Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: USP20 directly interacts with GPX4. ( A ) Immunoblot analysis of target proteins in sorafenib-resistant 769-P and A549 cells following 24 h treatment with specific deubiquitinase inhibitors. ML-323: 10 μM, GSK2643943A: 10 μM, XL177A: 10 μM, Spautin-1: 10 μM, IU1: 10 μM, AZ1: 10 μM, MF-094: 10 μM, LDN-57444: 10 μM, TCID: 10 μM, PR-619: 10 μM, BAY11-7082: 10 μM, EOAI3402143: 2.5 μM, ML364: 10 μM, b-AP15: 0.5 μM. ( B ) HEK293T cells were transfected with GST-GPX4 and Flag-USP. The cell lysate was applied with GST pull-down, then analyzed with immunoblot for indicated proteins. ( C ) 769-P and A549 cells were subjected to immunoprecipitate with anti-GPX4 antibodies, then analyzed with immunoblot for indicated proteins. ( D ) Overview of USP20 structures. ( E-F ) HEK293T cells transfected with the indicated USP20 structures and Flag-GPX4 were subjected to pull-down with GSH beads or immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( G ) 769-P and A549 cells were subjected to subcellular fractionation, then analyzed with immunoblot for indicated proteins. ( H ) Overview of GPX4 isoforms structures. ( I ) HEK293T cells transfected with the indicated GPX4 isoforms and GST-USP20 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( J ) HEK293T cells transfected with the GST-USP20 WT/C154S and Flag-GPX4 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins.

Article Snippet: Primary antibodies against USP20 (cat. no. 17491-1-AP; in ), HA-Tag (cat. no. 51064-2-AP), FLAG-Tag (cat. no. 20543-1-AP), GST-Tag (cat. no. 66001-2-Ig), β-Actin (cat. no. 66009-1-Ig), His-Tag (cat. no. 66005-1-Ig), GPX4(cat.no.67763-1-Ig), ACSL4 (cat. no. 22401-1-AP), SLC7A11 (cat. no. 26864-1-AP), FSP1 (cat. no. 20886-1-AP), FTH (cat. no. 11682-1-AP), Vinculin (cat. no. 26520-1-AP), MEK (cat. no. 11049-1-AP), TOMM20 (cat. no. 11802-1-AP), and H3 (cat. no. 17168-1-AP) were purchased from ProteinTech.

Techniques: Western Blot, Transfection, Fractionation

Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: USP20 removes GPX4 K48-linked poly-ubiquitination. ( A ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, GST-USP20 and His-Ub. ( B ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, GST-USP20 WT/C154S and His-Ub. ( C ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, and His-Ub under the condition of the indicated concentration of GSK2643943A. ( D ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in USP20 knockdown HEK293T cells transfected with Flag-GPX4 and His-Ub. ( E ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, HA-USP20 and His-Ub WT or His-Ub K48−only . ( F ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, HA-USP20 and His-Ub WT or His-Ub K48R .

Article Snippet: Primary antibodies against USP20 (cat. no. 17491-1-AP; in ), HA-Tag (cat. no. 51064-2-AP), FLAG-Tag (cat. no. 20543-1-AP), GST-Tag (cat. no. 66001-2-Ig), β-Actin (cat. no. 66009-1-Ig), His-Tag (cat. no. 66005-1-Ig), GPX4(cat.no.67763-1-Ig), ACSL4 (cat. no. 22401-1-AP), SLC7A11 (cat. no. 26864-1-AP), FSP1 (cat. no. 20886-1-AP), FTH (cat. no. 11682-1-AP), Vinculin (cat. no. 26520-1-AP), MEK (cat. no. 11049-1-AP), TOMM20 (cat. no. 11802-1-AP), and H3 (cat. no. 17168-1-AP) were purchased from ProteinTech.

