human embryonic kidney 293t cells  (ATCC)

Journal: Bio-protocol

Article Title: Detecting the Activation of Endogenous Small GTPases via Fluorescent Signals Utilizing a Split mNeonGreen: Small GTPase ActIvitY ANalyzing (SAIYAN) System

doi: 10.21769/BioProtoc.5557

Figure Lengend Snippet: (A) SAIYAN cells derived from BJ-5ta cells were treated with L-ascorbic acid phosphate and cultured for 2 days, followed by fixation and staining with anti-Sec16A and anti-ERGIC53 antibodies. Images were acquired using an Airyscan2 confocal microscope. Scale bars, 10 µm. (B) Magnified view of (A) showing that the mNG signal is distributed adjacent to the ER exit site marker Sec16A (red arrowheads) and along ERGIC-53-positive structures (blue arrowheads). Scale bars, 1 µm. ©2024 Maeda et al. [8]. Originally published in Journal of Cell Biology (2024), DOI: 10.1083/jcb.202403179. SAIYAN: Small GTPase ActIvitY Analyzing system.

Article Snippet: BJ-5ta cells (ATCC, catalog number: CRL-4001) 4.

Techniques: Derivative Assay, Cell Culture, Staining, Microscopy, Marker, Activity Assay

Journal: bioRxiv

Article Title: An ER–Inner Membrane Complex Bridge via TgVAP-TgVPS13A-TgDAT1 Drives Daughter Budding in Toxoplasma gondii

doi: 10.64898/2026.01.02.697355

Figure Lengend Snippet: (A) The schematic representation illustrating the DiCre-LoxP system used to conditionally deplete TgVPS13A. (B) PCR analysis confirming the N-terminal recombination of TgVPS13A involving the two LoxP sites. (C) IFA analysis showing the expression of TgVPS13A in parasites cultured with or without rapamycin (Rapa) for 96 h. (D) Plaque assays were conducted by infecting BJ-5ta cells with the specified parasites for 9 days, either in the presence or absence of Rapa. (E) Replication of the indicated parasites in host cells was assessed after 96 h of culture with or without Rapa. The average number of parasites per vacuole was calculated, and the data were analyzed using a two-way ANOVA, revealing statistical significance (****P < 0.0001). (F) IFA analysis of IMC biogenesis involved staining for TgIMC29 and TgGAPM3. The abnormal and normal PVs were counted from TgGAPM3. The data were analyzed for statistical significance by the unpaired t-test; ****P<0.0001. Schematic diagram of the assembly of early buds of daughter IMCs in TgVPS13A-deficient parasites. (G) Transmission electron microscopy analysis of daughter parasites treated with and without Rapa for 96 h was performed. In the images, ‘N’ indicates the nucleus. (H) IFA analysis of the IMC-associated cytoskeleton by examining the localization of TgIMC1 and Tgtubulin in TgVPS13A-deficient parasites. The abnormal and normal PVs were counted from H. The data were analyzed for statistical significance by the unpaired t-test; ****P<0.0001. (I) A schematic model of the IMC biogenesis and daughter cell budding in TgVPS13A-delepleted parasites. PL indicates Phospholipid. Green: rabbit anti-HA antibody, rabbit anti-TgIMC1 polyclonal antibodies, rabbit anti-TgTubulin polyclonal antibodies, and EGFP signal; Magenta: rabbit and mouse anti-TgSAG2 polyclonal antibodies; Blue: DAPI. Scale bars represent 2 µm.

Article Snippet: The hTERT-immortalized HFF cell line BJ-5ta (ATCC CRL-4001) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (CLARK, FB25015), 20% (v/v) Medium 199 basic (gibco, C11150500BT), and 1% penicillin/streptavidin.

Techniques: Expressing, Cell Culture, Staining, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: An ER–Inner Membrane Complex Bridge via TgVAP-TgVPS13A-TgDAT1 Drives Daughter Budding in Toxoplasma gondii

