Journal: Disease Models & Mechanisms

Article Title: A novel mouse model of anterior segment dysgenesis (ASD): conditional deletion of Tsc1 disrupts ciliary body and iris development

doi: 10.1242/dmm.028605

Figure Lengend Snippet: Conditional deletion of Tsc1 leads to an elevation of mTORC1 signalling in tissue domains that underlie anterior segment development. (A-H) Immunohistochemical analysis of pS6 S235/236 in coronal eye sections taken from control (A-D) and Lhx2-Cre:Tsc1 f/f mice (E-H). No difference in pS6 S235/236 activity is seen at E10.5 (A,E). A modest elevation of pS6 S235/236 is detected within the CM, POM and LE of mutant animals at E12.5 (B,F). The levels of pS6 S235/236 continue to be elevated at E14.5 (C,G) and E16.5 (D,H) within the CM of Lhx2-Cre:Tsc1 f/f mice, whereas pS6 S235/236 levels within the POM and LE are similar to controls. (I-P) Immunohistochemical analysis of pS6 S240/244 in coronal eye sections taken from control (I-L) and Lhx2-Cre:Tsc1 f/f mice (M-P). No difference in pS6 S240/244 activity is seen at E10.5 (I,M). A robust elevation of pS6 S240/244 is detected within the CM, POM and LE of mutant animals at E12.5 (J,N). The levels of pS6 S240/244 continue to be elevated at E14.5 (K,O) and E16.5 (L,P) within the CM of Lhx2-Cre:Tsc1 f/f mice, whereas pS6 S240/244 levels within the POM and LE are similar to controls. Scale bar: 25 µm (A-P). LP, lens pit.

Article Snippet: The following antibodies and dilutions were used: αSMA (1:200, Abcam, ab5694 and 1:500, Sigma-Aldrich, C6198), β-gal (1:500, MP Biomedical, 0855976); Cav3 (1:500, BD Biosciences, 610421), neuronal β-III-tubulin (1:1000, Covance, MRB-435), Pax6 (1:100, DSHB, Clone P3U1), pS6 S235/236 (1:100, Cell Signaling Technology, 4857), pS6 S240/244 (1:100, Cell Signaling Technology, 5364) and Raldh1 (1:4000, Abcam, ab96060).

Techniques: Immunohistochemical staining, Control, Activity Assay, Mutagenesis

Journal: Cell

Article Title: B cell-specific diversion of glucose carbon utilization reveals a unique vulnerability in B cell malignancies

doi: 10.1016/j.cell.2018.02.048

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal (4856) anti-p-S6 (S235/236) , Cell Signaling Technology , Cat# 4856.

Techniques: Western Blot, Recombinant, Flow Cytometry, IP Phosphatase Assay, TUNEL Assay, Ab Array, ATP Bioluminescent Assay, Expressing, Chromatin Immunoprecipitation, Sequencing, Next-Generation Sequencing, CRISPR, Disruption, Luciferase, Plasmid Preparation, Dominant Negative Mutation, Software

Journal: Endocrinology

Article Title: Basement Membrane Laminin α 2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

