Journal: Communications Chemistry

Article Title: Supercritical fluid chromatography-mass spectrometry enables simultaneous measurement of all phosphoinositide regioisomers

doi: 10.1038/s42004-022-00676-6

Figure Lengend Snippet: a – c MEFs were treated with 1 μM VPS34-IN, 200 nM Apilimod, or vehicle (DMSO) for 90 min, then PIPs were extracted and analyzed using SFC-MS/MS system. a The content of individual PIPs in MEFs (1 × 10 6 cells) treated with 1 μM VPS34-IN, 200 nM Apilimod or vehicle (DMSO) ( n = 3). b , c MRM chromatogram [987.6 → 605.6 ( b ) and 1117.6 → 627.6 ( c )] of extracts from MEFs (1 × 10 6 cells) treated with VPS34-IN, Apilimod or vehicle (DMSO). d – g MEFs were treated with 10 μM PAO, 1 μM A23187, or vehicle (DMSO) for 30 min, and then PIPs were extracted and analyzed using SFC-MS/MS system (see “Methods”). d , e The content of individual PIPs in MEFs (1 × 10 6 cells) treated with 10 μM PAO, 1 μM A23187 or vehicle (DMSO) ( n = 4). f , g Overlay of MRM chromatogram [987.6 → 605.6 ( f ) and 1117.6 → 627.6 ( g )] of extracts from MEFs (1 × 10 6 cells) treated with PAO, A23187, or vehicle (DMSO). h Contents of individual PIPs in MEFs (1 × 10 6 cells) transfected with the indicated siRNA ( n = 3). PIPs were extracted at 48 h after transfection. i , j Intracellular 38:4-PI(3,4,5)P 3 content ( i ) and species ( j ) in MEFs. MEF cells cultured in complete medium were serum-starved for 12 h (starved) and then treated with 10% FBS for 10 min (stimulated). PIPs were extracted from MEFs at each step and analyzed using SFC-MS/MS system. Data were collected using a QTRAP4500 mass spectrometer. Values are mean ± s.e.m. Data were analyzed by one-way ANOVA with Dunnett’s test. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs vehicle (DMSO). Data are from one set of experiments.

Article Snippet: The membranes were blocked with 5% (w/v) BSA in TTBS buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% (w/v) Tween 20] and incubated with an antibody to GAPDH (6C5; Calbiochem), monoclonal mouse anti-LPIAT1 antibody (clone: FT10) , VPS34 (sc-365404; Santa Cruz), Pikfyve (sc-100408; Santa Cruz), PI4Kα (12411-1-AP; Proteintech), and PI4Kβ (611816; BD laboratory).

Techniques: Tandem Mass Spectroscopy, Transfection, Cell Culture, Mass Spectrometry

Journal: eLife

Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

doi: 10.7554/elife.14023

Figure Lengend Snippet: Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:

Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

Techniques: Western Blot, Cell Culture, Staining, Fluorescence, Transduction, shRNA, Control, Transfection

Journal: eLife

Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

doi: 10.7554/elife.14023

Figure Lengend Snippet: Figure 5. Notch activity and autophagy regulate Treg suppressor function. (A) Representative, confocal images (central stack) of activated Tregs (upper panel), or T-effectors (lower panel) immune-stained for NIC (red) and Hoechst 33342 (blue). N: 15–20 cells/experiment. (B) Representative confocal (central stack) field views of activated Tregs immune-stained for Foxp3 (green) and Hoechst 33342 (blue). Tregs were derived from C57Bl/6 (wildtype, WT) or Cre negative (Cre-ve) or Cd4-Cre::Notch1lox/lox (Cre+ve) mice. (C) Real Time PCR quantification of genes enriched in Tregs, comparing activated Tregs from Cd4-Cre::Notch1lox/lox (open bars) and genetic control Cre-ive (black bars) mice. 5–6 mice are included in each group being compared. The data plotted is mean+/-SD. p**

Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

Techniques: Activity Assay, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Control, Expressing, Fluorescence, Generated, Isolation, Injection, Transduction, shRNA, Western Blot, Plasmid Preparation, Recombinant, In Vitro, Immunostaining, Flow Cytometry, Cell Culture