Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Venn diagram of proteins identified in SW620 cell-derived sMB-R (sMV-HD), sMV-LD and Exos. b Heatmap illustration of proteins identified in sMB-R (sMV-HD), sMV-LD and Exos. Proteins present in higher abundance in sMB-R (red) as compared to sMV-LD and Exos include conserved cytokinetic proteins as well as additional cytokinetic proteins. *Proteins uniquely identified in sMB-Rs. **Proteins enriched (fold change >2) in sMB-R compared to sMV-LD and Exos. c STRING-based protein-protein interaction network analysis of 928 enriched proteins in sMB-Rs (sMV-HD) compared to sMV-LD and Exos. The interactions were “evidence”-based, with “experiments” as active interaction source and interaction threshold set at 0.900 (highest confidence). Disconnected nodes in the network are hidden. Proteins identified under biological processes or molecular processes (Gene Ontology) are indicated. Centralspindlin complex components (RACGAP1 and KIF23/MKLP1) are also indicated. d EnrichmentMap of Reactome pathways enriched in 456 proteins commonly identified in SW620 cell-derived sMB-R proteome (2300 proteins) with the proteome of MB-Rs shed by Hela cells reported recently by Peterman et al. 2019 . e Mass spectrometry-based identification of KRAS peptides (UniProtKB ID RASK_HUMAN) in sMB-Rs. Two peptides (TEYKLVVVGAGGVGK and LVVVGAGGVGK) spanning Gly-12/ Val-12 substitution in KRAS protein. Peptide spectral profiles are displayed on the right. f Immunofluorescence microscopy of SW620 cells using anti-MKLP1 and anti-KRAS G12V antibodies. Nuclei (blue) were stained with Hoechst stain. White arrows indicate the position of MB and MB-Rs. Inset represents higher magnification. Scale bar, 10 µm. g Western blot analysis of exosomes, crude 10,000 x g sMVs, and isopycnic (iodixanol-density) gradient centrifugation fractions of sMV-LD and -HD/sMB-Rs using anti-KRAS G12V antibody.

Article Snippet: Cells were then incubated with primary antibodies (1:100) (mouse anti-MKLP1 (Santa Cruz Biotechnology), mouse anti-RACGAP1 (Santa Cruz), rabbit anti-KRAS G12V mutant specific (Cell Signalling), rabbit anti-RAB7 (Abcam) and rabbit anti-β-tubulin (Cell Signalling) in blocking solution for 1 h at room temperature.

Techniques: Derivative Assay, Mass Spectrometry, Immunofluorescence, Microscopy, Staining, Western Blot, Gradient Centrifugation

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Uptake of sMB-Rs by fibroblasts. Fluorescence microscopy analysis of NIH3T3 fibroblasts incubated with/without SW620 cell-derived sMB-Rs or Exos (50 µg ml −1 ) for 2 h using anti-MKLP1 and anit-RACGAP1 antibodies. b Uptake and accumulation of sMB-Rs in NIH3T3 fibroblasts was quantified by counting MKLP1 + puncta per cell; data represented as mean ± s.e.m. Nuclei (blue) were stained with Hoechst. Scale bar, 10 µm. c Internalisation of sMB-Rs by fibroblasts. Confocal microscopy of NIH3T3 fibroblasts incubated with sMB-Rs using anti-MKLP1 (in green) and anti-RAB7 (in red) antibodies. Confocal microscopy analysis along Z-axis (inset) reveal internalisation of sMB-Rs following uptake. Scale bar, 10 µm. d Intercellular transfer of sMB-R KRAS G12V into NIH3T3 cells. Fluorescence microscopy of NIH3T3 fibroblasts incubated with SW620 cell-derived sMB-Rs (5 µg) for 2 h using anti-KRAS G12V antibodies. Nuclei were stained with Hoechst stain (blue). Right panel represents fluorescence signals from left panel overlaid onto bright-field images. Inset represents enlarged image. Scale bar, 10 µm.

Article Snippet: Cells were then incubated with primary antibodies (1:100) (mouse anti-MKLP1 (Santa Cruz Biotechnology), mouse anti-RACGAP1 (Santa Cruz), rabbit anti-KRAS G12V mutant specific (Cell Signalling), rabbit anti-RAB7 (Abcam) and rabbit anti-β-tubulin (Cell Signalling) in blocking solution for 1 h at room temperature.

