Journal: Biotechnology and bioengineering

Article Title: Scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

doi: 10.1002/bit.27440

Figure Lengend Snippet: Plasmids and strain used in this study

Article Snippet: Plasmids pETM6 and pETM6-mCherry were a gift from Mattheos Koffas (Addgene plasmids #49795 and #66534). pLysS was obtained from NEB.

Techniques:

Journal: Cell reports

Article Title: Apical clathrin-coated pits control the location, timing, and scale of microvillar growth

doi: 10.1016/j.celrep.2025.116008

Figure Lengend Snippet: (A) Scanning electron microscopy (SEM) of native mouse small intestine along the full crypt-villus axis. Scale bar: 50 μm. (B) High magnification of the small intestinal crypt (scale bar: 10 μm) with magnification (base, middle, and transition [scale bars: 1 μm for solid box magnification and 500 nm for dashed box magnification]) showing the accumulation of microvilli along the transit-amplifying zone. (C) Native mouse small intestine stained for Villin, Dynamin2, Pacsin2, and clathrin light chain A (CLTA). Shown are single z plane confocal images of intestinal crypts. Scale bar: 10 μm. (D) Fold change in mean brush border intensity measurements for each marker from the base of the crypt relative to the crypt-villus transition zone. n = 20–25 crypts per marker, 3 wild-type mice. Kruskal-Wallis test with multiple comparisons; Pacsin2 *** p = 0.0007. CLTA *** p = 0.0003.

Article Snippet: DNMII-pmCherry-N1 (dynamin2) (human) , Addgene , #27689.

Techniques: Electron Microscopy, Staining, Marker

Journal: Cell reports

Article Title: Apical clathrin-coated pits control the location, timing, and scale of microvillar growth

doi: 10.1016/j.celrep.2025.116008

Figure Lengend Snippet: (A and C) Montage of a single microvillus growth event in CL4 cells expressing mCherry-espin and (A) pPLC-ΔPH-GFP (PIP(4,5)P 2 marker) and (C) EGFP-CLTA. Every column represents 30 s. Arrows mark the analyzed core bundle. Column width = 2.09 μm. (B and D) Average fluorescence intensity measurements over time. (B) n = 10 growth events from 6 cells across 3 independent experiments. (D) n = 21 growth events from 8 cells across 3 independent experiments. (E) Montage of a single microvillus growth event in CL4 cells expressing mCherry-espin and EGFP-Pacsin2. Each column represents 1 min. Arrows mark the analyzed core bundle. Column width = 2.09 μm. (F) Average fluorescence intensity measurements over time. n = 20 growth events from 10 cells across 3 independent experiments. (G) Montage of a single microvillus growth event in CL4 cells expressing mCherry-Dynamin2 and GFP-espin. Every column represents 1 min. Arrows mark the analyzed core bundle. Column width = 2.09 μm. (H) Average fluorescence intensity measurements over time. n = 10 growth events from 4 cells across 3 independent experiments.

Article Snippet: DNMII-pmCherry-N1 (dynamin2) (human) , Addgene , #27689.

Techniques: Expressing, Marker, Fluorescence

Journal: Nucleic Acids Research

Article Title: The human chromatin remodeling complex p400 restricts HIV-1 transcription in a Tat-dependent manner

doi: 10.1093/nar/gkaf1323

Figure Lengend Snippet: p400 specifically suppresses HIV Tat-dependent viral replication. (A) HIV GKO vector and its Tat-deficient variant (HIV GKO -ΔTat). (B) Workflow for stable Jurkat T-cell selection, HIV GKO infection, and analysis of viral replication. (C) Western blot of EP400, DMAP1, and BRD4 in stable Jurkat cells depleted by shRNA. (D-F) Representative flow cytometry plots of GFP expression (D) with quantification of GFP + % (E) and GFP MFI (F). Cells were infected at MOI = 0.1 (day 1), reactivated with PMA + TSA (day 3), and analyzed on day 4. HIV GKO -ΔTat infected cells were stimulated with 10 nM PMA + 2 µM TSA, while HIV GKO infected cells received 10% of that dose to avoid overstimulation. Data in (E-F) represent mean ± SEM from three experiments. Statistical significance was assessed by two-way ANOVA.

Article Snippet: HIV GKO was packaged in HEK293T cells by transfection of HIV GKO (Addgene, #112234) and pCMV-VSV-G (Addgene, #8454) expression plasmid at a ratio of 5:1.

