Journal: Technology in Cancer Research & Treatment

Article Title: Nipped-B-like Protein Sensitizes Esophageal Squamous Cell Carcinoma Cells to Cisplatin via Upregulation of PUMA

doi: 10.1177/1533033820960726

Figure Lengend Snippet: NIPBL is relevant to the growth of ESCC cells. A, Western blotting analysis of NIPBL expression in ESCC cell lines. GAPDH is shown as loading control. Normal esophageal squamous epithelial tissue from 2 patients, N1 and N2, were used as the control. B, Western blotting analysis of NIPBL expression in COLO-680N cells transfected with the NIPBL overexpressing vector. GAPDH is shown as loading control. C, Relative cell proliferation of COLO-680N with NIPBL overexpression was determined by the MTS assay. Cells were transfected with pEGFP-N1-FLAG vector or NIPBL recombinant vector respectively, and the relative cell proliferation was determined by MTS assay after transfection for 72 h. All experiments were repeated thrice and the representative results are shown. The statistical significance is p < 0.001 (Student’s t -test, *** represents p < 0.001). NIPBL expression in EC9706 cells transfected with NIPBL siRNA was determined by quantitative real-time PCR (D) and western blotting analysis (E). siRNA 1 and siRNA 2 are 2 different NIPBL siRNAs, whereas control is a non-targeting scrambled control siRNA. F, Relative cell proliferation in Eca-109 and EC9706 cells with NIPBL depletion was determined by MTS assay after transfection with NIPBL siRNA for 72 h. All experiments were repeated thrice and the representative results are shown. The statistical significance is p < 0.001 (Student’s t -test, *** represents p < 0.001).

Article Snippet: NIPBL ORF (1-8,094 bp) was cloned into the pEGFP-N1-FLAG vector (Addgene, Watertown, Massachusetts, USA).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Over Expression, MTS Assay, Recombinant, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: Regulation of N-degron recognin-mediated autophagy by the SARS-CoV-2 PLpro ubiquitin deconjugase

doi: 10.1080/15548627.2024.2442849

Figure Lengend Snippet: Reagents used in the investigation.

Article Snippet: pEGFP N1 Ub-CRT , Addgene , 69619; Marta Hallak lab.

Techniques: Recombinant, Diagnostic Assay, DC Protein Assay, Transfection, Mutagenesis, Plasmid Preparation, Gel Extraction, Purification, Cell Culture, shRNA

Journal: bioRxiv

Article Title: Respiratory viruses activate autophagy via the IFN-STAT1/STAT5B-SOCS1 axis

doi: 10.1101/2025.10.28.685013

Figure Lengend Snippet: A, Quantification of autophagosome levels by flow cytometry in HEK293T autophagy reporter cells (HEK293T GL) transiently expressing Cas9 and at least 2 individual gRNAs against indicated STATs. Treated with 10 pM IFN-β for 24h. n = 4-6± SEM. B, Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) transfected with siRNAs targeting STAT1 , STAT3 , STAT5B or ATG5 and treated with 1 nM IFN-β for 24h. BafA1 (625 µM) or Torin-1 (1 µM) for 4h used as controls. n = 4 ± SEM. C , Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) 24 h after treatment with increasing concentrations of IFN-β in presence or absence of 1 µM 5,15-DPP (STAT3 Inhibitor, STAT3i), 1 µM AS1517499 (STAT6 Inhibitor, STAT6i), 10 µM Fludarabine (STAT1 Inhibitor, STAT1i) or 100 µM STAT5 Inhibitor (STAT5i). n = 4 ± SEM. D, Area under the curve (AUC) analysis of the data in (C). E , Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) 24 h after treatment with increasing concentrations of IFN-γ and STAT5 Inhibitor (STAT5i, 100 µM). n = 4 ± SEM. F , Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) 24 h after treatment with increasing concentrations of IFN λ1 and STAT5 Inhibitor (STAT5i, 100 µM). n = 3 ± SEM. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. *, p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Constructs coding for human STAT1 (eGFP STAT1 WT, Addgene #12301, kindly gifted from Alan Perantoni), STAT3 (pEGFP-N1-STAT3, Addgene #111934, kindly gifted from Geert van den Bogaart) and STAT5B (MAC_N_STA5B, Addgene #167800, kindly gifted from Markku Varjosalo) were obtained from Addgene. pTwist-FLAG was generated by removing the ORF of TRIM3 from pTwist_3x_FLAG_optTRIM (purchased from Twist Biosciences) as described previously (Klute et al.). pTwist-V5 was generated by linearizing pTwist-FLAG by PCR using the pTwist-FLAG fwd and rev primers from , amplifying the V5-gBlock (IDT) and inserting it into the linearized pTwist ‘null’ vector using Gibson Assembly (New England Biolabs).

