = 10 WT, 11 IGFtg), 72 h CCI ( n = 11 WT, 11 IGFtg).

Article Snippet: Primary antibodies include: pS6 (1:100 rabbit polyclonal anti-pS6, Cat. #2215, Cell Signaling, Boston, MA), a well characterized downstream mTOR effector ( ); BrdU (1:1000 rat monoclonal anti-BrdU, Cat. #ab6326, Abcam, Boston, MA), a proliferation reporter ( ); the microtubule protein doublecortin (1:2500 rabbit polyclonal anti-Dcx, Cat. #ab18723, Abcam), a commonly used immature neuron marker ( ); proliferating cell nuclear antigen (1:500 mouse monoclonal anti-PCNA, Cat. #2586, Cell Signaling), a proliferation marker; NeuN (1:500 mouse monoclonal anti-NeuN, Cat. #MAB377, EMD Millipore Corporation, Burlington, MA), a marker for mature neurons; Iba1 (1:1,000 rabbit polyclonal anti-Iba1, Cat. #019-19741, FUJIFILM Wako Pure Chemical Corporation, Richmond, VA), a microglia marker; and glial fibrillary acidic protein (1:1,000 rabbit polyclonal anti-GFAP, Cat. #G9269, EMD Millipore or 1:1,000 chicken polyclonal anti-GFAP, Cat. #GFAP, Aves Labs, Inc., Davis, CA), an astrocyte marker.

Techniques: Activity Assay, Imaging, Transgenic Assay, Staining, Activation Assay, Immunostaining

Journal: Frontiers in Cell and Developmental Biology

Article Title: IGF1-Stimulated Posttraumatic Hippocampal Remodeling Is Not Dependent on mTOR

doi: 10.3389/fcell.2021.663456

Figure Lengend Snippet: Statistical analysis of regional pS6 immunolabeling data from the ipsilateral hippocampal dentate gyrus.

Article Snippet: Primary antibodies include: pS6 (1:100 rabbit polyclonal anti-pS6, Cat. #2215, Cell Signaling, Boston, MA), a well characterized downstream mTOR effector ( ); BrdU (1:1000 rat monoclonal anti-BrdU, Cat. #ab6326, Abcam, Boston, MA), a proliferation reporter ( ); the microtubule protein doublecortin (1:2500 rabbit polyclonal anti-Dcx, Cat. #ab18723, Abcam), a commonly used immature neuron marker ( ); proliferating cell nuclear antigen (1:500 mouse monoclonal anti-PCNA, Cat. #2586, Cell Signaling), a proliferation marker; NeuN (1:500 mouse monoclonal anti-NeuN, Cat. #MAB377, EMD Millipore Corporation, Burlington, MA), a marker for mature neurons; Iba1 (1:1,000 rabbit polyclonal anti-Iba1, Cat. #019-19741, FUJIFILM Wako Pure Chemical Corporation, Richmond, VA), a microglia marker; and glial fibrillary acidic protein (1:1,000 rabbit polyclonal anti-GFAP, Cat. #G9269, EMD Millipore or 1:1,000 chicken polyclonal anti-GFAP, Cat. #GFAP, Aves Labs, Inc., Davis, CA), an astrocyte marker.

Techniques: Immunolabeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: IGF1-Stimulated Posttraumatic Hippocampal Remodeling Is Not Dependent on mTOR

doi: 10.3389/fcell.2021.663456

Figure Lengend Snippet: IGF1 selectively enhances granule cell and hilar layer mTOR activity, without affecting delayed S6 activation in proliferating astrocytic in the molecular layer after severe injury. (A) Representative confocal images of immunoreactivity of phospho-S6 Ribosomal Protein (pS6, white) in wildtype (WT) and insulin-like growth factor 1 transgenic (IGFtg) mice following sham injury and 24 and 72 h after severe controlled cortical impact (CCI) injury. DAPI staining is shown as blue. Scale bar represents 100 μm. (B) Confocal images illustrating colocalization of pS6 (white) and proliferating cell nuclear antigen (PCNA, red) within astrocytes (GFAP, green) of the molecular layer (ML) in the hippocampus ipsilateral to impact at 72 h after severe CCI. Scale bar represents 100 μm. (C) In the granule cell layer (GCL) and the hilar layer (HL), IGFtg mice exhibited a significantly larger area of pS6 staining when compared to either injured WT mice or sham controls at 72 h after CCI. In the ML, both WT and IGFtg mice exhibited significant increases in pS6 levels at 72 h. pS6 + area is presented as a percent of the region of interest area. Data are presented as quartile box plots with min-max. One-way ANOVA, followed by post hoc tests: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 compared to Sham; # p < 0.05 compared to WT CCI. Group sizes: Sham ( n = 12; 7 WT, 5 IGFtg), 24 h CCI ( n = 9 WT, 10 IGFtg), 72 h CCI ( n = 9 WT, 10 IGFtg).