Techniques: Plasmid Preparation, Variant Assay, Selection, Infection, Western Blot, shRNA, Flow Cytometry, Expressing

Journal: Nucleic Acids Research

Article Title: The human chromatin remodeling complex p400 restricts HIV-1 transcription in a Tat-dependent manner

doi: 10.1093/nar/gkaf1323

Figure Lengend Snippet: p400 suppresses HIV Tat-dependent viral activation. (A) Western blot of Tat and Gag p55 expression in HIV GKO clone α10 and HIV GKO -ΔTat populations. Cells were treated with 10 nM PMA + 2 µM TSA to reactivate HIV gene expression. (B–D) GFP expression in HIV GKO clone α10 and HIV GKO -ΔTat populations depleted of EP400, DMAP1, or BRD4, shown by flow plots (B), mRNA analysis of host genes (C), and HIV transcripts (D). Cells were transduced with retroviral vectors expressing gene-specific shRNAs, followed by puromycin selection (1 µg/mL) for 4 days before analysis. HIV GKO -ΔTat cells were stimulated with 10 nM PMA + 2 µM TSA, while HIV GKO clone α10 cells received 10% of that dose to avoid overstimulation. (E–H) X-ChIP-qPCR analysis of total RNAPII in HIV GKO clone α10 cells (E) and HIV GKO -ΔTat cells (F), and of RNAPII-Ser2P (G) and RNAPII-Ser5P (H) in HIV GKO clone α10 cells following depletion of EP400, DMAP1, or BRD4.

Article Snippet: HIV GKO was packaged in HEK293T cells by transfection of HIV GKO (Addgene, #112234) and pCMV-VSV-G (Addgene, #8454) expression plasmid at a ratio of 5:1.

Techniques: Activation Assay, Western Blot, Expressing, Gene Expression, Transduction, Retroviral, Selection, ChIP-qPCR

Journal: Nucleic Acids Research

Article Title: The human chromatin remodeling complex p400 restricts HIV-1 transcription in a Tat-dependent manner

doi: 10.1093/nar/gkaf1323

Figure Lengend Snippet: Recruitment of p400 to transcriptionally active HIV loci is Tat-independent. (A-B) N-ChIP for RNAPII (A) and Tat (B) in Jurkat cells with HIV GKO or HIV GKO -ΔTat. (C-D) N-ChIP for EP400 in HIV GKO (C) or HIV GKO -ΔTat (D) populations. (E-F) N-ChIP for DMAP1 in HIV GKO (E) or HIV GKO -ΔTat (F) populations. For all experiments, cells were treated with 10 nM PMA + 2 µM TSA to reactivate HIV gene expression. Data represent mean ± SEM from three independent experiments.

Article Snippet: HIV GKO was packaged in HEK293T cells by transfection of HIV GKO (Addgene, #112234) and pCMV-VSV-G (Addgene, #8454) expression plasmid at a ratio of 5:1.

Techniques: Gene Expression

Journal: Nucleic Acids Research

Article Title: The human chromatin remodeling complex p400 restricts HIV-1 transcription in a Tat-dependent manner

doi: 10.1093/nar/gkaf1323

Figure Lengend Snippet: p400 restricts Tat-dependent HIV gene expression. (A) Flow cytometry plot of GFP expression in Jurkat T cells infected with HIV GKO Tat mutant viruses. Viral integration was gated on mKO2 + cells. (B) Quantification of GFP MFI in HIV GKO -integrated populations in (A). (C-D) GFP MFI of Tat-mutated HIV GKO virus in stable Jurkat T cells depleted of EP400, DMAP1, or BRD4. Cells were infected at MOI = 0.3 (day 1), treated with TNF-α (day 3), and analyzed on day 4. (E) Properties of alanine substitutions in the Tat basic domain: DMAP1 binding (from Fig. ), transactivation activity (from B), and HIV activation after EP400 or DMAP1 depletion (from C). (F) Model illustrating the role of the p400 complex in HIV transcriptional regulation. Data in (B–D) represent mean ± SEM from three experiments. Statistical significance was assessed by two-way ANOVA.

Article Snippet: HIV GKO was packaged in HEK293T cells by transfection of HIV GKO (Addgene, #112234) and pCMV-VSV-G (Addgene, #8454) expression plasmid at a ratio of 5:1.

Techniques: Gene Expression, Flow Cytometry, Expressing, Infection, Mutagenesis, Virus, Binding Assay, Activity Assay, Activation Assay