Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae

doi: 10.3389/fcimb.2022.921410

Figure Lengend Snippet: Dot blot using anti-GP63 (upper row) confirmed the presence of this GPI-anchored extracellular vesicle (EV) marker in EVs of both species and dot blot using anti-GFP (lower row) confirmed the presence of this cytosolic EV marker within EVs of both species. (A, F) L. tarentolae GFP+ extract as a positive control for tEV (35 µg protein in total). (B, G) L. tarentolae GFP+ EVs or tEV (1 µg in total). (C, H) L. major GFP+ extract as a positive control for mEV (35 µg in total). (D, I) L. major GFP+ EVs or mEV (1 µg in total). (E, J) Negative control (phosphate-buffered saline). The middle row depicts a separate dot blot using normal mouse sera as the primary antibody, which serves as a negative control. (a) L. tarentolae GFP+ extract (same as A, F ). (b) L. tarentolae GFP+ EVs or tEV (similar to B, G ). (c) L. major GFP+ extract (same as C, H ). (d) L. major GFP+ EVs or mEV (similar to D, I ). PC, positive control; tEV, L. tarentolae EV; mEV, L. major EV; NC, negative control.

Article Snippet: The GP63 antibody was provided by the Pasteur Institute of Iran, the anti-GFP-HRP goat polyclonal antibody was obtained from Acris antibodies GmbH (Germany), and the beta actin monoclonal antibody was purchased from ProteinTech Group Inc. (Chicago, IL, USA).

Techniques: Dot Blot, Marker, Positive Control, Negative Control

Journal: Communications Biology

Article Title: Plasmodium berghei liver stage parasites exploit host GABARAP proteins for TFEB activation

doi: 10.1038/s42003-024-07242-x

Figure Lengend Snippet: a HeLa cells constitutively expressing TFEBmCh were infected with Pb GFP sporozoites. Samples were fixed at 6, 15, 24, 30, and 48 hpi and stained with anti-GFP (green) and anti-RFP (red) antibodies to enhance the signals. Samples were analyzed with a widefield fluorescent microscope. Parasites are labeled with a white asterisk. Scale bar 50 µm. Note that in infected cells, TFEBmCh locates to the host cell nucleus whereas in non-infected cells TFEBmCh is in the cytoplasm. b HeLa cells expressing TFEBmCh were grown in full medium (control) or in starvation medium (EBSS) for 2 h. Samples were fixed and stained with anti-RFP antibodies to enhance the TFEB signal here shown in gray. Only in starved cells TFEB localizes to the cell nucleus. c Quantification of the experiment described in ( a ) and ( b ). Cells were fixed at indicated time points and stained with anti-GFP (only infected cells) and anti-RFP antibodies, pictures were taken with a widefield fluorescent microscope. Fluorescence intensity in the nucleus and the cell cytoplasm was measured and the ratio nuclear/cytoplasmic was calculated for each cell. A ratio above 1 indicates more nuclear than cytoplasmic TFEB, a ratio lower than 1 indicates more cytoplasmic than nuclear TFEB. Note that starved and Pb -infected cells have a ratio above 1 which means activated TFEB. Pictures were analyzed using Fiji. N > 30 for each cell line in each experiment. The graph depicts mean and SD of the pooled data of two independent experiments. P -values were calculated using a one-way ANOVA test. d mTORC1 activity in Pb -infected HeLa cells. Non-infected and Pb mCh-infected cells were fixed 24 hpi and stained with anti-pS6(Ser240/244) or with anti-p4E-BP1 antibodies to visualize phosphorylated substrates of the mTOR kinase. Note that mTOR is active in Pb -infected cells at the same level as in non-infected cells. Pictures were taken with a widefield fluorescent microscope and fluorescence intensity was measured using Fiji. N > 60 for each sample in each experiment. The graph depicts mean and SD of the pooled data of two independent experiments. P -values were calculated using a Student’s t test. e Control experiment for experiment described in ( d ). mTORC1 activity in non-treated, starved (EBSS, 2 h) and Torin 1 (200 nM, 2 h) treated HeLa cells. Cells were fixed and stained with anti-pS6(Ser240/244) or with anti-p4E-BP1 antibodies to visualize phosphorylated substrates of the mTOR kinase. Note that Torin 1 and EBSS inhibit mTOR kinase. Experiment was performed as described in ( d ).

Article Snippet: Primary antibodies used were, rat monoclonal anti-red antibody (Chromotek 5f8; 1:2000), rabbit polyclonal anti-GFP antibody (Origene, SP3005P; 1:1000), rabbit monoclonal anti-GFP antibody (Cell Signaling, CS2956; 1:1000), mouse monoclonal anti-GFP antibody (Roche 11814460001; 1:1000), rabbit monoclonal anti-pS6Ser240/244 antibody (Cell Signaling, CS5364; 1:1000), rabbit polyclonal anti-pS6Ser235/236 antibody (Cell Signaling, CS2211; 1:1000), rabbit monoclonal anti-p4E-BP1Thr37/46 antibody (Cell Signaling, CS2855; 1:1000), mouse monoclonal anti-V5 antibody (Invitrogen R960-25, 1:1000), rabbit anti-UIS4 antiserum (1:5000), anti-GABL1 ankyron protein (Proimmune, Ankyron1191, AH50324; 1:500), mouse monoclonal anti-HA antibody (Santa Cruz, SC-7392, 1:500).