Techniques: Fluorescence, Microscopy, Incubation, Derivative Assay, Staining, Confocal Microscopy

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion does not affect Rab5 recruitment to the endosomes and ligand-bound EGFR-positive endosomes HeLa cells expressing control or CPAP shRNA were treated with AF555-EGF ligand for the indicated time points, stained, and subjected to 4-color imaging by confocal microscopy. Images representing the detection of Rab5 and EEA1 (A), Rab5 and CD63 (B), and Rab5 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Right (A–C): single Z-plane of images. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab5 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Bottom right graph in (C): relative integrated fluorescence intensity values of Rab5 staining quantified in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Scale bars: 10 μm. p values were not statistically significant. Zoomed images correspond to the dashed inset boxes of the indicated images. Note: (B) and (C) present data from the same experiments where 4-color imaging was done and convey information on two different aspects based on three markers at a time. Since the visuals of the same cell can help with more reliable interpretation of the data, images of the same cell were used, where possible, for (B) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (B) and (C) is intentional.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to late endosomes are restored in CPAP-depleted cells upon HRS, but not TSG101, overexpression (A) Schematic of the experimental strategy using control and CPAP-specific siRNA-treated HeLa cells with and without GFP-HRS or GFP-TSG101 expression. (B and C) Control and CPAP-specific siRNA-treated HeLa cells were subjected to mock or GFP-HRS or GFP-TSG101 vector transfection for 24 h, treated with untagged EGF for 60 min, and stained for Rab5 and Rab7 (B) or treated with AF555-EGF for 30 min and stained for Rab7 (C) and imaged by Lightning super resolution microscopy. Left: representative single Z-plane of images showing localization of Rab7 on Rab5-positive puncta (B) and AF555-EGF on Rab7-positive puncta (C) in cells with and without GFP-HRS or GFP-TSG101 expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in (B) and EGF-positive (red) puncta containing Rab7 (green, pseudo-color) in (C) in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗<0.05, ∗∗ <0.01, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Transfection, Staining, Super-Resolution Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to MVB are restored in CPAP- and HRS-depleted cells upon exogenous CPAP expression (A) Schematic of the experimental strategy using control and CPAP- and HRS-specific siRNA-treated HeLa cells with and without siRNA-resistant GFP-CPAP expression. (B) Airyscan super-resolution microscopy images showing cells stained for Rab5 and Rab7 at 60 min time point. Left: representative single Z-plane of images showing localization of Rab5- and Rab7-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (C) Confocal microscopy images showing cells stained for CD63 and AF555-EGF at 60 min time point. Left: representative single Z-plane of images showing localization of CD63 − and EGF-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63-positive (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed for (B) and (C). Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗<0.001, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Journal of cell science

Article Title: TRPC6 channels promote dendritic growth via the CaMKIV-CREB pathway.

doi: 10.1242/jcs.026906

Figure Lengend Snippet: Fig. 5. TRPC6-induced dendritic growth depends on CaMKIV activity. (A) Total lysates of cells treated as indicated blotted with antibodies to phosphorylated CaMKIV, total CaMKIV, phosphorylated CaMKIIα or total CaMKIIα. (B) Quantification of CaMKIV phosphorylation in the neurons transfected with the indicated constructs. *P<0.05 versus ctrl. (C) Representative images of the neurons transfected at 3 DIV for 4 days with wild-type TRPC6 (WTC6) and the dominant-negative CaMKIV mutant, or shTRPC6 RNAi construct and the constitutive active CaMKIV mutant. Scale bar: 20 μm. (D,E) Quantification of total dendritic tips (D) and total dendritic length (E) of the neurons shown in C. **P<0.01 versus ctrl. All data are the means ± s.e.m. n.s., not significant.

Article Snippet: Rabbit anti-TRPC1 antibody (Alomone Labs, 1:200), rabbit anti-TRPC3 antibody (Montell, 1:500), rabbit anti-TRPC4 antibody (Alomone Labs, 1:500), rabbit antiTRPC5 antibody (Sigma, 1:200), rabbit anti-TRPC6 antibody (Alomone Labs, 1:500), mouse anti-α-tubulin antibody (Sigma, 1:1000), rabbit anti-phosphoCaMKIIα (Thr286) antibody (Santa Cruz, 1;500), mouse anti-CaMKIIα antibody (Cell Signaling, 1;1000), rabbit anti-phophoCaMKIV (Thr 196) antibody (Santa Cruz, 1:100) and rabbit anti-CaMKIV antibody (Cell Signaling, 1;500) were used as primary antibodies.

Techniques: Activity Assay, Phospho-proteomics, Transfection, Construct, Dominant Negative Mutation, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Polar interactions formed by Arg 249 with neighbouring residues in p53 WT . b Change in polar contacts with neighbouring residues due to mutation of Arg 249 to serine in p53 R249S . b Superimposed view of the mutation site in p53 WT and p53 R249S . Residues of p53 WT are shown in green and for p53 R249S they are shown in pink. Change in the structure at 249th position is highlighted by a circle. Residues that acquired significantly different conformations are shown in stick representation. c Root mean square deviations of residues in p53 WT and p53 R249S . (a) RMSD of Arg 248 during the course of MD simulation in p53 WT (green) and in p53 R249S (red). (b) RMSD of residues from 249 to 271 in p53 WT (green) and p53 R249S (red)

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Difference in the molecular surface near Y220 position in p53 WT and p53 Y220C mutant. a Size of cavity in p53 WT . b Mutation from Tyr to Cys at 220th position rearranges the conformation of surrounding residues, especially located in the loop region. Widening of the loop near 220th position and removal of the Tyr side chain creates a larger cavity in p53 Y220C protein. c Superimposition of the two molecular surfaces reveals the stringency of the p53 WT (green color) as compared to the p53 Y220C (red color). b Water network near 220th residue in p53 WT and p53 Y220C protein. a Tyr 220 stabilizes the residues of surrounding loops with the help of water molecules. b Cys 220 stabilizes the cavity by solvating it with water molecules. Binding of the withanolides with p53 WT near Tyr 220. Wi-A c and Wi-N d were found to interact with the surface residues near Tyr 220 as no deep cavity was present in p53 WT