Techniques: Flow Cytometry, Expressing, Transfection

Journal: bioRxiv

Article Title: Respiratory viruses activate autophagy via the IFN-STAT1/STAT5B-SOCS1 axis

doi: 10.1101/2025.10.28.685013

Figure Lengend Snippet: A, Representative confocal immunofluorescence images of HeLa autophagy reporter cells (HeLa GL) transiently expressing FLAG-tagged (red) and V5-tagged (yellow) STAT1, STAT3 or STAT5 in combination or FLAG-tagged TRIM32 (scale bar, 10 μm) (left panel). Quantification of the number of autophagosomes (= eGFP-LC3B positive puncta) per cell in the images in the left panel. n= 9-63 cells ± SEM Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. B, Co-immunoprecipitation of FLAG-tagged STAT1 and STAT5B from whole cell lysates of HEK293T cells transiently expressing FLAG-tagged and V5-tagged STAT1 and STAT5B or V5-tagged STAT1 or STAT5B alone. Immunoblots stained with anti-V5, anti-FLAG or anti-GAPDH. C, Exemplary images of Proximity Ligation Assay (PLA) of STAT1 and STAT2 or STAT5B (left panel) in HDF hTERT cells with or without treatment with 1 nM IFN-β for 1h. PLA signal (red). DAPI, nuclei (blue). Quantification of the number of PLA spots per nucleus per image of the left panel with single antibody controls (right panel). n = 15-21 tiles ± SEM. Student’s t-test with Welch’s correction. D, Representative confocal immunofluorescence images of NHLF cells treated with 1 nM of IFN-β for 1h and stained with α-STAT1 (green) and α-STAT5B (red), and DAPI (blue, nuclei) (scalebar 25 µm). E, Quantification of the ratio of nuclear to cytosolic intensity of STAT1 and STAT5B in the images in (D). n= 19-20 cells ± SEM. Student’s t-test with Welch’s correction. *, p<0.05, ** p<0.01, *** p<0.001

Article Snippet: Constructs coding for human STAT1 (eGFP STAT1 WT, Addgene #12301, kindly gifted from Alan Perantoni), STAT3 (pEGFP-N1-STAT3, Addgene #111934, kindly gifted from Geert van den Bogaart) and STAT5B (MAC_N_STA5B, Addgene #167800, kindly gifted from Markku Varjosalo) were obtained from Addgene. pTwist-FLAG was generated by removing the ORF of TRIM3 from pTwist_3x_FLAG_optTRIM (purchased from Twist Biosciences) as described previously (Klute et al.). pTwist-V5 was generated by linearizing pTwist-FLAG by PCR using the pTwist-FLAG fwd and rev primers from , amplifying the V5-gBlock (IDT) and inserting it into the linearized pTwist ‘null’ vector using Gibson Assembly (New England Biolabs).

Techniques: Immunofluorescence, Expressing, Immunoprecipitation, Western Blot, Staining, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