Techniques: Ubiquitin Proteomics, Transfection, Concentration Assay, Knockdown

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 directly interacts with DDX41 and regulates its phosphorylation. ( A ) HCECs transfected with either pcDNA3.1 (Flag) or pcDNA3.1 PIM1 (Flag-PIM1) were stimulated with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. Cell lysates were immunoprecipitated using anti-Flag beads, and bound proteins were separated by SDS-PAGE and visualized by silver staining. ( B ) A GST pull-down assay was performed to examine direct binding between PIM1 and DDX41. ( C ) Endogenous interaction between PIM1 and DDX41 in HCECs was assessed by co-IP. HEK293T cells were cotransfected with Flag-PIM1 and Myc-DDX41 for 48 hours. (D) Co-IP was carried out with anti-Flag beads, and Myc-DDX41 was detected with anti-Myc antibody. ( E ) Reciprocal co-IP was performed using anti-Myc beads, and Flag-PIM1 was probed with anti-Flag antibody. ( F ) HCECs were transfected with NC siRNA (siNC) or PIM1 siRNAs (siPIM1-1 and siPIM1-2) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours. DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and DDX41 antibodies. ( G ) HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours, followed by DDX41 immunoprecipitation and immunoblotting with indicated antibodies. ( H ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA (siPIM1-2), and/or pcDNA3.1-PIM1 (PIM1) for 24 hours, then treated with A. fumigatus hyphae for 12 hours. Co-IP of DDX41 was performed, and associated proteins were analyzed by Western blot. ( I ) Quantification of protein levels in ( H ). ( J ) HCECs were pretreated with SGI-1776 (0 and 4 µM) for 12 hours prior to 12-hour stimulation with A. fumigatus hyphae. Co-IP assay was performed using anti-DDX41 antibody and immunoblotted with the antibodies indicated. ( K ) Quantification of protein levels in ( J ). ( L ) Recombinant His-PIM1 and GST-DDX41 were incubated in reaction buffer with or without phosphatase for 30 minutes. The protein levels of p-Ser, His-PIM1, and GST-DDX41 were detected by Western blot. Quantification of protein levels in F, G, and L is shown in A–C. Data are presented as the mean ± SD; ** P < 0.01; n = 3.

Article Snippet: Antibodies targeting PIM2 (25865-1-AP, 1:1000), PIM3 (33202-1-AP, 1:1000), β-actin (20536-1-AP, 1:50,000), GST (10000-0-AP, 1:2000), MYC (16286-1-AP, 1:2000), Flag (66008-4-Ig, 1:2000), His (66005-1-Ig, 1:2000), STING (19851-1-AP, 1:2000), IRF3 (11312-1-AP, 1:1000), and TBK1 (28397-1-AP, 1:1000) were sourced from Proteintech (Wuhan, China).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, SDS Page, Silver Staining, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Western Blot, Recombinant, Incubation

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet: (A) DRaCALA competition binding assay with CdgR showing the competitive binding of radiolabeled [ 32 P]c-di-AMP with different nucleotides (1 mM) compared to the absence of competitor (none). SbtB was used as a positive control for c-di-AMP binding assays (+) . The fraction of bound-[ 32 P]c-di-AMP to CdgR represents the mean ± SD relative to the no competitor control. (B) DRaCALA titration binding assay for unlabeled c-di-AMP and c-di-GMP, showing the efficiency of c-di-GMP in competing with [ 32 P]c-di-AMP compared to c-di-AMP. (C-F) ITC analysis of c-di-GMP (C) , c-di-AMP (D) , c-di-GMP (in presence of 250 µM c-di-AMP) (E) , and c-di-AMP (in presence of 125 µM c-di-GMP) (F) binding to CdgR. Upper panels show the raw ITC data in the form of heat produced during the titration of c-di-GMP/c-di-AMP on CdgR protein; lower panels show the binding isotherms and the best-fit curves according to the one binding site model.

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Binding Assay, Positive Control, Control, Titration, Produced

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet:

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet: A: Phototaxis assays of the cdgR::Km mutant strain in response to white light. Synechocystis wild-type and cdgR::Km mutant strains were cultured on BG11 agar plates (0.5% (w/v)) supplemented with 11 mM glucose and 10 mM TES buffer (pH 8.0). The cells were then unidirectionally illuminated with 5 μmol photons m −2 s −1 of white light. Images were captured after 14 days of incubation. The white dashed lines indicate the initial spotting areas. B and C: Single-cell motility of the cdgR::Km mutant strain. Cells of Synechocystis wild type and the cdgR::Km mutant were illuminated with unidirectional red light (40 µmol photons m -2 s -1 ) on BG11 agarose plates under a microscope, and the displacement of the cells over a 3 min time-frame was captured 2 min after the onset of illumination. Raw tracks of moving cells were determined using Fijís TrackMate plugin. The velocity and directionality of the moving cells were analyzed using R software. B: Mean speed of wild-type and cdgR::Km cells. C: The mean resultant length from a Rayleigh test ( r ) and the number of tracked motile cells ( n ) are shown. D: Phototaxis assays of the cdgR::Km mutant strains in response to 5 µmol photons m -2 s -1 of blue light. E: Flocculation assay of the cdgR::Km strain under white light. Aggregation values of Synechocystis wild type (white boxes, n = 12) and cdgR::Km (grey boxes, n = 24) are displayed before and after two days of incubation. A two-sample t -test assuming equal variances was applied F: Transformation efficiency of the cdgR::Km mutant strain. These experiments determined the transformation efficiency of Synechocystis wild-type (n = 24) and the cdgR::Km mutant strains (n = 32). The number of colony-forming units (cfu) was counted after 12 days, and the transformation efficiency was normalized to the optical density at 750 nm (OD 750nm ) and the amount of DNA used. For each transformation, one microgram of pJET -ΔcrhR -SpecR plasmid DNA was used, conferring spectinomycin resistance. A Welch′s t -test was applied. Wild type, WT.