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Interactions of Wi-A with p53 Y220C near Cys 220. a Binding pose of Wi-A within the binding site near Cys 220 b 2D representation of the interactions of Wi-A with p53 Y220C . Interactions of Wi-N with p53 Y220C protein structure. c Binding pose of Wi-N within the binding cavity of p53 Y220C near Cys 220 d 2D representation of the interactions of Wi-N with surrounding residues within the cavity of p53 Y220C . Charge complementarity of Wi-A and Wi-N with the binding cavity. Wi-A e and Wi-N f both were fitting inside the cavity according to the charge distribution within it. White region represents the hydrophobic region whereas blue and red represents the hydrophilic region

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A furnished wild type p53 function in mutant p53 (p53 Y220C ) horboring hepatoma cells. a Viability assay of human hepatocarcinoma with wild type p53 (HuH-6), mutant p53 (HuH-7), and telomerized human cells bearing p53 mutants (p53 V143A , p53 R249S and p53 R273H ). b Comparison of response of HuH-6 and HuH-7 cells to Wi-A. Dose response was observed for both the cell lines. HuH-7 showed stronger cytotoxicity to Wi-A. c Western blot showed reduction in mortalin and increase in p53 in cells treated with 1 μM Wi-A in the p53 mutants, p53 V143A and p53 R273H ; p53 R249S cells possessed low expression that remained undetected on these blots. In contrast to increase in the expression of p53 V143A and p53 R273H , mutant p53 Y220C protein expression was decreased in Wi-A treated cells. d Dose dependent decrease in mutant p53 Y220C protein expression in Wi-A treated cells. e Immunostaining of mortalin and p53 (40 x magnification) in control and Wi-A (0.5 μM) showing increase in nuclear p53 V143A and p53 R273H . HuH-6 (p53 WT ) cells showed increase in nuclear p53 staining. In contrast, HuH-7 (p53 Y220C ) cells exhibited decrease in p53 nuclear staining

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Viability Assay, Comparison, Western Blot, Expressing, Immunostaining, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced restoration of wild type p53 and induction of senescence in HuH-7 cells. a Wild type p53 reporter activity in mock (untransfected), control (transfected and untreated) and Wi-A (transfected and Wi-A treated) cells. Luciferase reporter assays driven either by p53 consensus binding sites (PG13-Luc) or by a p21 WAF-1 promoter (WWP) showed an increase in Wi-A treated cells. b Flow cytometry analysis revealed G 2 cell cycle phase arrest in HuH-7 cells. c Immunostaining of p21 WAF-1 in HuH-6 and HuH-7 control and Wi-A treated cells showing increase in p21 WAF-1 expression in the latter. d Senescence-associated β-galactosidase staining was observed in Wi-A treated HuH-6 and HuH-7 cells (10 x phase magnification). e Wi-A treated HuH-6 and HuH-7 cells showed enhanced staining for nuclear heterochromatin protein HP1γ

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Activity Assay, Control, Transfection, Luciferase, Binding Assay, Flow Cytometry, Immunostaining, Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced apoptosis of HuH-7 cells. a Annexin-V staining revealed induction of early apoptosis in Wi-A treated cells. b Apoptosis in Wi-A treated HuH-7 cells was marked by cleavage of caspase3, increase in p21 WAF-1 and phosphorylated p53. c Wi-A treated cells showed Single Strand Breaks as determined by a comet assay, 40 X magnification. d Wi-A rich extract (AL-βCD) treated HuH-6 (p53 WT ) and HuH-7 (p53 Y220C ) cells showed increase and decrease in nuclear p53, respectively. e Increase in the expression of p21 WAF-1 was observed in AL-βCD treated HuH-6 and HuH-7 cells. f HuH-7 cells showed strong cytotoxicity to AL-βCD

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Staining, Single Cell Gel Electrophoresis, Expressing