doi: 10.1210/en.2016-1630

Figure Lengend Snippet: Antibodies Used for Different Experiments in This Report

Article Snippet: The use of rats for experiments reported herein was approved by the Rockefeller University Institutional Animal Care and Use Committee with Protocol Numbers 12-506 and 15-780-H. Antibodies were obtained commercially and listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Host Species Vendor Catalog Number Working Dilution IB IF/IHC Actin Goat Santa Cruz Biotechnology sc-1616 1:300 Akt Rabbit Cell Signaling Technology 9272 1:1000 p -Akt1 S473 Rabbit Cell Signaling Technology 4060 1:1000 p -Akt2 S474 Rabbit Cell Signaling Technology 8599 1:1000 Arp3 Mouse Sigma-Aldrich A5979 1:3000 1:50 α -tubulin Mouse Abcam ab7291 1:1000 1:300 β -catenin Rabbit Thermo Fisher Scientific 71-2700 1:250 1:100 β -tubulin Rabbit Abcam ab6046 1:1000 CAR Rabbit Santa Cruz Biotechnology sc-15405 1:200 1:50 Detyrosinated α -tubulin Rabbit Abcam ab48389 1:1000 Dia1 Goat Santa Cruz Biotechnology sc-10885 1:200 EB1 Mouse BD Biosciences 610534 1:200 EB1 Rabbit Santa Cruz Biotechnology sc-15347 1:200 Eps8 Mouse BD Biosciences 610143 1:5000 1:50 Laminin α 2 Mouse Millipore MAB1922 1:300 1:300 (tissue) 1:100 (cell) MARK2 Rabbit Proteintech 15492-1-AP 1:2000 MARK4 Rabbit Proteintech 20174-1-AP 1:2000 mTOR Rabbit Cell Signaling Technology 2983 1:2000 N -cadherin Mouse Thermo Fisher Scientific 33-3900 1:200 1:100 Raptor Rabbit Cell Signaling Technology 2280 1:1000 rpS6 Rabbit Cell Signaling Technology 2217 1:1000 p -rpS6 S235/236 Rabbit Cell Signaling Technology 4858 1:1000 p -rpS6 S240/244 Rabbit Cell Signaling Technology 5364 1:1000 ZO-1 Rabbit Thermo Fisher Scientific 61-7300 1:250 1:100 Goat IgG-HRP Bovine Santa Cruz Biotechnology sc-2350 1:3000 Rabbit IgG-HRP Bovine Santa Cruz Biotechnology sc-2370 1:3000 Mouse IgG-HRP Bovine Santa Cruz Biotechnology sc-2371 1:3000 Rabbit IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific A-21429 1:250 Mouse IgG-Alexa Fluor 488 Goat Thermo Fisher Scientific A-11029 1:250 Mouse IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"A21422","term_id":"583525","term_text":"A21422"}} A21422 1:250 Open in a separate window Abcam, Cambridge, MA; Cell Signaling Technology, Danvers, MA; Santa Cruz Biotechnology, Santa Cruz, CA; Sigma-Aldrich, St. Louis, MO; Invitrogen, Life Technologies, Carlsbad, CA; Proteintech, Chicago, IL; BD Biosciences, San Jose, CA; Millipore Corp., Billerica, MA.

Techniques:

Journal: Endocrinology

Article Title: Basement Membrane Laminin α 2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

doi: 10.1210/en.2016-1630

Figure Lengend Snippet: Knockdown of laminin α2 disrupts the Sertoli cell TJ-permeability barrier function through mTORC1-Akt signaling. Primary Sertoli cells cultured for 3 days were transfected with laminin α2 (Lam α2) vs nontargeting negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and cultured for additional 24 hours (for IF) or 48 hours (for IB). (a) Studies by IB illustrated laminin α2 was silenced by ∼50%. Knockdown of laminin α2 had no effect on the protein levels of TJ (e.g., CAR and ZO-1) and basal ES proteins (e.g., N-cadherin and β-catenin). Laminin α2 knockdown, however, induced an upregulation of p-rpS6 S235/236 and p-rpS6 S240/244 (the activated form of rpS6) and downregulated p-Akt1 S473 and p-Akt2 S474, but not the total rpS6 or total Akt. Each bar in the histogram is a mean ± SD of 6 independent experiments. ** P < 0.01 by Student t test. Changes in the steady-state level of p-rpS6 S235/236 and p-rpS6 S240/244 vs p-rpS6, and also p-Akt1 S473 and pAkt2 S474 vs total Akts, were shown in Supplemental Fig. 1. (b) Knockdown of laminin α2 perturbed the Sertoli cell TJ-permeability barrier function. Each data point is a mean ± SD of triplicate bicameral units of a representative experiment from a total of n = 3 independent experiments which yielded similar results. *P < 0.05; **P < 0.01 by Student t test. (c) Studies by IF confirmed laminin α2 expression in Sertoli cell cytosol but laminin α2 also prominently localized at the cell-matrix interface, consistent with the notion that laminin α2 is a basement membrane protein. Knockdown of laminin α2 by using specific shRNA considerably reduced its expression (red fluorescence) in Sertoli cells. Laminin α2 knockdown also caused mislocalization of BTB-associated proteins (e.g., CAR, ZO-1, N-cadherin, and β-catenin), such that these proteins no longer localized tightly at the cell-cell interface (see white brackets in cells transfected with control shRNA), but diffusely localized at the site (yellow brackets in cells transfected with laminin α2-specific shRNA), illustrating these proteins were being redistributed from the near the cell surface to the cell cytosol. Sertoli cell nuclei were visualized by DAPI (blue). The expression of GFP (green) illustrated successful transfection because GFP is an integrated component of the vector DNA. Scale bar, 30 μm. Histograms on the right panel summarized IF results for each target protein shown on the two left panels with each bar represents a mean ± SD of three experiments. Fluorescence at two opposite sides of two adjacent cells from 50 randomly selected cells were scored and analyzed from each experiment as described in Materials and Methods, and a total of three independent experiments were done, which yielded similar results. **P < 0.01 by Student t test.