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Mutagenesis, Cell Culture, Incubation, Single Cell, Microscopy, Software, Flocculation, Transformation Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet: A: Phototaxis assays of the Δ cdgR (Cfp1) mutant strain in response to white light. Synechocystis wild type, Δ cdgR (Cfp1) and complementation strain (C-Δ cdgR ) were cultivated on BG11 agar plates (0.7% (w/v)) supplemented with 10 mM glucose and 12.5 mM TES buffer (pH 8.0). The cells were then unidirectionally illuminated with 5 μmol photons m -2 s -1 of white light for four days. The dashed lines indicate the initial spotting areas. Three independently generated complementation clones (#1-3) were used. B: BG11 agar (0.7% (w/v)) supplemented with 10mM glucose and 12.5 mM TES buffer (pH 8.0) was filled in a (24 cm x 24 cm) square plates for phototaxis assay. All the indicated strains were inoculated on the plate at different distances from the white, red, green or blue LED light source, which was placed laterally to provide unidirectional illumination. The position of the initial spot was marked by grey dashed lines and the light intensity (μmol photons m -2 s -1 ) was specified by the number on the right. C: Quantification of the relative run length from phototaxis assay from B . light intensity was indicated.

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Mutagenesis, Generated, Clone Assay

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet: A: Volcano plot showing the differentially expressed genes (DEGs) in the ΔcdgR mutant compared to the wild type determined by RNA-seq. DEGs were defined as genes with [log 2 fold change] >1 and log 10 (FDR)<0.05. B-E: Expression of minor pilin genes in the cdgR::Km mutant strain. The cdgR::Km mutant strain was examined for the expression of minor pilin genes. After 24 h of exposure to white-light illumination (75 µmol photons m -2 s -1 ), total RNA was extracted from cells grown in BG11 medium. Three micrograms of RNA were hybridized with radioactively labeled RNA probes targeting the pilA5 mRNA ( B ) and the 5’-UTR of the pilA9 mRNA ( C ). A double-stranded DNA probe that hybridized with Synechocystis 16S rRNA was used as a loading control. Densitometric quantification determined the relative levels of pilA5 ( D ) and pilA9 mRNA ( E ), which were normalized to 16S rRNA levels. Two biological replicates, each with two technical replicates, were performed for the wild type. Four biological replicates, each with two technical replicates, were used for the cdgR::Km mutant experiment.

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Mutagenesis, RNA Sequencing, Expressing, Labeling, Control

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet: (A). The effect of high concentration (B) and low concentration (C) of c-di-GMP and c-di-AMP on the CdgR-SyCRP1 complex; and the effect of cAMP on the CdgR-SyCRP1 complex in absence or presence of c-di-GMP (D) . The complex is indicated by higher mass of 90-120 kDa.

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet: (A). The effect of high concentration (B) and low concentration (C) of c-di-GMP and c-di-AMP on the CdgR-SyCRP2 complex; and the effect of cAMP on the CdgR-SyCRP2 complex in absence and presence of different concentration of c-di-GMP (D) . The complex is indicated by higher mass of 90-120 kDa.

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Regulation of cyanobacterial type IV pilus-dependent functions by interaction between a c-di-GMP receptor and two transcription factors

doi: 10.64898/2026.03.27.713163

Figure Lengend Snippet: Under high c-di-GMP conditions (e.g., blue light-dependent cyclase activity of Cph2), CdgR does not bind to the transcription factors SyCRP1 and SyCRP2. Therefore, they can bind to DNA and repress or activate the expression of minor pilin genes. Under low cellular c-di-GMP concentrations, CdgR inactivates both transcription factors by binding to them. In the absence of CdgR, the pilA5-pilA6 operon, which encodes minor pilins important for DNA uptake, is repressed, and the cells are not transformable. Overaccumulation of pilA9-pilA12 mRNA in the cdgR mutant leads to enhanced phototactic movement. Other unknown functions of CdgR beyond the control of the two CRP-like transcription factors, as well as the contribution of other nucleotide second messengers, are likely.

Article Snippet: Anti-His-tag antibody at a dilution of 1:2000 was used to detect the immobilized His-tagged CdgR and SyCRP2, while anti-GST-tag antibody (10000-0-AP, Proteintech) at a dilution of 1:2000 was used to detect the GST-tagged SyCRP1-GST and SyCRP2-GST co-eluted with the immobilized CdgR.

Techniques: Activity Assay, Expressing, Binding Assay, Mutagenesis, Control