Article Snippet: Primary antibodies include: pS6 (1:100 rabbit polyclonal anti-pS6, Cat. #2215, Cell Signaling, Boston, MA), a well characterized downstream mTOR effector ( ); BrdU (1:1000 rat monoclonal anti-BrdU, Cat. #ab6326, Abcam, Boston, MA), a proliferation reporter ( ); the microtubule protein doublecortin (1:2500 rabbit polyclonal anti-Dcx, Cat. #ab18723, Abcam), a commonly used immature neuron marker ( ); proliferating cell nuclear antigen (1:500 mouse monoclonal anti-PCNA, Cat. #2586, Cell Signaling), a proliferation marker; NeuN (1:500 mouse monoclonal anti-NeuN, Cat. #MAB377, EMD Millipore Corporation, Burlington, MA), a marker for mature neurons; Iba1 (1:1,000 rabbit polyclonal anti-Iba1, Cat. #019-19741, FUJIFILM Wako Pure Chemical Corporation, Richmond, VA), a microglia marker; and glial fibrillary acidic protein (1:1,000 rabbit polyclonal anti-GFAP, Cat. #G9269, EMD Millipore or 1:1,000 chicken polyclonal anti-GFAP, Cat. #GFAP, Aves Labs, Inc., Davis, CA), an astrocyte marker.

Techniques: Activity Assay, Activation Assay, Transgenic Assay, Staining

Journal: bioRxiv

Article Title: Single cell proteomics of tumor compartments identifies differential kinase activities defining sensitivity to mTOR-PI3-kinase inhibition

doi: 10.1101/2021.01.06.425147

Figure Lengend Snippet: Bone metastases growth requires c-Met activity 2a: Dimensional reduction of the expression of all analyzed epitopes in single tumor cells in the PC3 mouse tumor metastasis model using viSNE shows a large fractured island consisting of dispersed primary tumor cells (black) and CTCs (red), and liver (green), lung (yellow) and bone (blue) metastatic cells. 2b: Signaling intermediates of the PI3K/mTOR- and MAPK-pathways and RTKs are elevated in bone metastases. Median epitope levels across single tumor cells isolated from the primary tumor and lung, liver and bone metastases as well as CTCs per mouse are shown in box plots. Each dot represents the median intensity of the epitope quantified (across single cells from each of the tumor compartment) for a single mouse. Ki67 q=0.3088/p=0.0702, pS6, pGSK3 q=0.002/p=0.0007, EGFR q=0.0098/p=0.0047, p4EBP1 q<0.0001/p<0.0001, pErk q<0.0001 p<0.0001, c-Met q=0.0294/p=0.0.0094 by one-way ANOVA, corrected for multiple comparisons by FDR, Benjamini, Krieger and Yekutieli. 2c: PI3K/mTOR index (measured as the sum of standardized pS6, p4EBP1, pAkt and pGSK3β levels) was significantly elevated in PC3 bone metastatic cells compared to primary tumor cells, CTCs, and metastatic cells in other sites. q<0.0001 by one-way ANOVA, corrected for multiple comparisons by FDR, Benjamini, Krieger and Yekutieli, whereas MAPK- and cytokine-signaling pathway signatures were not significantly different . 2d: viSNE of all analyzed epitopes in single tumor cells from the CE1-4 mouse prostate cancer model showing primary tumor cells (black), CTCs (red), and liver (green), lung (yellow) and bone (blue) metastatic cells. 2e: RTK index (measured as the sum of standardized EGFR, HER2, HER3, c-Met and IGF1R levels) is significantly elevated in CE1-4 bone metastatic cells compared to primary tumor cells, CTCs, and metastatic cells in other sites. q<0.0001 by one-way ANOVA, corrected for multiple comparisons by FDR, Benjamini, Krieger and Yekutieli. 2f: PI3K/mTOR index (measured as the sum of standardized pS6, p4EBP1, pAkt and pGSK3β levels) is significantly elevated in CE1-4 bone metastatic cells compared to primary tumor cells, CTCs, and metastatic cells in other sites. q<0.0001 by one-way ANOVA, corrected for multiple comparisons by FDR, Benjamini, Krieger and Yekutieli. 2g: mTOR and PI3K activities are elevated in CTCs isolated from the blood of mCRPC patients . Gene expression signatures associated with mTOR and PI3K activation are significantly higher in single cell RNA-sequencing data derived from 77 individual CTCs collected from 13 mCRPC patients (mean 6 CTCs per patient), compared with RNA-sequencing of 12 primary prostate cancers at single cell resolution. p<0.0023 and p<0.001 respectively, by Mann Whitney test. 2h: c-Met protein expression is significantly elevated prostate cancer bone metastases compared with primary tumors. Quantification of c-Met protein expression by IHC of primary human prostate cancer (n=6) and prostate cancer bone metastases (n=5). p=0.0066 by t-test. 2i: Suppression of c-Met impairs prostate tumor growth in the bone. c-Met depleted (using shRNAs against c-Met) and control PC3 (1000 cells each) and CE1-4 (250 cells each) were injected into the tibia of mice and their growth was monitored over 4 weeks and 16 weeks, respectively. Left panel showing bioluminescence signal over time and right panel showing number of tibiae with and without tumor growth observed at the final time point. p<0.01 by t-test. c-Met knockdown impaired tumor growth in the bone in both models. p=0.0188 for PC3, p=0.0114 for CE1-4 by t-test.