Article Snippet: The use of rats for experiments reported herein was approved by the Rockefeller University Institutional Animal Care and Use Committee with Protocol Numbers 12-506 and 15-780-H. Antibodies were obtained commercially and listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Host Species Vendor Catalog Number Working Dilution IB IF/IHC Actin Goat Santa Cruz Biotechnology sc-1616 1:300 Akt Rabbit Cell Signaling Technology 9272 1:1000 p -Akt1 S473 Rabbit Cell Signaling Technology 4060 1:1000 p -Akt2 S474 Rabbit Cell Signaling Technology 8599 1:1000 Arp3 Mouse Sigma-Aldrich A5979 1:3000 1:50 α -tubulin Mouse Abcam ab7291 1:1000 1:300 β -catenin Rabbit Thermo Fisher Scientific 71-2700 1:250 1:100 β -tubulin Rabbit Abcam ab6046 1:1000 CAR Rabbit Santa Cruz Biotechnology sc-15405 1:200 1:50 Detyrosinated α -tubulin Rabbit Abcam ab48389 1:1000 Dia1 Goat Santa Cruz Biotechnology sc-10885 1:200 EB1 Mouse BD Biosciences 610534 1:200 EB1 Rabbit Santa Cruz Biotechnology sc-15347 1:200 Eps8 Mouse BD Biosciences 610143 1:5000 1:50 Laminin α 2 Mouse Millipore MAB1922 1:300 1:300 (tissue) 1:100 (cell) MARK2 Rabbit Proteintech 15492-1-AP 1:2000 MARK4 Rabbit Proteintech 20174-1-AP 1:2000 mTOR Rabbit Cell Signaling Technology 2983 1:2000 N -cadherin Mouse Thermo Fisher Scientific 33-3900 1:200 1:100 Raptor Rabbit Cell Signaling Technology 2280 1:1000 rpS6 Rabbit Cell Signaling Technology 2217 1:1000 p -rpS6 S235/236 Rabbit Cell Signaling Technology 4858 1:1000 p -rpS6 S240/244 Rabbit Cell Signaling Technology 5364 1:1000 ZO-1 Rabbit Thermo Fisher Scientific 61-7300 1:250 1:100 Goat IgG-HRP Bovine Santa Cruz Biotechnology sc-2350 1:3000 Rabbit IgG-HRP Bovine Santa Cruz Biotechnology sc-2370 1:3000 Mouse IgG-HRP Bovine Santa Cruz Biotechnology sc-2371 1:3000 Rabbit IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific A-21429 1:250 Mouse IgG-Alexa Fluor 488 Goat Thermo Fisher Scientific A-11029 1:250 Mouse IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"A21422","term_id":"583525","term_text":"A21422"}} A21422 1:250 Open in a separate window Abcam, Cambridge, MA; Cell Signaling Technology, Danvers, MA; Santa Cruz Biotechnology, Santa Cruz, CA; Sigma-Aldrich, St. Louis, MO; Invitrogen, Life Technologies, Carlsbad, CA; Proteintech, Chicago, IL; BD Biosciences, San Jose, CA; Millipore Corp., Billerica, MA.

Techniques: Knockdown, Permeability, Cell Culture, Transfection, Negative Control, shRNA, Expressing, Membrane, Fluorescence, Control, Plasmid Preparation

Journal: Endocrinology

Article Title: Basement Membrane Laminin α 2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

