Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sar1 membrane association is efficiently detected using the Split mNeonGreen system. (A) Schematic representation of the SAIYAN system. The membrane-spanning regions of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG (mNG 1–10 ) were expressed in cells. In addition, Sar1A constructs with a FLAG-tag and a glycine linker fused to the 11th strand of mNG (mNG 11 ) were also expressed. Upon Sar1A activation, mNG 1–10 and mNG 11 combined to form the complete mNG proteins, inducing mNG signals. (B) HA-mNG 1–10 cells transfected with the indicated Sar1A constructs were fixed and stained with anti-Sec16-C and anti-FLAG antibodies. Scale bar = 10 µm. (C) Quantification of mNG intensity from B (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Membrane, Construct, FLAG-tag, Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Validation of SAIYAN technology. (A) Doxycycline-inducible HeLa cells expressing the membrane-spanning region of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG (HA-mNG 1–10 cells) were either non-transfected or transfected with Sar1A constructs with a FLAG tag and a glycine linker fused to the 11th strand of mNG (Sar1A-FLAG-mNG 11 ). The cells were fixed and stained with anti-HA and anti-PDI antibodies. Scale bar = 10 µm. (B) HA-mNG 1–10 cells, treated with or without doxycycline, were transfected with Sar1A-FLAG-mNG 11 . The cells were fixed and stained with anti-HA and anti-FLAG antibodies. Scale bar = 10 µm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Expressing, Membrane, Transfection, Construct, FLAG-tag, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Production of Sar1A/SAIYAN cells. (A) Doxycycline-inducible stable cell lines expressing the membrane-spanning regions of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG were established using a lentiviral system and G418 selection (HA-mNG 1–10 cells). Stable cells were subsequently electroporated with Cas9 protein, sgRNA, and ssDNA to facilitate the knock-in of FLAG-mNG 11 into the Sar1A locus of the genome. Cells were treated with doxycycline for 24 h and further sorted via FACS to isolate single cells exhibiting mNG signals into 96-well plates. The expanded cell population was then collected and subjected to genomic sequencing. Positive clones were identified and selected for further analysis (Sar1A/SAIYAN cells). (B) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fixed and stained with anti-HA and anti-PDI antibodies. Boxed areas in the middle panels are shown at high magnification in the bottom panels. Scale bars: 10 µm (main), 5 µm (magnification). (C) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fixed and stained with anti-HA and anti-FLAG antibodies. Boxed areas in the middle panels are shown at high magnification in the bottom panels. Scale bar:10 µm (main), 5 μm (magnification). (D) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with an anti-Sec16-C antibody. Scale bar = 10 µm. (E) Quantification of mNG intensity from D (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells. (F) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sar1A, and anti-GAPDH antibodies. (G) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fractionated via centrifugation. The lysates, the supernatants, and the pellets were subjected to SDS-PAGE, followed by western blotting with anti-FLAG, Sar1A, HA, ERK1, and calnexin antibodies. Source data are available for this figure: .

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Stable Transfection, Expressing, Membrane, Selection, Knock-In, Genomic Sequencing, Clone Assay, Staining, Transfection, SDS Page, Western Blot, Centrifugation

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sar1A/SAIYAN (HeLa) cells proliferate and secrete normally. (A) HeLa and Sar1A/SAIYAN (HeLa) cells were treated with or without doxycycline for 24 h, and cell viability was measured and normalized using untreated HeLa cells as control. Error bars represent the means ± SEM. n = 4. (B) Sar1A/SAIYAN (HeLa) cells, treated with or without doxycycline, were fixed and stained with anti-Sec16-C and anti-GM130 antibodies. Scale bars = 10 µm. (C) Sar1A/SAIYAN (HeLa) cells treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry. RUSH chase was started with biotin addition, and live imaging was performed. Scale bars = 10 μm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Control, Staining, Transfection, Imaging

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: ER exit site organization is required for the efficient activation of Sar1A. (A, C, E, and G) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with anti-Sec16-C antibodies. Scale bar = 10 µm. (B, D, F, and H) Quantification of mNG signals from A, C, E, and G, respectively (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Western blotting analysis of Sar1A/SAIYAN (HeLa) cells on , , and . (A) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec12, and anti-GAPDH antibodies. (B) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-cTAGE5 CC1, anti-Sec12, and anti-GAPDH antibodies. (C) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-TANGO1 CC1, and anti-GAPDH antibodies. (D) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec16-N, and anti-GAPDH antibodies. (E) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec23A (11D8), and anti-GAPDH antibodies. (F) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec31A (rabbit), and anti-GAPDH antibodies. (G) Sar1A/SAIYAN (HeLa) cells were stably expressed using mCherry-tagged Sec23A constructs as indicated. Cells were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec23A (11D8), anti-Sec31A (rabbit), and anti-GAPDH antibodies. Source data are available for this figure: .