Article Snippet: The following primary antibodies were used for western blotting: c-Met (Cell Signaling; 1:1000), pS6 (Fluidigm; 1:1000), Total S6 (Cell Signaling; 1:1000), p4EBP1 (Fluidigm; 1:500), Total 4EBP1 (Cell Signaling; 1:1000), pAKT308 (Cell Signaling; 1:1000); pAKT473 (Fluidigm; 1:1000), Total Akt (Cell Signaling; 1:1000), pGSK3ß (Cell Signaling; 1:500), Total GSK3ß (Cell Signaling; 1:1000), ß-actin (Cell Signaling; 1:1000).

Techniques: Activity Assay, Expressing, Isolation, Gene Expression, Activation Assay, RNA Sequencing, Derivative Assay, MANN-WHITNEY, Control, Injection, Knockdown

Journal: bioRxiv

Article Title: Single cell proteomics of tumor compartments identifies differential kinase activities defining sensitivity to mTOR-PI3-kinase inhibition

doi: 10.1101/2021.01.06.425147

Figure Lengend Snippet: The primary tumors and bone metastases derived from the xenograft models shown were stained for c-Met, pS6 or p4EBP1 (DAB) and counterstained with hematoxylin. Representative images are shown in left. Scale bars represent 50 µm. Quantification of c-Met, pS6 and p4EBP1 levels show significantly increased expression for all three epitopes in bone metastases when compared to primary tumors (right panels). c-Met: p=0.0182 by Mann-Whitney test, pS6: p=0.0386, and p4EBP1 p=0.0019 by one-way ANOVA, Holm-Sidak’s multiple comparisons test.

Article Snippet: The following primary antibodies were used for western blotting: c-Met (Cell Signaling; 1:1000), pS6 (Fluidigm; 1:1000), Total S6 (Cell Signaling; 1:1000), p4EBP1 (Fluidigm; 1:500), Total 4EBP1 (Cell Signaling; 1:1000), pAKT308 (Cell Signaling; 1:1000); pAKT473 (Fluidigm; 1:1000), Total Akt (Cell Signaling; 1:1000), pGSK3ß (Cell Signaling; 1:500), Total GSK3ß (Cell Signaling; 1:1000), ß-actin (Cell Signaling; 1:1000).

Techniques: Derivative Assay, Staining, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Single cell proteomics of tumor compartments identifies differential kinase activities defining sensitivity to mTOR-PI3-kinase inhibition

doi: 10.1101/2021.01.06.425147

Figure Lengend Snippet: Dual PI3K/mTOR inhibitor treatment shows heterogeneous drug responsiveness of tumor deposits. 3a: Mouse model outlining the experiment to collect single cells from the primary and metastatic tumors and CTCs from untreated and BEZ235-treated mice harboring orthotopic PC3 tumor xenografts. CTCs were enriched from the mouse blood processed through the CTC-iChip. 3b: GFP and luciferase labeled PC3 cells were injected into the prostates of immune-compromised mice and 2-week old tumors were treated with BEZ235 or GDC0941 (n=6 mice for BEZ235; n=4 mice for GDC0941). BEZ235-treatment significantly suppressed tumor growth (week 5 p=0.3571, week 6 p=0.0032, week 7 p<0.0001, week 8 p<0.0001, two-way ANOVA, Dunnett correction). GDC0941 had no effect on tumor growth (week 5 p=0.9579, week 6 p=0.4257, week 7 p=0.9294, week 8<0.001, two-way ANOVA, Dunnett correction). Untreated tumor growth is shown as control (n=7 mice). 3c: BEZ235-treatment eliminates tumor growth in the bone. The bar graph shows that the percentage of mice with bone metastases in the untreated group is significantly higher than in the BEZ235-treated group (p=0.047 by Two-tailed Fisher’s exact test). 3d: PI3K/mTOR indices (measured as the sum of standardized pS6, p4EBP1, pAkt and pGSK3β levels) of single tumor cells enriched from the primary tumors, CTCs and lung and liver metastases collected from mice untreated or treated with BEZ235. PI3K/mTOR indices in primary tumor cells and CTCs were significantly decreased upon drug treatment (p<0.0001 by Mann-Whitney test); lung and liver metastases showed no significant change upon BEZ235 treatment. 3e-3g: pS6 and p4EBP1 staining of primary tumors resected from untreated control (CTR) and BEZ235-treated animals. Representative images are shown in and . Scale bar represents 100 µm. Quantification of staining indices in shows that BEZ235 treatment significantly reduced pS6 and p4EBP1 levels in primary tumors (n=3 mice and n=17 tumor areas for pS6, n=2 mice and n=18 tumor areas for p4EBP1). p<0.001 for p4EBP1, p=0.0011 for pS6 by Mann-Whitney test. 3h. PI3K/mTOR indices primary tumor-derived cells and CTCs treated with the PI3K inhibitor, GDC0941. Untreated controls are shown (CTR). GDC0941 significantly increased PI3K/mTOR levels in the primary tumor cells (p<0.001 by Mann-Whitney test), whereas the levels in CTCs did not change.