Techniques: Expressing, Infection, Staining, Microscopy, Labeling, Control, Fluorescence, Activity Assay

Journal: Communications Biology

Article Title: Plasmodium berghei liver stage parasites exploit host GABARAP proteins for TFEB activation

doi: 10.1038/s42003-024-07242-x

Figure Lengend Snippet: a SopF inhibits TFEB nuclear translocation in Pb -infected HeLa cells. TFEBmCh, SopF expressing HeLa cells were infected with Pb GFP sporozoites. 24 hpi cells were fixed and stained with anti-GFP (green) and anti-RFP (red) antibodies to enhance the signals. Samples were analyzed with a widefield fluorescence microscope. Parasites are labeled with a white asterisk. Scale bar 50 µm. Note, in Pb -infected WT cells TFEB always localizes to the host cell nucleus, whereas in SopF expressing cells TFEB is cytoplasmic, even in infected cells. b Pb induced TFEB nuclear translocation depends on ATG16L1-WD40 domain. HeLa cells lacking ATG16L1, reconstituted with either ATG16L1-ΔWD40-BFP or with ATG16L1-β-BFP all expressing TFEBmCh, were infected with Pb GFP sporozoites. 24 hpi cells were treated as described in ( a ). Parasites are labeled with a white asterisk. Scale bar 50 µm. Note that nuclear TFEB can be found only in ATG16L1-KO cells expressing the WT ATG16L1-β-BFP. In ATG16L1-KO cells or KO cells reconstituted with ATG16L1-ΔWD40-BFP TFEB is trapped in the cytoplasm. c GABARAPs are indispensable for Pb induced TFEB nuclear translocation. TFEBmCh expressing HeLa cells lacking all 3 LC3s (LC3A, LC3B, LC3C), or all 3 GABARAPs (GAB, GABL1, GABL2) or lacking all LC3s and all GABs were infected with Pb GFP. 24 hpi cells were treated as described in ( a ). Parasites are labeled with a white asterisk. Scale bar 50 µm. Note that TFEB nuclear translocation only happens in the cell line expressing GABARAPs. d Quantification of the experiments described in ( a ), ( b ), and ( c ). All cell lines constitutively express TFEBmCh and were infected with Pb GFP. Cells were fixed 24 hpi and stained with anti-GFP (only infected cells) and anti-RFP antibodies, pictures were taken with a widefield fluorescence microscope. Fluorescence intensity in the nucleus and the cell cytoplasm was measured and the ratio nuclear/cytoplasmic was calculated for each cell A ratio above 1 indicates more nuclear than cytoplasmic TFEB, a ratio lower than 1 indicates more cytoplasmic than nuclear TFEB. Pictures were analyzed using Fiji. N > 30 for each cell line in each experiment. The graph depicts mean and SD of the pooled data of two independent experiments. P -values were calculated using a one-way ANOVA test. Note that Pb induced TFEB nuclear translocation depends on the ATG16L1-WD40 domain and on GABARAPs and can be inhibited by SopF.

Article Snippet: Primary antibodies used were, rat monoclonal anti-red antibody (Chromotek 5f8; 1:2000), rabbit polyclonal anti-GFP antibody (Origene, SP3005P; 1:1000), rabbit monoclonal anti-GFP antibody (Cell Signaling, CS2956; 1:1000), mouse monoclonal anti-GFP antibody (Roche 11814460001; 1:1000), rabbit monoclonal anti-pS6Ser240/244 antibody (Cell Signaling, CS5364; 1:1000), rabbit polyclonal anti-pS6Ser235/236 antibody (Cell Signaling, CS2211; 1:1000), rabbit monoclonal anti-p4E-BP1Thr37/46 antibody (Cell Signaling, CS2855; 1:1000), mouse monoclonal anti-V5 antibody (Invitrogen R960-25, 1:1000), rabbit anti-UIS4 antiserum (1:5000), anti-GABL1 ankyron protein (Proimmune, Ankyron1191, AH50324; 1:500), mouse monoclonal anti-HA antibody (Santa Cruz, SC-7392, 1:500).