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule approach for the enrichment of plasma-circulating H3-K27M-mutant nucleosomes (A) Experimental scheme: nucleosomes are extracted from HEK293 cells ectopically expressing either WT or mutant H3, and the DNA is fluorescently labeled with Cy3 by Klenow polymerase and Cy3-dATP. Next, labeled H3-K27M nucleosomes are captured on PEG-streptavidin surface covered with biotinylated antibodies targeting the H3-K27M mutant histone (α-H3-K27M) and imaged by TIRF imaging. Nucleosomes containing WT H3 do not bind the microscope coverslip and are washed prior to imaging. (B) Representative TIRF images of Cy3 signal, corresponding to surface-bound nucleosomes, extracted from HEK293 cells expressing WT or mutant histone. Nucleosomes extracted from cells expressing H3-K27M were loaded also on a surface absent of α-H3-K27M antibodies (middle), to demonstrate antibody-dependent capture. Numbers within images represent counted spots; each spot corresponds to a single surface-bound nucleosome. Scale bar applies to all three images. Nucleosomes extracted from cells expressing mutant H3 show higher binding to surface coated with α-H3-K27M antibodies. (C) Quantification of the experiment described in (B). Data are presented as the mean ± SD of 50 fields of view (FOVs) per sample. (D) Experimental scheme: cfNuc from plasma are immobilized on a PEG-streptavidin covered with biotinylated α-H3-K27M antibody. Captured H3-K27M nucleosomes are detected by incubation with fluorescently labeled antibody followed by TIRF imaging. (E) Representative TIRF images of different antibodies targeting the indicated histone PTMs, while incubated on surfaces enriched for plasma-circulating H3-K27M nucleosomes (as described in D). Antibodies were tested on plasma samples from healthy individuals, as well as patients with DMG carrying WT H3 or H3-K27M. Numbers within images represent counted spots; each spot corresponds to a single surface-bound nucleosome. Scale bar applies to all images. H3K27ac and H3K36me2 antibodies are suitable for the detection of surface-bound H3-K27M nucleosomes. (F and G) Quantification of the single-molecule signal obtained for H3K27Ac antibody when incubated with and without H3-K27M enriched cfNuc from different plasma samples. Data are presented as the mean ± SD of 50 FOVs per sample.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Expressing, Labeling, Imaging, Microscopy, Binding Assay, Incubation

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule detection of mutant nucleosomes in plasma differentiates patients with H3-K27M from those with H3 WT DMG (A) Plasma samples of patients with DMG carrying WT H3 or H3-K27M mutant (light blue and dark blue, respectively) and plasma of healthy adults or children (light gray and dark gray, respectively) were analyzed as described in Figure 2 D. Mutant nucleosomes captured on surface were detected by H3K27ac antibody. Each bar represents a subject, and data are presented as the mean of the normalized H3K27ac counts ±SD of 50 FOVs per sample. (B) Boxplot representation of the data in (A), with samples grouped as follows: DMG H3-K27M ( n = 52), H3 WT glioma ( n = 16), healthy adults ( n = 21), and healthy children ( n = 29). Boxplot limits indicate 25%–75% quantiles, the middle lines indicate the median, and the upper and lower whiskers denote the largest and smallest values, respectively, no further than 1.5× the interquartile range from the hinge. p values were calculated by Wilcoxon rank-sum exact test and adjusted by Bonferroni correction for multiple comparisons. ∗∗ p value <0.01, ∗∗∗ p value <0.001; ns, non-significant. (C) Receiver operating characteristic (ROC) curve discriminates between plasma samples containing WT H3 nucleosomes ( n = 65) and H3-K27M-mutant nucleosomes ( n = 52) using a naive Bayes model. The area under the curve (AUC) for H3-K27M-H3K27ac normalized counts (blue line), while the red dashed diagonal line indicates expected curve for random classification. Blue shaded area represents 95% confidence interval. (D) H3-K27M-H3K27ac classifier performance; each bar represents the mean value of repeated ( n = 10,000) 4-fold cross-validation across all samples.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Detection of plasma-circulating mutant p53 in patients with DMG (A) Experimental scheme: biotinylated capture antibodies targeting total p53 protein (the WT and mutant form) are anchored to a PEG-streptavidin surface (α-p53). Plasma-circulating p53 protein molecules are captured on surface, followed by incubation with two distinct fluorescently labeled p53 antibodies: an antibody targeting all forms of p53 (“total p53,” red) and an antibody specific to the mutant conformation of p53 (“mutant p53,” green). (B) Representative TIRF images of the indicated p53 antibodies incubated on surfaces enriched for plasma-circulating p53 proteins. Scale bar applies to all images. (C) A cohort of 19 plasma samples of H3-K27M DMG patients was analyzed as described in (A) (13 samples harbor TP53 mutations and 6 harbor WT TP53). Principal component analysis (PCA) with the following input parameters: normalized counts of total p53 and mutant p53, and the ratio between mutant and total p53. Sample groups are color-coded according to known TP53 status; each dot represents one plasma sample. (D) The ratio between mutant and total p53 signal for each sample is shown. Each bar represents a sample, color-coded according to known TP53 status. Data are presented as the mean ± SD of 50 FOVs per sample. (E) Boxplot representation of the data in (D) grouped according to TP53 status (mutant p53 n = 13, WT-p53 n = 6). Boxplot limits indicate 25%–75% quantiles, the middle lines indicate the median, and the upper and lower whiskers denote the largest and smallest values, respectively, no further than 1.5× the interquartile range from the hinge. p values were calculated by Wilcoxon rank-sum exact test. ∗∗∗ p value <0.001.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Incubation, Labeling