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Western Blot, Transfection, SDS Page, Stable Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sec23A and Sec31A depletion exerts opposite effects on the activation of Sar1A in living cells. (A and C) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with anti-Sec16-C antibodies. Scale bar = 10 µm. (B and D) Quantification of mNG signals from A and C, respectively (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells. (E) Sar1A/SAIYAN (HeLa) cells transfected with the indicated plasmids were fixed and processed for microscopic analysis. Scale bar = 10 µm. (F) Quantification of mNG signals from E (A.U.). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Each CLSD mutant of Sec23A exhibits different properties on Sar1 activation. (A) Sar1A/SAIYAN (HeLa) cells stably expressing mCherry-tagged Sec23A constructs, as indicated, were fixed and processed for microscopic analysis. Scale bar = 10 µm. (B) Quantification of mNG signals from A (A.U.). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Mutagenesis, Activation Assay, Stable Transfection, Expressing, Construct

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: DPD treatment accumulates collagen I within the ER of Sar1A/SAIYAN (BJ-5ta) cells. Sar1A/SAIYAN (BJ-5ta) cells were treated with DMSO or 0.5 mM DPD and incubated for 16 h. Cells were fixed and stained with an anti-collagen I antibody. Scale bar = 10 µm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Activated Sar1 prevails in the ERGIC region of Sar1A/SAIYAN (BJ-5ta) cells. (A–O) Sar1A/SAIYAN (BJ-5ta) cells were fixed and stained with anti-Sec16-C (A), anti-ERGIC53 (B), anti-Sec23 (5H2) (C), anti-Sec24B (D), anti-Sec24D (E), anti-p125A (F), anti-TANGO1-CT (G), anti-Sec12 (H), anti-TFG (I), anti-Sec13 (J), anti-Sec31A (mouse) (K), anti-β-COP (L), anti-GM130 (M), anti-PDI (N), and anti-Rab1A (O) antibodies. Images were captured using Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (A–L) Right; top: Magnification of the indicated regions is on the left. Right; bottom: Magnification of the indicated regions on the upper. (P) Pearson’s correlation coefficient was used to quantify the degree of colocalization. n = 5. Cyan; outer COPII coats, blue; inner COPII coats, purple; ER exit site resident proteins, red; ERGIC proteins, orange; COPI protein, magenta; ER and Golgi proteins. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Triple staining of Sar1A/SAIYAN (BJ-5ta) and parental BJ-5ta reveals the organization of the ER-Golgi interface of collagen-secreting cells. (A–C) Sar1A/SAIYAN (BJ-5ta) cells were fixed and stained with anti-Sec16-C and anti-ERGIC53 (A), anti-Sec23 (5H2), and anti-ERGIC53 (B), and anti-Rab1A and anti-ERGIC53 (C) antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (D) BJ-5ta cells were fixed and stained with anti-Sec16-C, anti-Sec23 (5H2), and anti-ERGIC53 antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (A–D) (right; top) Magnification of the indicated regions is on the left. (right; bottom) Double staining of the magnified region on the top.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining, Double Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Quantification of Pearson’s correlation coefficient to quantify the degree of colocalization in Sar1A/SAIYAN (HeLa) cells. Sar1A/SAIYAN (HeLa) cells were fixed and stained with anti-Sec16-C, anti-ERGIC53, anti-Sec23, anti-Sec24B, anti-Sec24D, anti-p125A, anti-TANGO1-CT, anti-Sec12, anti-TFG, anti-Sec13, anti-Sec31A (mouse), anti-β-COP, anti-GM130, anti-PDI, and anti-Rab1A antibodies. Images were captured using the Airyscan2. n = 5. Cyan; outer COPII coats, blue; inner COPII coats, purple; endoplasmic reticulum (ER) exit site resident proteins, red; ERGIC proteins, orange; COPI protein, magenta; ER and Golgi proteins. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Reticular pattern of activated Sar1 signals diminished with DPD treatment in Sar1A/SAIYAN (BJ-5ta) cells. (A) Sar1A/SAIYAN (BJ-5ta) cells were treated with DMSO or 0.5 mM DPD and incubated for 16 h. Cells were fixed and stained with anti-Sec16-C antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (B) Pearson’s correlation coefficient was quantified to assess the degree of colocalization. n = 5. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Incubation, Staining

