Journal: EMBO Reports

Article Title: Targeting monocytic Occludin impairs transendothelial migration and HIV neuroinvasion

doi: 10.1038/s44319-024-00190-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The initial plasmid backbone used was the pLenti-spCas9-T2A-Crimson-P2A-puro (kindly provided by Dr. Feng Zheng) and derived versions of this plasmid were also generated to express mNeonGreen (Addgene #122183), TagRFP-T (Addgene #122200) or iRFP670 (Addgene # 122182) fluorescent proteins.

Techniques: Isolation, Recombinant, Sequencing, Knock-Out, DNA Ligation, Transfection, Cell Viability Assay, Blocking Assay, Protease Inhibitor, Virus, Software, Electroporation, Imaging, Polymer, Microscopy, Flow Cytometry, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: ER-phagy mediates the anti-tumoral synergism between HDAC inhibition and chemotherapy

doi: 10.1186/s12964-025-02198-9

Figure Lengend Snippet: ISX and Gemcitabine synergistically induce ER-stress ( A ) Scheme of the IRE1-selective ER-stress reporter. ( B ) Immunofluorescence images of Panc1 cells transiently transfected with IRE1 reporter (green), treated with DMSO, 20 µM ISX, 1.0 µM Gem and the combination ISX/Gem (I/G) for 48h. Nuclei appear blue (DAPI). Scale bar: 100µm. Shown is one representative of n=3. ( C ) Quantification of Panc1 cells transiently expressing IRE1 reporter. Cells were treated as described in ( B ) or with 1.0 μM Thapsigargin (Th) for 48h, analyzed using the number of positive cells per viewing field normalized to the values of the DMSO samples. Each dot represents one image (from n=2 independent experiments; mean ± SD). ( D ) Rel. mRNA expression of ER-stress genes selective for the IRE1 pathway ( XBP1s , DNAJB9 ). Panc1 cells were treated as described in ( C ) (mean of n=3 ± SD). ( E ) Scheme of the PERK-selective ER-stress reporter. ( F ) Immunofluorescence pictures of Panc1 cells transiently expressing PERK reporter (red) and treated as in ( B ). Nuclei appear blue (DAPI). Scale bar: 100µm. Shown is one representative of n=3. ( G ) Quantification of PERK pathway activity in Panc1 cells stably expressing a PERK reporter construct. Treatment as in ( C ). Shown are the numbers of positive cells per viewing field normalized to the DMSO condition. Each dot represents one analyzed viewing field (mean ± SD of n=2). ( H ) Rel. mRNA expression of ER-stress genes selective for the PERK pathway ( CHOP , PERK ) in Panc1 cells treated as described in ( C ) Quantification of ATF6 activity in Panc1 cells transiently expressing an ATF6 luciferase reporter, treated as described in ( C ) (mean of n=3 ± SD). ( K ) Rel. mRNA expression of HERPUD1 , an ER-stress gene selective for the ATF6 pathway (Panc1 cells treated as in ( B ); mean of n=3 ± SD)

Article Snippet: The following plasmids were used: IRE1 reporter/pLHCX-XBP1 mNeonGreen NLS (Addgene 115971; David Andrews), PERK reporter/pLHCX-ATF4 mScarlet NLS (Addgene, 115,970; David Andrews), ER-Keima reporter/GST-Keima-cb5 (Addgene, 137,755; Carol Mercer), mCherry-LC3B (Addgene 40827, David Rubinsztein), ATF6 reporter/p5xATF6-GL3 (Addgene, 11,976, Ron Prywes).

Techniques: Immunofluorescence, Transfection, Expressing, Activity Assay, Stable Transfection, Construct, Luciferase

Journal: Cell Communication and Signaling : CCS

Article Title: ER-phagy mediates the anti-tumoral synergism between HDAC inhibition and chemotherapy