doi: 10.1210/en.2016-1630

Figure Lengend Snippet: Knockdown of laminin α2 disrupts the Sertoli cell F-actin organization through mTORC1-rpS6-Akt signaling. Primary Sertoli cells cultured for 3 days were transfected with laminin α2 (Lam α2) vs nontargeting negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours (for IF). For IB, transfected cells were cultured for additional 24 hours and then treated with rapamycin (100 ng/mL) for 24 hours before cells were harvested for lysate preparation. (a) Studies by IF confirmed that transfection of Sertoli cells with laminin α2 shRNA successfully knockdown laminin α2, as the expression of laminin α2 was considerably diminished in the laminin α2 silenced cells. However, rapamycin blocked the disruptive effect of laminin α2 knockdown on F-actin organization. Knockdown of laminin α2 caused truncation and defragmentation of F-actin network, whereas rapamycin rescued this disruptive effect, supporting the notion that laminin α2 exerts its effects through mTORC1 signaling. GFP expression (green fluorescence) illustrated successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm. (b) Studies by IB confirmed that a knockdown of laminin α2 activated mTORC1-rpS6-Akt signaling by upregulating p-rpS6 S235/236 and p-rpS6. S240/244 and downregulating p-Akt1 S473 and p-Akt2 S474, but not total rpS6 and Akts (i.e., Akt1, 2, and 3). Rapamycin inactivated p-rpS6/p-Akt pathway by abolishing the laminin α2 knockdown-induced up- and downregulation of p-rpS6 and p-Akt1/2, but rapamycin had no effect on mTOR, raptor, total rpS6, and Akt levels. Actin and α-tubulin served as the protein loading control.

Article Snippet: The use of rats for experiments reported herein was approved by the Rockefeller University Institutional Animal Care and Use Committee with Protocol Numbers 12-506 and 15-780-H. Antibodies were obtained commercially and listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Host Species Vendor Catalog Number Working Dilution IB IF/IHC Actin Goat Santa Cruz Biotechnology sc-1616 1:300 Akt Rabbit Cell Signaling Technology 9272 1:1000 p -Akt1 S473 Rabbit Cell Signaling Technology 4060 1:1000 p -Akt2 S474 Rabbit Cell Signaling Technology 8599 1:1000 Arp3 Mouse Sigma-Aldrich A5979 1:3000 1:50 α -tubulin Mouse Abcam ab7291 1:1000 1:300 β -catenin Rabbit Thermo Fisher Scientific 71-2700 1:250 1:100 β -tubulin Rabbit Abcam ab6046 1:1000 CAR Rabbit Santa Cruz Biotechnology sc-15405 1:200 1:50 Detyrosinated α -tubulin Rabbit Abcam ab48389 1:1000 Dia1 Goat Santa Cruz Biotechnology sc-10885 1:200 EB1 Mouse BD Biosciences 610534 1:200 EB1 Rabbit Santa Cruz Biotechnology sc-15347 1:200 Eps8 Mouse BD Biosciences 610143 1:5000 1:50 Laminin α 2 Mouse Millipore MAB1922 1:300 1:300 (tissue) 1:100 (cell) MARK2 Rabbit Proteintech 15492-1-AP 1:2000 MARK4 Rabbit Proteintech 20174-1-AP 1:2000 mTOR Rabbit Cell Signaling Technology 2983 1:2000 N -cadherin Mouse Thermo Fisher Scientific 33-3900 1:200 1:100 Raptor Rabbit Cell Signaling Technology 2280 1:1000 rpS6 Rabbit Cell Signaling Technology 2217 1:1000 p -rpS6 S235/236 Rabbit Cell Signaling Technology 4858 1:1000 p -rpS6 S240/244 Rabbit Cell Signaling Technology 5364 1:1000 ZO-1 Rabbit Thermo Fisher Scientific 61-7300 1:250 1:100 Goat IgG-HRP Bovine Santa Cruz Biotechnology sc-2350 1:3000 Rabbit IgG-HRP Bovine Santa Cruz Biotechnology sc-2370 1:3000 Mouse IgG-HRP Bovine Santa Cruz Biotechnology sc-2371 1:3000 Rabbit IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific A-21429 1:250 Mouse IgG-Alexa Fluor 488 Goat Thermo Fisher Scientific A-11029 1:250 Mouse IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"A21422","term_id":"583525","term_text":"A21422"}} A21422 1:250 Open in a separate window Abcam, Cambridge, MA; Cell Signaling Technology, Danvers, MA; Santa Cruz Biotechnology, Santa Cruz, CA; Sigma-Aldrich, St. Louis, MO; Invitrogen, Life Technologies, Carlsbad, CA; Proteintech, Chicago, IL; BD Biosciences, San Jose, CA; Millipore Corp., Billerica, MA.