doi: 10.1101/2022.06.12.495801

Figure Lengend Snippet: a. A diagram showing the effect of Latrunculin A (Lat A) and Jasplakinolide and example STED micrographs showing the localization of filamentous actin (F-actin) relative to the active zone in neurons treated with DMSO (control), Lat A, and Jasplakinolide. Active zone is marked by anti-Bassoon antibody and its secondary antibody conjugated with Alexa594. F-actin binding EGFP-UtrCH is expressed in neurons and stained with GFP-antibody and its secondary antibody conjugated with Atto646. b. Cumulative plots showing distribution of F-actin signals against the active zone boundary and intensity of F-actin signals. The active zone boundary was defined by Bassoon signals. See Supplementary Table 2 for detailed statistical analysis. c. A schematic showing the lateral membrane compression model for ultrafast endocytosis and basic values used for simulations. The lateral membrane pressure exerted by exocytosis is predicted to compress the plasma membrane against the stiff periactive zone membrane and induce pit formation at the interface between actin-free and actin-enriched regions, or at the endocytic zone. d. Schematics showing the initial conditions of simulations from top-down view (left) and orthogonal view (right). The initial length of active zone (blue) is set at 500 nm. The width of F-actin band (purple) is set at 50 nm. Here, the active zone refers to the actin-free membrane area that includes not only the vesicle fusing area but also the endocytic zone. In contrast, the periactive zone is represented by the F-actin band where actin cortex impinges upon the membrane. The center of the active zone is set as (x, y) = (0, 0). One fusing vesicle (light blue circle) is placed at (D, 0), while two other vesicles are placed at (C, -C) and (-C, -C) such that three vesicles would form an isosceles triangle. As the initial condition, we set C = D = 60 nm. e. Snapshots from simulations, showing the evolution of membrane curvature within the active zone over time. Three fusing vesicles are organized with C = D = 60 nm. At 58 ms, simulations reach the steady state, with 2 endocytic pits forming at the boundary between active zone and actin-enriched region. f. Plot showing the depth of exocytic pits and endocytic pits as a function of time. g. Plot showing the resulting membrane curvature as a function of the spatial arrangement of fused vesicles. Distances among vesicles are modulated by changing C and D, depicted in d. h. Plot showing the dependence of successful endocytic pit formation on bending moduli of active zone and periactive zone membranes. The colored areas indicate successful formation of endocytic pits. i. Snapshots from simulations, showing the evolution of membrane shape within the active zone as a function of tension. As shown in the plot, the membrane area conservation was relaxed at 2.5 ms; consequently, the membrane tension decreases from 0.6 pN/nm to 0.3 pN/nm. This reduction in tension mimicks the expected tension change that occurs with exocytosis. Simulations reach the steady sate at 5 ms without inducing any curvature.

Article Snippet: To label the endocytic zone, we used a plasmid expressing Dynamin1xA C-terminally tagged to GFP, purchased from addgene (phsDyn1xA-EGFP-N1, #120313).

Techniques: Control, Binding Assay, Staining, Membrane, Clinical Proteomics

Journal: bioRxiv

Article Title: Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

doi: 10.1101/2022.06.12.495801

Figure Lengend Snippet: a. A schematic showing the protein structural elements of Epsin1. Epsin1 contains ENTH domain, NPF and DPW. The C-terminal domain, marked light red, interacts with F-actin, while the ENTH domain, marked light blue, interacts with plasma membrane. b. Example STED micrographs showing the localization of Epsin 1 relative to the active zone in wild-type neurons. Active zone is marked by anti-Bassoon antibody and its secondary antibody conjugated with Alexa594. Epsin1 stained with Epsin1-antibody and its secondary antibody conjugated with Atto646. c. The distribution of Epsin1 signals against the active zone boundary. The active zone boundary was defined by Bassoon signals. See Methods for the analysis method. d. Example confocal fluorescence micrographs showing F-actin signals at wild-type or Epsin1 knock-down (KD) neurons. F-actin binding EGFP-UtrCH is expressed in neurons and stained with GFP-antibody and its secondary antibody conjugated with Atto646. False-colored images of the bottom panels show relative fluorescence intensity of EGFP-UtrCH. e. The normalized intensity of F-actin signals from neurons expressing scramble (scr) shRNA or Epsin1 shRNA, measured by Airyscan. Signals are normalized the fluorescence signals in axons. f. Example electron micrographs showing wild-type and Epsin1 KD synapses unstimulated or stimulated with a single electrical pulse (1 ms) and frozen 100 ms or 1 s later. Black arrow: endocytic pit. Black arrowhead: ferritin-positive endosomes. g,h. Number of endocytic pits at 100 ms after stimulation (g) or ferritin-positive structures at 1 s after stimulation (h) in neurons expressing scramble shRNA (scrRNA) or Epsin1 shRNA. Mean and 95% confidential interval are shown. Brown-Forsythe and Welch ANOVA analysis, with Games-Howell multiple comparisons test. ****p<0.0001. p values are only shown for direct comparison between unstimulated and stimulated neurons treated with the same drug. See Supplementary Table 2 for the detailed numbers for each sample.

Article Snippet: To label the endocytic zone, we used a plasmid expressing Dynamin1xA C-terminally tagged to GFP, purchased from addgene (phsDyn1xA-EGFP-N1, #120313).

Techniques: Clinical Proteomics, Membrane, Staining, Fluorescence, Knockdown, Binding Assay, Expressing, shRNA, Comparison