Journal: bioRxiv

Article Title: SUMM4 complex couples insulator function and DNA replication timing control

doi: 10.1101/2021.10.02.462895

Figure Lengend Snippet: a , Physical interactions of recombinant EGG, SUUR and WDE. Untagged EGG (green arrowhead) was co-expressed with FLAG-SUUR (red arrowheads, p130 and p65) or WDE-FLAG (purple arrowhead) in Sf9 cells and purified by FLAG affinity chromatography. EGG forms a specific complex with WDE but not SUUR. b , Recombinant FLAG-SUUR(K59A) and FLAG-ISWI expressed in Sf9 cells. See legend to . c , Protein composition of in vitro reconstituted chromatin. Oligonucleosomes prepared from plasmid DNA and core histones with (+H1) or without H1 (–H1) were analyzed by SDS-PAGE and Coomassie staining. Positions of BSA, H1 and core histone bands are indicated on the right; molecular mass markers (kDa) are shown on the left. d , Micrococcal nuclease (MNase) analysis of reconstituted chromatin. Partial digestion with five different dilutions of MNase was performed on H1-free (– H1) and H1-containing (+H1) oligonucleosomes. Deproteinated DNA fragments were analyzed by agarose gel electrophoresis and stained with ethidium. Note the increased nucleosome repeat length in (+H1) lanes consistent with H1 incorporation. Triangles at the top indicate increasing MNase concentrations; 123 bp ladder was used as a molecular mass marker. e , Chromatosome stop assay. Oligonucleosomes assembled with or without H1 were subjected to partial MNase digestion, and DNA was analyzed by agarose gel electrophoresis and ethidium bromide staining. Positions of the core particle and chromatosome DNA are indicated by arrowheads. DNA fragment sizes in the 20-bp DNA ladder marker are shown. f , EpiCypher ® EpiDyne ® - PicoGreen™ assay design. EpiDyne nucleosomes encompass a restriction site shielded by the initial nucleosome position but exposed for Dpn II cleavage upon remodeling (sliding or displacement). Biotinylated substrates are immobilized on streptavidin magnetic beads. Digest by Dpn II releases the substrates from beads, and supernatant is quantified by PicoGreen™ (dsDNA detection reagent) fluorescence. g , Titration of Drosophila ISWI remodeling activity using terminally (6-N-66) or centrally (50-N-66) positioned mononucleosomes. Early reaction time points were separately plotted to indicate linear ranges. RFU, relative fluorescence units. h , Early remodeling rates for ISWI were calculated by linear regression analyses of data in respective linear ranges. ISWI exhibits a stronger remodeling activity with a centrally positioned nucleosome substrate. i , Titration of human BRG1 remodeling activity. Data are presented as in ( g ). j , Early remodeling rates for BRG1 were calculated and plotted as in ( h ). BRG1 does not exhibit a bias towards remodeling centrally or terminally positioned nucleosomes.

Article Snippet: Full-length recombinant human BRG1 (SMARCA4, EpiCypher) was produced in Sf9 cells and obtained from EpiCypher, Inc.

Techniques: Recombinant, Purification, Affinity Chromatography, In Vitro, Plasmid Preparation, SDS Page, Staining, Agarose Gel Electrophoresis, Marker, Picogreen Assay, Magnetic Beads, Fluorescence, Titration, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: RREB-1 is a transcriptional repressor of HLA-G.

doi: 10.4049/jimmunol.0902053

Figure Lengend Snippet: FIGURE 8. In situ binding of RREB-1 or HDAC1 to the HLA-G pro- moter in a repressive and active-type chromatin. A and B, ChIP performed with JEG-3 (HLA-G ) and M8 (HLA-G ) cells using anti-RREB-1 and anti-HDAC1 Abs on distal and proximal promoter regions (A) Abs target- ing RNA polymerase II (RNApolII), acetylated histone H3 (AcH3), and phosphorylated histone H3 (AcH3 P) on proximal promoter region (B). Immunoprecipitated HLA-G promoter regions are analyzed on agarose gels by semiquantitative HLA-G-specific PCRs targeting proximal and distal HLA-G promoter. Input chromatin (Input) used as PCR control and IgG () are shown. The absence of RREB-1 and HDAC1 binding observed in JEG-3 cells and the absence of RNA polymerase II, acetylated histone H3, and phosphorylated histone H3 binding in M8 cells validate the specificity of Abs used in ChIP assays.

Article Snippet: ChIP assays were performed as previously described (51) using antiRREB-1 from Rockland; anti-acetylated histone H3 (06-599) and antiphosphorylated Ser10 histone H3 (07-081) from Upstate Biotechnology Associates; and anti-RNApolII (C-21) and anti-HDAC1 (H-51) from Santa Cruz Biotechnology.

Techniques: In Situ, Binding Assay, Immunoprecipitation, Control

Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A and B) Cells natively co-expressing Ist1-HaloTag and either GFP-Rab5a (A) or GFP-Rab4b (B) were imaged live using confocal microscopy following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification showing the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bars, 5 μm; inset bars, 2 μm. (C and D) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against Hrs (C) or CHMP1B (D) following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bars, 5 μm; inset bars, 2 μm. (E) Cells natively co-expressing Ist1-HaloTag and labeled with the JF650-HaloTag ligand were exposed to Alexa Fluor 555-EGF for 9 min and imaged live using confocal microscopy. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm; inset bar, 2 μm.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Labeling, Expressing, Confocal Microscopy, Marker, Staining

Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against Snx15 following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm; inset bar, 2 μm. (B) Representative images of natively expressed Ist1-HaloTag in control cells and cells lacking Snx15 following labeling with the JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under each condition is also shown (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm. (C) Quantification of CHMP1B levels in control cells and cells depleted of CHMP1A, CHMP1B, or both CHMP1A and CHMP1B, based on immunoblot analysis ( n = 3). Error bars represent the mean ± SEM. ****P < 0.0001 and ***P < 0.001, as calculated using a one-way ANOVA and Tukey’s post hoc test. (D) Representative images of natively expressed Ist1-HaloTag in control cells treated with a scrambled siRNA (Mock) or siRNAs targeting CHMP1 isoforms following labeling with JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under the conditions shown is also provided (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm. (E) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against CHMP4B following labeling with JF650-HaloTag ligand and treatment with either a scrambled siRNA (Mock) or siRNAs targeting CHMP1A and CHMP1B. Representative images are shown (left) with quantification highlighting CHMP4B fluorescence intensity under each condition (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm; inset bar, 2 μm.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Expressing, Staining, Labeling, Marker, Control, Western Blot, Fluorescence

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test