Techniques: Knockdown, Cell Culture, Transfection, Negative Control, shRNA, Expressing, Fluorescence, Control

Journal: Endocrinology

Article Title: Basement Membrane Laminin α 2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

doi: 10.1210/en.2016-1630

Figure Lengend Snippet: A schematic drawing that illustrates the likely signaling pathway by which laminin α2 at the basement membrane regulates Sertoli cell BTB dynamics in the seminiferous epithelium of rat testes. Based on the findings reported herein, it is likely that an 80-kDa fragment is released from laminin α2 chain in the basement membrane, mediated by MMP-9 earlier reported in the rat testis (58). Once released, this fragment is being transported across the seminiferous epithelium. When the level of this 80-kDa laminin fragment is reduced—such as following its knockdown by RNAi as reported herein or mediated by its intrinsic downregulation mediated by miRNAs (e.g., endogenouosly produced siRNA) or its reduced spatiotemporal expression during the epithelial cycle—rpS6 is also activated through an induction of p-rpS6, which, in turn, downregulates p-Akt1/2. The activation of this mTORC1/rpS6/Akt1/2 signaling pathway induces BTB remodeling, causing a gradual disassembly of the “old” BTB above the preleoptotene spermatocytes connected in clones. Both TJ and basal ES proteins at the “old” BTB undergo endocytosis, transcytosis and recycling as earlier reported (67–69) to facilitate the assembly of the “new” BTB behind the preleptotene spermatocytes. This thus facilitates remodeling of the BTB at stage VIII of the epithelial cycle to support the transport of preleptotene spermatocytes across the immunological barrier, illustrating the physiological significance of laminin α2 chain in this series of cellular event.

Article Snippet: The use of rats for experiments reported herein was approved by the Rockefeller University Institutional Animal Care and Use Committee with Protocol Numbers 12-506 and 15-780-H. Antibodies were obtained commercially and listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Host Species Vendor Catalog Number Working Dilution IB IF/IHC Actin Goat Santa Cruz Biotechnology sc-1616 1:300 Akt Rabbit Cell Signaling Technology 9272 1:1000 p -Akt1 S473 Rabbit Cell Signaling Technology 4060 1:1000 p -Akt2 S474 Rabbit Cell Signaling Technology 8599 1:1000 Arp3 Mouse Sigma-Aldrich A5979 1:3000 1:50 α -tubulin Mouse Abcam ab7291 1:1000 1:300 β -catenin Rabbit Thermo Fisher Scientific 71-2700 1:250 1:100 β -tubulin Rabbit Abcam ab6046 1:1000 CAR Rabbit Santa Cruz Biotechnology sc-15405 1:200 1:50 Detyrosinated α -tubulin Rabbit Abcam ab48389 1:1000 Dia1 Goat Santa Cruz Biotechnology sc-10885 1:200 EB1 Mouse BD Biosciences 610534 1:200 EB1 Rabbit Santa Cruz Biotechnology sc-15347 1:200 Eps8 Mouse BD Biosciences 610143 1:5000 1:50 Laminin α 2 Mouse Millipore MAB1922 1:300 1:300 (tissue) 1:100 (cell) MARK2 Rabbit Proteintech 15492-1-AP 1:2000 MARK4 Rabbit Proteintech 20174-1-AP 1:2000 mTOR Rabbit Cell Signaling Technology 2983 1:2000 N -cadherin Mouse Thermo Fisher Scientific 33-3900 1:200 1:100 Raptor Rabbit Cell Signaling Technology 2280 1:1000 rpS6 Rabbit Cell Signaling Technology 2217 1:1000 p -rpS6 S235/236 Rabbit Cell Signaling Technology 4858 1:1000 p -rpS6 S240/244 Rabbit Cell Signaling Technology 5364 1:1000 ZO-1 Rabbit Thermo Fisher Scientific 61-7300 1:250 1:100 Goat IgG-HRP Bovine Santa Cruz Biotechnology sc-2350 1:3000 Rabbit IgG-HRP Bovine Santa Cruz Biotechnology sc-2370 1:3000 Mouse IgG-HRP Bovine Santa Cruz Biotechnology sc-2371 1:3000 Rabbit IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific A-21429 1:250 Mouse IgG-Alexa Fluor 488 Goat Thermo Fisher Scientific A-11029 1:250 Mouse IgG-Alexa Fluor 555 Goat Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"A21422","term_id":"583525","term_text":"A21422"}} A21422 1:250 Open in a separate window Abcam, Cambridge, MA; Cell Signaling Technology, Danvers, MA; Santa Cruz Biotechnology, Santa Cruz, CA; Sigma-Aldrich, St. Louis, MO; Invitrogen, Life Technologies, Carlsbad, CA; Proteintech, Chicago, IL; BD Biosciences, San Jose, CA; Millipore Corp., Billerica, MA.

Techniques: Membrane, Knockdown, Produced, Expressing, Activation Assay, Clone Assay