Article Snippet: The following primary antibodies were used for western blotting: c-Met (Cell Signaling; 1:1000), pS6 (Fluidigm; 1:1000), Total S6 (Cell Signaling; 1:1000), p4EBP1 (Fluidigm; 1:500), Total 4EBP1 (Cell Signaling; 1:1000), pAKT308 (Cell Signaling; 1:1000); pAKT473 (Fluidigm; 1:1000), Total Akt (Cell Signaling; 1:1000), pGSK3ß (Cell Signaling; 1:500), Total GSK3ß (Cell Signaling; 1:1000), ß-actin (Cell Signaling; 1:1000).

Techniques: Luciferase, Labeling, Injection, Control, Two Tailed Test, MANN-WHITNEY, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Single cell proteomics of tumor compartments identifies differential kinase activities defining sensitivity to mTOR-PI3-kinase inhibition

doi: 10.1101/2021.01.06.425147

Figure Lengend Snippet: Epithelial and mesenchymal tumor cell populations exhibit differential PI3K/mTOR activity. 4a: EpCAM (brown-DAB) and GFP (red-alkaline phosphatase) staining of primary tumors resected from untreated and BEZ235-treated mice. Sections were counterstained with hematoxylin. Clusters of EpCAM low cells (marked with hatched lines) pre-exist within EpCAM high cell populations in the untreated tumors. A EpCAM high cell cluster is marked with an arrow in the high magnification image of the inset. BEZ235-treated tumors have a prevalence of EpCAM low cells with very few EpCAM positive cells (marked by arrows in the higher magnification inset). Scale bar represents 50µm. 4b: Quantification of EpCAM intensity in the untreated and drug-treated primary tumors following immunohistochemical staining with an EpCAM antibody. p<0.001 by Mann-Whitney test. 4c: The bar graph shows the percentage of EpCAM low cells in untreated and BEZ235-treated primary tumor (PT) cells (p=0.037 by t-test) and in CTCs (p=0.0014 by t-test). EpCAM low is defined by the median level of EpCAM in all primary tumor cells. 4d: SPADE map of primary tumor cells and CTCs from untreated and BEZ235-treated mice shows two major tumor cell populations defined by EpCAM expression: EpCAM high (red) and EpCAM low (blue). The EpCAM high cells are further classified into three subclasses based on the expression levels: EpCAM high -RTK high -pAkt high , EpCAM hghi -pS6 high -p4EBP1 high , EpCAM hi -pAkt mid ) and mesenchymal subpopulations into four subclasses (EpCAM very-low -pS6 low , EpCAM very-low -pAkt low , EpCAM low pAkt mid , EpCAM low pS6 low ). The number of tumor cells within each compartment is illustrated using circles, the size of which corresponds to the number of cells. 4e: Median Ki67 and PI3K/mTOR levels were compared in SPADE nodes of untreated primary tumors that had low or high median EpCAM levels. EpCAM high nodes harbored significantly higher Ki67 (p=0.0003 by Mann-Whitney test) and PI3K/mTOR index (p<0.0001 by Mann-Whitney test).

Article Snippet: The following primary antibodies were used for western blotting: c-Met (Cell Signaling; 1:1000), pS6 (Fluidigm; 1:1000), Total S6 (Cell Signaling; 1:1000), p4EBP1 (Fluidigm; 1:500), Total 4EBP1 (Cell Signaling; 1:1000), pAKT308 (Cell Signaling; 1:1000); pAKT473 (Fluidigm; 1:1000), Total Akt (Cell Signaling; 1:1000), pGSK3ß (Cell Signaling; 1:500), Total GSK3ß (Cell Signaling; 1:1000), ß-actin (Cell Signaling; 1:1000).

Techniques: Activity Assay, Staining, Immunohistochemical staining, MANN-WHITNEY, Expressing