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule measurements of mutant nucleosomes as a potential proxy for tumor growth (A) Experimental scheme for parallel measurements of cell viability and H3-K27M-mutant nucleosomes released to culture media. Cells were plated as monolayer, and a day later media was replaced to initiate the experiment. Media was collected for single-molecule measurements and replaced to account for newly released nucleosomes between examined time points. Viability measurements were conducted from a parallel culture. (B) Viability measurements and H3-K27M-H3K27ac single-molecule measurements are shown for untreated culture (left) and 5 μM ONC201-treated culture (right). The drug was replenished every 2 days. For single-molecule measurements, mean ± SD of 40 FOVs for each time point is shown. For CellTiter-Glo measurements, mean ± SE of three technical repeats for each time point is shown. (C) Clinical features, initial MRI, and MRI of disease at clinical progression of DMG patient UMPED 128. (D–J) Analysis of serial plasma samples from patients undergoing ONC201/206 treatment. Single-molecule measurements (normalized to first data point) of mutant nucleosomes (H3-K27M-H3K27ac) were performed on serial time point plasma samples plotted along with tumor cross-sectional area according to MRI. Green bars: ONC201/206 mainline treatment. Purple bars: radiation treatment. Brown bars: other treatment modalities that are detailed in Table S1 . Radiographic progression is marked with a black asterisk placed next to the corresponding MRI data point. Day 0 corresponds to the initiation of treatment. (K) Heatmap showing the correlation between the direction of change for the indicated features (see ). For the majority of patients, tumor MRI measurements show the highest correlation with the single-molecule measurements of H3-K27M-H3K27ac in plasma. The correlation was calculated based on interpolated data points to account for measurements not taken at exactly the same time. See also mutant p53 and ctDNA data in Figure S4 .

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet:

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Recombinant, Conjugation Assay, Antibody Labeling, Software

Journal: Oncology Reports

Article Title: Acquirement of DNA copy number variations in non-small cell lung cancer metastasis to the brain

doi: 10.3892/or.2015.4188

Figure Lengend Snippet: Expression of EGFR protein in formalin-fixed and paraffin-embedded tissues by IHC staining. (A and E) H&E staining of primary tumor in lung and secondary metastasis tumor in the brain. (B and F) Overexpression of EGFR was detected both in primary tumor in lung and secondary metastasis tumor in the brain. (C and G) Secondary tumor showed stronger staining of E746-A750del specific short in-frame deletions in 19 exon of EGFR than in primary tumor. (D and H) L858R mutant in 21 exon of EGFR was not detectable in either the primary or the secondary tumors. EGFR, epidermal growth factor receptor; IHC, immunohistochemistry.

Article Snippet: The sections (5 mm-thick) were incubated at 4°C in a humid chamber overnight with mouse monoclonal antibody against human epidermal growth factor receptor (EGFR; ready to use, catalog MAB-0196; Maixin Fuzhou, China), rabbit monoclonal antibody against short in-frame deletions in exon 19 (E746-A750del Specific) of EGFR (1:100, catalog #5747), and rabbit monoclonal antibody against L858R mutant in 21 exon of EGFR (1:100, catalog #3197) (both from Cell Signaling Technology, Boston, MA, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Over Expression, Mutagenesis

Journal: Theranostics

Article Title: Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition

doi: 10.7150/thno.60028

Figure Lengend Snippet: ATF4 transcriptionally upregulates DDIT4 upon inhibition of glutaminolysis to inactivate mTOR. (A) Venn diagrams for the intersection of the candidate genes derived from ATF4 potential target genes, differentially expressed genes upon glutaminolysis inhibition, and hallmarks genes of mTORC1. (B) DDIT3, ASNS, DDIT4, NUPR1, PHGDH mRNA expression of cells treated by 968 for 24 h was detected by qRT-PCR. (C) DDIT4 expression of cells treated by 968 for 24 h was detected by western blotting. (D) The effect of DDIT4 knockdown on mTORC1 activity of cells treated by 968 for 24 h was detected by western blotting. p-mTOR, p-p70S6K, p-S6, DDIT4 band density was quantified and expressed as fold change, compared with the control, by arbitrarily setting the control value as 1. (E) The effect of DDIT4 knockdown on 968-induced cell apoptosis was measured by Flow Cytometry. The result presents the proportion of apoptotic cells. (F) The expression of cleaved PARP after DDIT4 knockdown and 968 treatment was detected by western blotting, cleaved PARP band density was quantified and expressed as fold change, compared with the control, by arbitrarily setting the control value as 1. (G) ATF4 enrichment at the DDIT4 promoter was determined with ChIP assay. (H) Schematic representation of ATF4 and dominant-negative ATF4ΔN, an ATF4 mutant lacks the N-terminal transcriptional activation domain was shown. The transcription activation ability of ATF4 and dominant-negative ATF4ΔN on the DDIT4 promoter was measured with luciferase assay. (I) The change of ATF4 enrichment at the DDIT4 promoter after 968 treatment was determined with ChIP assay. (J) DDIT4 protein expression after ATF4 knockdown during 968 treatment was detected by western blotting. ATF4, DDIT4 band density was quantified and expressed as fold change, compared with the control, by arbitrarily setting the control value as 1. (K) DDIT4 mRNA expression after ATF4 knockdown during 968 treatment was detected by qRT-PCR. Data are shown as the means ± SD from three experiments. For all experiments, statistical significance was assessed by Student's t -tests, * P < 0.05.