Journal: bioRxiv

Article Title: Improved long-transcript representation in Oxford Nanopore direct RNA sequencing with UltraMarathonRT

doi: 10.64898/2025.12.10.693495

Figure Lengend Snippet: (A) Stability of a 7.6 kb HCV small genome RNA fragment was tested by incubation in the reaction buffers for UltraMarathonRT, Maxima H Minus, SuperScript IV, and Induro at their recommended reaction temperatures for 0, 10, 20, and 30 minutes, respectively. No reverse transcription was performed. The RNA samples were then analyzed using the Agilent™ 2100 Bioanalyzer™ system. (B) Amplification of human RNAs with lengths ranging from 1.2 kb to 20 kb using UltraMarathonRT Two-Step RT-PCR Kit. Oligo(dT) primed reverse transcription was performed using 100 ng of Hela total cellular RNA. PCR Amplification of cDNA was performed using gene-specific primers for GAPDH, ACTB, PGK1, UBE4B, SRGAP2C, XRN1, HERC1, & SMAD2.

Article Snippet: RT-PCR was performed on 100 ng of HeLa total cellular RNA (Takara TM , Cat#636543) using UltraMarathonRT ® Two-Step RT-PCR Kit (RNAConnect TM , Cat#R1005) according to the manufacturer recommendations.

Techniques: Incubation, Reverse Transcription, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Gels

Article Title: Enzyme Responsive Vaginal Microbicide Gels Containing Maraviroc and Tenofovir Microspheres Designed for Acid Phosphatase-Triggered Release for Pre-Exposure Prophylaxis of HIV-1: A Comparative Analysis of a Bigel and Thermosensitive Gel

doi: 10.3390/gels8010015

Figure Lengend Snippet: Cytotoxic effect of the varying concentrations of maraviroc and tenofovir microspheres, negative and positive control on the HeLa cell lines. Results are given as mean ± SD, n = 3.

Article Snippet: Cell lines: HeLa cells and fetal bovine serum were purchased from Cell Lines Service GmbH, Germany.

Techniques: Positive Control

Journal: bioRxiv

Article Title: Outer membrane vesicles produced by pathogenic strains of Escherichia coli block autophagic flux and exacerbate inflammasome activation

doi: 10.1101/2021.04.20.440604

Figure Lengend Snippet: Autophagy flux blocked by OMVs HlyF WT at lysosomal fusion step. ( A ) Confocal images of DiFluo Hela cells deprived with HBSS or treated for 3hr with 50μM chloroquine or with OMVs from BL21 HlyF WT 5μg/mL. Images of LC3-GFP fluorescence (green), LC3-RFP fluorescence (red), and the overlay with DAPI (blue) which shows co-localization of GFP- and RFP-positive puncta. Scale bar = 10μm. 50 cells were analyzed per condition. Images representative of 3 independent experiments. ( B ) Images acquired by ISX of DiFluo Hela cells deprived with HBSS or treated for 3hr with 50μM chloroquine or with OMVs from BL21 HlyF WT 5μg/mL. On the left panel, brightfield channel (BF), on the medium panel, LC3-GFP channel images cells according to their fluorescence in GFP in green and on the right panel LC3-RFP channel images cells according to their fluorescence in RFP in red. Scale bars = 7 μm. Images representatives of 3 independent experiments. ( C ) Quantification of BDI (GFP and RFP) by ISX of DiFluo Hela cells deprived with HBSS or treated for 3hr with chloroquine 50μM or with OMVs from BL21 HlyF WT 5μg/mL. Each square represents the ratio of the intensity of RFP and GFP within a cell. The graph shows the mean of each condition. Experiment reproduced 3 times independently. ****p< 0.0001 t test, n.s. = non-significant. ( D ) Confocal images of LC3-GFP (green), LysoTracker Red (red) and DAPI (blue) fluorescence in LC3-GFP Hela cells. The cells were incubated with OMVs from BL21 HlyF WT 5μg/mL for 3hr and LysoTracker Red for 1hr. 100 cells were analyzed per condition. Scale bar = 10μm. Arrows indicates colocalization foci. Images representative of 3 independent experiments.

Article Snippet: HeLa-Difluo hLC3 cells were maintained in growth medium supplemented with the selection antibiotic Zeocin 200μg/mL (Invivogen) .

Techniques: Fluorescence, Incubation