doi: 10.64898/2026.01.02.697355

Figure Lengend Snippet: (A) IFA was performed to examine the colocalization of TgVAP with TgVPS13A-N 1-1392 before and after division. The parasites were transfected with a plasmid expressing TgTgVPS13A-N1-1392-SmFP-Myc, under the control of the native promoter. (B) Co-IP was performed using 3×FLAG-tagged TgVAP and 3×HA-tagged TgVPS13A-N928-1392aa. Cells were transiently transfected with plasmids expressing 3×FLAG-tagged TgVAP and 3×HA-tagged TgVPS13A-N928-1392aa. The resulting cell lysates were subjected to immunoprecipitation and then blotted with anti-FLAG and anti-HA antibodies. (C) A schematic diagram of inserting a 6×HA-AID* tag at the N-terminal of TgVAP. (D) PCR identification of the 6×HA-AID* tag inserted at the N-terminal of TgVAP. (E) The expression of TgVAP was examined using western blot analysis. The parasites were cultured in the presence of IAA for 24 h in BJ-5ta cells and then subjected to the analysis. WT refers to the wild type. TgVAP expression was detected using anti-HA antibodies. (F) IFA analysis of TgVAP expression in parasites with and without IAA treatment for 24 h. (G) Plaque assays were conducted by infecting BJ-5ta cells with the parasites for 9 days, both in the presence and absence of IAA. (H) The parasites were cultured with or without IAA for 48 h to assess the biogenesis of the IMC in daughter cells, using TgIMC29 for staining. The EGFP tag was inserted at the C-terminus of TgIMC29 in the parasites. (I) IFA analysis showing the impact of TgIMC1 and Tgtubulin in TgVAP-deficient parasites with and without IAA treatment for 48 h. (J) A schematic diagram of the effect of TgVAP on IMC biogenesis. Magenta: rabbit anti-HA antibody, mouse and rabbit anti-TgSAG2 polyclonal antibodies; Green: rabbit anti-HA antibody, mouse anti-Myc antibody, rabbit anti-TgTubulin polyclonal antibodies, EGFP signal and anti-TgIMC1 polyclonal antibodies. Scale bars measure 2 μm in the merged panels and 0.2 μm in the zoomed-in panels.

Article Snippet: The hTERT-immortalized HFF cell line BJ-5ta (ATCC CRL-4001) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (CLARK, FB25015), 20% (v/v) Medium 199 basic (gibco, C11150500BT), and 1% penicillin/streptavidin.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Cell Culture, Staining

Journal: bioRxiv

Article Title: An ER–Inner Membrane Complex Bridge via TgVAP-TgVPS13A-TgDAT1 Drives Daughter Budding in Toxoplasma gondii

doi: 10.64898/2026.01.02.697355

Figure Lengend Snippet: (A) PCR identification of the 12×HA-AID* tag inserted at the N-terminal of TgDAT1. (B) Expression of TgDAT1 was examined using western blot analysis. The parasites were cultured in the presence of IAA for 24 h in BJ-5ta cells and then subjected to the analysis. WT refers to the wild type. TgDAT1 expression was detected using anti-HA antibodies. (C) Plaque assays were performed by infecting BJ-5ta cells with the parasites for 9 days in the presence or absence of IAA. (D) The biogenesis of the daughter IMC was investigated by examining the localization of TgIMC29 and TgGAPM3 in TgDAT1-deficient parasites. An EGFP tag or a mCherry tag was inserted at the C-terminal of TgIMC29 or TgGAPM3 in the 12×HA-AID*-TgDAT1 cell line respctively. Parasites were treated with IAA for 48 h. The abnormal and normal PVs were counted from TgGAPM3. The data were analyzed for statistical significance by the unpaired t-test; ****P<0.0001. Schematic diagram of the assembly of early buds of daughter IMCs in the TgDAT1-KO parssites. (E) IFA analysis showing the assembly of TgTubulin in TgDAT1-deficient parasites. The abnormal and normal PVs were counted. The data were analyzed for statistical significance by the unpaired t-test; ns. (F) The biogenesis of the early IMC was investigated by staining with localization of TgAC9 in TgDAT1-deletion strain. Parasites were transfected with plasmids expressing TgAC9-3×MYC under the control of the the GRA1 promoter. The abnormal and normal PVs were counted. The data were analyzed for statistical significance by the unpaired t-test; ****P<0.0001. (G) A model of TgDAT1 and the potential sites of salt bridges predicted by AlphaFold and Chimera. (H) IFA analysis showing the localization of TgDAT1 mutants at the IMC. Parasites were transfected with plasmids expressing mutated versions of TgDAT1-SmFP-MYC under the control of the native promoter, and those stably expressing these mutants were selected using pyrimethamine. (I) Plaque assays were performed by infecting BJ-5ta cells with TgDAT1 mutants and wild-type parasites for 8 days, both in the presence and absence of IAA. (J) IFA showing the localization of TgGAPM3 in salt-bridge mutants of TgDAT1. An endogenous mCherry tag was inserted into the C-terminus of TgGAPM3 in parasites. The abnormal and normal PVs were counted. The data were analyzed for statistical significance by the unpaired t-test; ****P<0.0001. (K) A model indicating abnormal IMC biogenesis in TgDAT1-deficient parasites. Magenta: rabbit anti-TgSAG2 polyclonal antibodies, mCherry, rabbit anti-HA; Green: EGFP signal, rabbit anti-TgSAG2, rabbit anti-TgTubulin polyclonal antibodies, and mouse anti-MYC; Blue: DAPI. Scale bars: 2 μm.

Article Snippet: The hTERT-immortalized HFF cell line BJ-5ta (ATCC CRL-4001) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (CLARK, FB25015), 20% (v/v) Medium 199 basic (gibco, C11150500BT), and 1% penicillin/streptavidin.

Techniques: Expressing, Western Blot, Cell Culture, Staining, Transfection, Control, Stable Transfection