Article Snippet: DDIT4 (rabbit, 10638-1-AP) and YTHDF2 (rabbit, 24744-1-AP) polyclonal antibodies were purchased from Proteintech.

Techniques: Inhibition, Derivative Assay, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Activity Assay, Control, Flow Cytometry, Dominant Negative Mutation, Mutagenesis, Activation Assay, Luciferase

Journal: Theranostics

Article Title: Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition

doi: 10.7150/thno.60028

Figure Lengend Snippet: Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition. (A-B) Timeline showing that mice were treated with AOM and with 2% DSS as indicated. From day 30 on, the mice began to be treated with respective drugs until sacrifice. On day 100, mice were euthanized and the tissue was collected as shown in (B). (C) Numbers of tumors per mouse were counted (n = at least 4, * P < 0.05). (D) Tumor size in mm 3 /tumor were measured (n = at least 4, * P < 0.05). (E) Body weights of mice were recorded (n = at least 4, * P < 0.05). (F) IHC analysis of ATF4 expression in normal tissue, DMSO and 968 treated tumors tissue. (G) Working model: during glutaminolysis inhibition, FTO enhances ATF4 expression by reducing YTHDF2-mediated mRNA decay. ATF4 transcriptionally upregulates DDIT4 to inhibit mTORC1 activity and promote pro-survival autophagy. Finally, targeting the ATF4 pathway by autophagy inhibition combined with asparagine inhibitor is synthetic lethality with glutaminolysis inhibitor in CRC. Data are shown as the means ± SD (C, D, E). For all experiments, statistical significance was assessed by Student's t -tests, * P < 0.05. Scale bars are 100 μm in (F).

Article Snippet: DDIT4 (rabbit, 10638-1-AP) and YTHDF2 (rabbit, 24744-1-AP) polyclonal antibodies were purchased from Proteintech.

Techniques: Inhibition, Expressing, Activity Assay

Journal: Nature Communications

Article Title: Rab27a regulates the transport of influenza virus membrane proteins to the plasma membrane

doi: 10.1038/s41467-025-61587-3

Figure Lengend Snippet: a A549 cells were transfected with siNC or siRNAs targeting Rab3b (siRab3b-1, siRab3b-2), Rab6a (siRab6a-1, siRab6a-2), Rab11a (siRab11a-1, siRab11a-2), Rab17 (siRab17-1, siRab17-2), Rab23a (siRab23a-1, siRab23a-2), Rab25 (siRab25-1, siRab25-2), Rab27a (siRab27a-1, siRab27a-2), Rab37 (siRab37-1, siRab37-2), or Rab38 (siRab38-1, siRab38-2) for 24 h. The cells were then infected with HM virus (MOI, 10), and HA protein levels on the cell surface were quantified by flow cytometry at 4 hpi. b A549 cells transfected with siNC or siRab27a (siRab27a-1, siRab27a-2) for 24 h were infected with HM virus (MOI, 0.1). c–e Rab27a KO and A549-Cas9 cells were infected with HM virus (MOI, 0.1), SH13/H9N2 virus (MOI, 0.01), or PR8/H1N1 virus (MOI, 0.01). f–h A549 cells were transfected with 2 μg/ml exogenous Rab27a or an empty vector as a negative control for 24 h. The cells were then infected with HM virus (MOI, 0.1), SH13 virus (MOI, 0.01), or PR8 virus (MOI, 0.01). i A549-Cas9, Rab27a KO, and Rab27a KO cells stably expressing Flag-Rab27a-WT, Flag-Rab27a-T23N (dominant-negative mutant), or Flag-Rab27a-Q78L (constitutively active mutant) were infected with HM virus (MOI, 0.1). Rab27a and NP protein expression levels were examined by western blot ( b , f , g , h , and i ), with GAPDH or β-actin serving as a loading control. Viral titers in the supernatants were quantified using a TCID 50 assay on MDCK cells ( b–i ). Data represent the means ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t -test.

Article Snippet: The antibodies used in this study and their sources are as follows: Rabbit polyclonal antibodies: Rab37, Rab3b, Rab6a, Rab11a, Rab17, Rab23, Rab25, Rab27a, Rab27b, SYTL4, SQSTM1, ATB4B, TNG46, GM130 (Proteintech; 13051-1-AP, 15774-1-AP, 10187-2-AP, 20229-1-AP, 17501-1-AP, 11101-1-AP, 13189-1-AP, 16868-1-AP, 13412-1-AP, 12128-1-AP, 18420-1-AP, 15131-1-AP, 13573-1-AP, 11308-1-AP), Rab38 (Abways, AY3825), SYTL1 (Bethyl, A305-648A-T), BECN1 (ABclonal Biotechnology, A7353), Phospho-ATG4B (Ser316) (Affinity Biosciences, AF3505), ATG16L1 (HUABIO, ET7106-65), and IAV M2, NP, NS1, and HA (GeneTex; GTX125951, GTX30852, GTX125990, GTX127357).