doi: 10.1186/s12964-025-02198-9

Figure Lengend Snippet: The impact of HDACi/Gem on apoptosis and its link to clinical data. ( A ) Rel. mRNA expression of FAM134B and CHOP (mean of n=3 ± SD). Panc1 cells were treated with DMSO or the combination of 20 µM ISX and 1.0 µM Gem for different time periods. ( B ) Rel. mRNA expression of FAM134B and PERK (mean of n=3 ± SD). Panc1 cells were treated with DMSO, 20 µM ISX, 1.0 µM Gem, dual treatment (I/G), or 1.0 µM Thapsigargin (Th) in calcium-free or calcium-containing DMEM medium for 48h. ( C ) Flow cytometric quantification of IRE1 activity in Panc1 cells stably expressing an IRE1 reporter construct. Treatment as in ( B ) (n=3 ± SD). ( D ) Caspase 3/7 activity in Panc1 cells exposed to ISX (I, 20 µM) and Gem (G, 1.0 µM) for various time periods. Staurosporine (1.0 µM) was included as a positive control. The mean ± SD of two representative experiments measured in triplicates is shown. ( E ) Caspase 3/7 activity in Panc1 cells treated with the indicated drugs for 60h. ISX (I, 20 µM); Gem (G, 1.0 µM); IRE1-inh. 4µ8c (30µM); PERK-inh. ISRIB (1.0µM); ATF6-inh. Ceapin-A7 (6.0µM). Shown is the mean (± SD) of two representative experiments measured in quadruplicate each. Each dot represents one biological sample. ( F ) Western blot analysis of FAM134B protein levels in Panc1 cells treated for 48h with DMSO, 20 µM Loperamide, or 5/10 µM Nelfinavir. Shown is one representative experiment of n=5. β-Actin was used as a loading control. ( G , H , I ) FAM134B mRNA expression in bulk tissue from PDAC patients and corresponding healthy pancreatic tissue. Each dot represents one patient. ( J , K ) Kaplan-Meier plots representing the overall survival of patients with PDAC as a function of FAM134B expression (Scan-split; log-rank p). ( L ) Scheme illustrating the ER-phagy-induced mechanism by which HDACi and Gemcitabine synergize in their anti-tumor effectiveness

Article Snippet: The following plasmids were used: IRE1 reporter/pLHCX-XBP1 mNeonGreen NLS (Addgene 115971; David Andrews), PERK reporter/pLHCX-ATF4 mScarlet NLS (Addgene, 115,970; David Andrews), ER-Keima reporter/GST-Keima-cb5 (Addgene, 137,755; Carol Mercer), mCherry-LC3B (Addgene 40827, David Rubinsztein), ATF6 reporter/p5xATF6-GL3 (Addgene, 11,976, Ron Prywes).

Techniques: Expressing, Activity Assay, Stable Transfection, Construct, Positive Control, Western Blot, Control

Journal: Viruses

Article Title: The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 in Intracellular Trafficking and Pathogenesis

doi: 10.3390/v17040522

Figure Lengend Snippet: The SARS-CoV-2 ORF3a is expressed in organelles of the secretory pathway and at the cell-plasma membrane. COS-7 cells were co-transfected with the empty pcDNA.3.1(+) vector or vectors expressing SARS-CoV-2 ORF3a-HA protein and vectors expressing markers for the rough endoplasmic reticulum (RER; ERmoxGFP), cis/medial Golgi (mNeonGreen-Giantin), trans Golgi network (TGN38 EGFP), and mitochondria (4xmts-mNeonGreen). In other cultures, COS-7 cells were transfected with the pcDNA.3.1(+) vectors expressing SARS-CoV-2 HA-ORF3a and immunostained with antibodies against other intracellular organelles (ERGIC, or Golgin 97) as described in the . At 48 h post-transfection, cells were fixed, permeabilized, and blocked. The coverslips were reacted with a mouse monoclonal antibody against the HA-tag of HA-ORF3a and with a rabbit antibody against ERGIC53 (ERGIC) or Golgin 97 ( trans- Golgi) followed by appropriate secondary antibodies, as described in the . Coverslips were washed, counter-stained with DAPI (1 μg/mL), and mounted. Cells were examined using a Leica TCS SPE confocal microscope, using a 100X objective with a 2X digital zoom and the Leica Application Suite X (LAS X) software package. A minimum of 100 cells were examined per staining, with the micrographs shown being representative. ( A ). Cells transfected with vectors expressing HA-ORF3a and ERmoxEGFP. ( B ). Cells transfected with a vector expressing HA-ORF3a and immunostained with antibodies against ERGIC-53 and HA. ( C ). Cells transfected with vectors expressing HA-ORF3a and mNeonGreen-Giantin. ( D ). Cells transfected with the vector expressing HA-ORF3a and immunostained with antibodies against Golgin 97. ( E ). Cells transfected with vectors expressing HA-ORF3a and TGN38 EGFP. ( F ). Cells transfected with the vector expressing HA-ORF3a and 4xmts-mNeon Green.

Article Snippet: These included (1) ER-moxGFP for the rough endoplasmic reticulum (RER), Addgene catalog #68072 (a gift from Eric Snapp); (2) mNeonGreen-Giantin for cis-medial Golgi; Addgene catalog #98880 (a gift from Dorus Gadella); (3) TGN38 EGFP for the trans-Golgi network; Addgene catalog #128148 (a gift from Jennifer Lippincott-Schwartz); (4) 4xmts-mNeonGreen for mitochondria; Addgene catalog #98876 (a gift from Dorus Gadella); and (5) LAMP-1-mNeonGreen for lysosomes; Addgene #98882 (a gift from Dorus Gadella).

Techniques: Membrane, Transfection, Plasmid Preparation, Expressing, Staining, Microscopy, Software