Techniques: Translocation Assay, Infection, Expressing, Staining, Fluorescence, Microscopy, Labeling

Journal: Communications Biology

Article Title: Plasmodium berghei liver stage parasites exploit host GABARAP proteins for TFEB activation

doi: 10.1038/s42003-024-07242-x

Figure Lengend Snippet: a All 3 GABARAP proteins localize to Pb PVM. HeLa WT cells were transiently transfected with GFP-GABARAPs and approximately 15 h post transfection infected with Pb mCh sporozoites. 6 hpi infected cells were fixed and stained with anti-GFP antibodies (green) to enhance the GABARAP signal and anti-UIS4 antibodies (magenta) to visualize the Pb PVM. DNA was stained with Dapi (cyan). Images were taken with a confocal laser scanning microscope. Scale bar 5 µm. Note that GABARAP, GABARAPL1, and GABARAPL2 clearly localize to the P. berghei PVM. b Quantification of ( a ). Graph shows the Pearson’s correlation coefficient (PCC) for UIS4 and GFP-GABARAPs. PCC was calculated using the Coloc2 tool of FIJI. N = 5 parasites. Each dot represents one parasite, each red dot represents the parasites shown in ( a ). c PVM localization of GABARAPs depends on ATG16L1. HeLa cells lacking ATG16L1 were transiently transfected with GFP-GABARAPs and treated as described in ( a ). Images were taken with a confocal laser scanning microscope. Scale bar 5 µm. Note that none of the GABARAP proteins localizes to the Pb PVM in ATG16L1-KO cells. d Quantification of the experiments described in ( a ) and ( c ). The graph shows the percentage of GABARAP-positive parasites in HeLa WT and ATG16L1-KO cells. Only UIS4-positive parasites were counted. The graph depicts the mean and SD of two independent experiments. P -values were calculated using a Student’s t test. N \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge$$\end{document} ≥ 70 per experiment and cell line. e GABARAPs are needed for nuclear translocation of TFEB in Pb -infected cells. Quantification of the TFEB signal in GAB-3KO cells and GAB-3KO cells transiently transfected with each of the GFP-GABARAPs all constitutively expressing TFEBmCh. Cells were fixed 24 hpi and stained with anti-GFP and anti-RFP antibodies, pictures were taken with a widefield fluorescence microscope. Fluorescence intensity in the nucleus and the cell cytoplasm was measured and the ratio nuclear/cytoplasmic was calculated for each cell. A ratio above 1 indicates more nuclear than cytoplasmic TFEB, a ratio lower than 1 indicates more cytoplasmic than nuclear TFEB. Note that all GABARAPs are proficient to activate TFEB upon Pb infection. Pictures were analyzed using Fiji. N > 30 for each cell line in each experiment. The graph depicts mean and SD of the pooled data of two independent experiments. P -values were calculated using a one-way ANOVA test.

Article Snippet: Primary antibodies used were, rat monoclonal anti-red antibody (Chromotek 5f8; 1:2000), rabbit polyclonal anti-GFP antibody (Origene, SP3005P; 1:1000), rabbit monoclonal anti-GFP antibody (Cell Signaling, CS2956; 1:1000), mouse monoclonal anti-GFP antibody (Roche 11814460001; 1:1000), rabbit monoclonal anti-pS6Ser240/244 antibody (Cell Signaling, CS5364; 1:1000), rabbit polyclonal anti-pS6Ser235/236 antibody (Cell Signaling, CS2211; 1:1000), rabbit monoclonal anti-p4E-BP1Thr37/46 antibody (Cell Signaling, CS2855; 1:1000), mouse monoclonal anti-V5 antibody (Invitrogen R960-25, 1:1000), rabbit anti-UIS4 antiserum (1:5000), anti-GABL1 ankyron protein (Proimmune, Ankyron1191, AH50324; 1:500), mouse monoclonal anti-HA antibody (Santa Cruz, SC-7392, 1:500).

Techniques: Transfection, Infection, Staining, Laser-Scanning Microscopy, Translocation Assay, Expressing, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

doi: 10.1101/163279

Figure Lengend Snippet: a) Pictures of Malpighian tubules with esg ts -driven expression of GFP (control) and indicated transgenes. Quantification of esg+ cells is given on each picture (see also Supplementary Figure 3f). (b) Drawing of the DIAP1 locus. Exons are represented in cyan, the DIAP1-4.3 enhancer in dark green and the insertion site of the DIAP1-LacZ reporter (J5C8) is in red. Regions bound in ChIP-seq (MACS peaks) by Svb and Yki are indicated in green and magenta,respectively. (c) Svb co-immuno-precipitates with Yki in S2 cells. Svb::GFP and Yki::HA, or a mutated form of Yki substituting the WW domains (YkiWW::HA), were expressed in S2 cells. Protein immuno-precipitated by anti-GFP were blotted with anti-GFP and anti-HA antibodies. (d) esg ts was used to drive the expression of yki , svb REP ( ovoA ), and yki together with svb REP in RNSCs. The expression of DIAP1 was followed by the activity of DIAP1-lacZ . (e) Expression of the DIAP1-4.3-GFP enhancer was followed by immuno-staining against GFP (green) and Hindsight (Hnt, red) revealing the antagonistic influence of Yki and OvoA on RNSCs.

Article Snippet: Immuno-precipitated samples were separated by SDS-PAGE and transferred to PVDF membranes, then blotted using anti-GFP (TP401, Acris Antibodies, 1:10000) and anti-HA (Covance, 1:2.000) antibodies.

Techniques: Expressing, ChIP-sequencing, Activity Assay, Immunostaining