Techniques: Transfection, Infection, Virus, Flow Cytometry, Plasmid Preparation, Negative Control, Stable Transfection, Expressing, Dominant Negative Mutation, Mutagenesis, Western Blot, Control, Two Tailed Test

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a A schematic model showing the domain composition of MIWI and trajectory of the 5′ and 3′ ends of piRNA anchored with MID and PAZ-domain, respectively. The Y569/K573 and Y346/Y347 conserved in PIWI proteins are required for the 5′ end or 3′ end piRNA loading capacity of MIWI. b RNA co-IP assay of MIWI-associated-piRNAs (top) in wild-type (lane 1), Miwi YY/YY (lane 2), Miwi YK/YK (lane 3), and Miwi −/− testes (lane 4), with anti-MIWI IB as a loading reference (bottom). c , d Immunostaining of MIWI ( c , red) and TDRKH ( d , red) on testis sections from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/ − mice using regular microscopy. Left: representative staining images of indicated mouse testis sections, scale bar, 20 μm; right, the enlargement of yellow framed regions, scale bar, 5 μm. The developmental stages of spermatocytes and spermatids were distinguished according to γH2AX (green) and DAPI (greyscale) staining. White arrows indicated MIWI ( c ) or TDRKH ( d ) aggregations. PS pachytene spermatocytes, DS diplotene spermatocytes, RS round spermatids. e Co-immunostaining of MIWI (red) and TDRKH (green) on testis sections from adult wildtype, Miwi YY/YY and Miwi YK/YK mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites; yellow and white open arrowheads respectively indicated the unique localization of MIWI and TDRKH at non-colocalization sites. Scale bar, 10 μm. The results shown are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Co-Immunoprecipitation Assay, Immunostaining, Microscopy, Staining, Confocal Microscopy

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a , b Co-IP assay of the association of MIWI and TDRKH in mouse testes from 18 dpp wildtype, Miwi YY/YY , and Miwi YK/YK mice. Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting]. c RNA co-IP assay of MIWI-interacting piRNAs in anti-MIWI (lane 1) and anti-TDRKH IP pellets (lane 2) from adult wildtype mouse testes, with anti-MIWI IB as a loading reference (bottom). d Anti-MIWI unloaded preferably pulled down piRNA-unloaded MIWI (left) and TDRKH (right) in adult mouse testicular lysate. Left, RNA co-IP assays using anti-MIWI unloaded and control anti-MIWI antibodies in adult wildtype mouse testicular lysate, with anti-MIWI IB as loading references. Right, co-IP assay of the association of MIWI and TDRKH using anti-MIWI unloaded (lane 3) and control anti-MIWI antibodies (lane 4) in adult wildtype mouse testicular lysate, with testicular lysate (lane 1) and IgG IP (lane 2) serving as positive and negative controls, respectively. Quantification of blot intensity of TDRKH is shown in parentheses [the one anti-MIWI IP (lane 4) is set as 1.0 after normalization with MIWI blotting]. e RNase A treatment enhanced the MIWI-TDRKH interaction in wild-type mouse testes. Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from RNase A-untreated (lane 4) is set as 1.0 after normalization with MIWI blotting]. f Transfection of piRNA attenuated the MIWI-TDRKH interaction in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cells. OE overexpression. Quantification of blot intensity of TDRKH is shown in parentheses [the one with RNase A-untreated and piRNA-free condition in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cell lysates (lane 2) is set as 1.0 after normalization with MIWI blotting]. g Schematic diagram showing that piRNA loading facilitates the dissociation of MIWI from TDRKH. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Co-Immunoprecipitation Assay, Control, Transfection, Over Expression, Western Blot

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a Co-immunostaining of TDRKH (red) and TDRD6 (green) on testis sections from adult wildtype mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). PS pachytene spermatocytes, DS diplotene spermatocytes, RS round spermatids. Scale bar, 10 μm. b Co-immunostaining of MIWI (red) and TDRKH (green) on testis sections from adult wildtype and Tdrd6 − /− mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites; yellow and white open arrowheads respectively indicated the unique localization of MIWI and TDRKH at non-colocalization sites. Scale bar, 10 μm. c , d Co-IP assay of the association of MIWI with TDRD6 and TDRKH in mouse testes from adult wildtype ( c and d , lanes 3 and 4), Miwi YY/YY ( c , lanes 5 and 6) and Miwi YK/YK ( d , lanes 5 and 6) mice. Anti-MIWI IP pellets ( c and d , lanes 4 and 6) were immunoblotted by the indicated antibodies, with testicular lysate ( c and d , lanes 1 and 2) and IgG IP ( c and d , lanes 3 and 5) serving as positive and negative controls, respectively. Quantification of blot intensity of indicated proteins in anti-MIWI IP pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting]. e Co-immunostaining of MIWI (red) and TDRD6 (green) on testis sections from adult wildtype, Miwi YY/YY and Miwi YK/YK mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites. Scale bar, 10 μm. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Immunostaining, Confocal Microscopy, Co-Immunoprecipitation Assay, Control, Western Blot

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a RNase A treatment barely altered MIWI methylation and MIWI-TDRD6 interaction in the adult testicular lysate. Quantification is shown in parentheses [the one from RNase A-untreated (lane 2) is set as 1.0 after normalization with MIWI blotting]. b Co-IP assay of the association of MIWI (lane 2) or arginine methylation-deficient MIWI R-K mutant (lane 3) with TDRD6 in co-transfected HEK293T cells. Quantification is shown in parentheses [the one with wildtype MIWI (lane 2) is set as 1.0 after normalization with MIWI blotting]. c Co-IP assay of the effect of methyltransferase inhibitor methylthioadenosine (MTA, Sigma, D5011) on the MIWI-TDRD6 interaction in co-transfected HEK293T cells. Quantification is shown in parentheses [the one with DMSO treatment (lane 1) is set as 1.0 after normalization with MIWI blotting]. d , e piRNA loading-deficient mutations impaired arginine methylation of MIWI in mouse testes. Quantification is shown in parentheses [the one from the wildtype control mouse (lane 2) is set as 1.0 after normalization with MIWI blotting]. f Anti-MIWI unloaded antibody pulled down less methylated MIWI and TDRD6 in adult wildtype mouse testicular lysate (lane 3) compared with control anti-MIWI antibody (lane 4). Quantification is shown in parentheses [the one anti-MIWI IP (lane 4) is set as 1.0 after normalization with MIWI blotting]. g Sequential co-IP showing that TDRKH is mainly associated with unmethylated MIWI. Quantification is shown in parentheses [the first anti-MIWI IP (lane 2) is set as 1.0 after normalization with MIWI blotting]. h TDRKH reduced MIWI methylation in co-transfected HEK293T cells. Quantification is shown in parentheses [the one without TDRKH transfection (lane 1) is set as 1.0 after normalization with MIWI blotting]. i Schematic diagram showing that piRNA loading promotes MIWI dissociation from TDRKH, leading to the exposure of the N-terminal of MIWI for arginine methylation by PRMT5 to enhance the MIWI-TDRD6 interaction. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Methylation, Co-Immunoprecipitation Assay, Mutagenesis, Transfection, Control, Western Blot

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a All tested Miwi YY/YY and Miwi YK/YK males were infertile. b Testes from adult Miwi YY/YY and Miwi YK/YK mice were moderately reduced compared with wildtype control. Left, a representative image of testes from indicated mice; right, the average weight of testes from wildtype, Miwi YY/YY ( p = 0.033) and Miwi YK/YK ( p = 0.014) mice ( n = 6, data are represented as mean ± SD, P values were calculated using two-tailed Student’s t -test, *p < 0.05). c PAS staining of the testis (top) and H&E staining of the epididymis (bottom) sections from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− mice. Scale bar, 30 μm. d Acrosome staining (ACRV1, red) of testis sections from wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi − /− mice using regular microscopy. Developmental stages of the seminiferous tubules were distinguished according to γH2AX (green) and DAPI (grayscale) staining. Scale bar, 10 μm. e TUNEL assays (red) of testis sections from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/ − mice using regular microscopy, with nuclei counterstained by DAPI (blue). Scale bar, 30 μm. Results shown in c – e are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Control, Two Tailed Test, Staining, Microscopy, TUNEL Assay

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: a Detection of piRNA expression in adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. b The length distribution of small RNAs from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. Data were normalized by miRNA reads (21–23 nt). c Nucleotide distributions at the first position in the piRNAs from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. d Genomic annotation of the piRNAs from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. The percentage of mapped reads is shown. e Scatter plot of total piRNA reads mapped to individual piRNA clusters from adult wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− testes. Data were normalized by miRNA reads (21–23 nt). f Western blotting of MIWI and MILI expression in testes from wildtype, Miwi YY/YY , Miwi YK/YK , and Miwi −/− mice with indicated ages. β-actin served as a loading control. Quantification of blot intensity of MIWI is shown in parentheses (the one in wildtype testis is set as 1.0 after normalization with β-actin). Results shown in a and f are representative of three independent experiments, and small RNA-seq experiments shown in b – e with two replicates. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Expressing, Western Blot, Control, RNA Sequencing

Journal: Nature Communications

Article Title: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

doi: 10.1038/s41467-024-46664-3

Figure Lengend Snippet: Upon its expression in mid-pachytene spermatocytes, MIWI protein is recruited to the IMC for piRNA processing via interacting with TDRKH through its unmethylated N-terminus, while piRNA loading induces a conformational change of MIWI and, in turn, weakens its interaction with TDRKH, leading to its release from the IMC. Meanwhile, the disassociation of MIWI with TDRKH simultaneously results in the arginine residues in its N-terminus exposed for methylation by PRMT5, thereby enhancing TDRD6 binding to prime its localization in the CB for piRNA function.

Article Snippet: Moreover, we serendipitously discovered that a commercially available rabbit monoclonal anti-MIWI antibody (ABclonal, Catalog No.: A3490, whose specificity was confirmed through immunoblotting of either epitope-deleted MIWI Δaa701-801 mutant, or MIWI protein in wildtype or Miwi −/− mouse testes; Fig. S ) selectively immunoprecipitated piRNA-unloaded MIWI protein in mouse testes as well as in transfected HEK293T cells (Figs and S ).

Techniques: Expressing, Methylation, Binding Assay