d4476  (Selleck Chemicals)

Journal: Advanced Science

Article Title: Restoring Iron Homeostasis via Smoothened Inhibition: A Novel Strategy Against Hearing Loss

doi: 10.1002/advs.202520749

Figure Lengend Snippet: SMO inhibition restored iron homeostasis to alleviate ferroptosis via PKC‐ATF2‐IRP1‐FPN/TFRC axis. (A) Western blot analysis of FPN. (B) Quantitative analysis of cochlear HCs after neomycin and SANT‐1 treatment with different siRNAs. (C) Diagram showing the regulation of FPN expression at the transcriptional and post‐translational levels. (D) Western blot analysis of IRPs. (E) qPCR analysis of Irp1 , Irp2 , Fbxl5 , and Hamp1 . (F) qPCR analysis of Irp1 and Irp2 expression at various time points post‐neomycin damage. (G) Western blot analysis of ATF2(phosphor T71). (H) Predicted binding motif of p‐ATF2. (I) The binding tracks for p‐ATF2 at the genomic loci of Irp1 and Irp2 identified by calling peaks in the CUT&TAG‐seq data. (J) Top: Comparison of phosphorylation sites in human and mouse ATF2. Bottom: Schematic of overexpression of the phospho‐mimetic ATF2 mutant, with threonine 51 and 53 mutated to aspartic acid to mimic constitutive phosphorylation. (K,L) Validation of the overexpression effects of ATF2 and p‐ATF2 plasmids by Western blot. (M) The predicted p‐ATF2 binding sites within the promoter region of Irp1 and their specific locations. (N) Binding of p‐ATF2 to the promoter of Irp1 in HEI‐OC1 cells analyzed by dual luciferase assay. (O) qPCR validation of the efficacy of siRNA‐mediated knockdown of Atf2 . (P) Atf2 knockdown abolished neomycin‐induced Irp1 expression, as analyzed by dual luciferase assay. (Q,R) p‐ATF2 expression in cochlear explants after SAG treatment with indicated protein kinase inhibitors. MK‐2206, AKT inhibitor (AKTi). D4476, CK1 inhibitor (CK1i). Dasatinib, SRC inhibitor (SRCi). Dactolisib, PI3K inhibitor (PI3Ki). GSK180736A, GRK2 inhibitor (GRK2i). Bisindolylmaleimide II (BisII), PKC inhibitor (PKCi). H89, PKA inhibitor (PKAi). (S) Quantitative analysis of HCs after SAG treatment with or without indicated protein kinase inhibitors. (T) Quantitative analysis of HCs after RSL3 treatment with or without PKCi. Statistical analysis: one‐way analysis of variance (ANOVA) with Bonferroni's multiple comparison was employed for (F); two‐tailed Student's t ‐test was employed for (L), (O), (P), (R) and (S); two‐way ANOVA with a post‐hoc Student Newman–Keuls test was employed for (B), (E), (N) and (T). ns means no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The compounds used in this study included neomycin (Selleck; 0.2 m m for the experiment in Figure , 1 m m for other neomycin‐challenged cochlear explants, and 2 m m for HEI‐OC1 cell experiments), gentamicin (Selleck; 0.2 m m ), tobramycin (Selleck; 0.2 m m ), amikacin (Selleck; 0.5 m m ), kanamycin (Selleck; 0.5 m m ), SAG (Selleck; 0.05–10 μ m ), SANT‐1 (Selleck; 10 and 20 μ m ), cyclopamine (Selleck; 10 and 20 μ m ), taladegib (Selleck; 10 and 20 μ m ), glasdegib (Selleck; 10 and 20 μ m ), sonidegib (Selleck; 10 and 20 μ m ), vismodegib (Selleck; 10 and 20 μ m ), RSL3 (Selleck; 5 μ m ), ferrostatin‐1 (Selleck; 10 μ m ), ammonium ferric citrate (FAC; Selleck; 0.1–10 m m ), deferoxamine (DFO; Selleck; 100–500 μ m ), D4476 (MCE; 100 μ m ), H89 (Selleck; 20 μ m ), MK‐2206 (MCE; 5 μ m ), GSK180736A (Selleck; 5 μ m ), dactolisib (Selleck; 10 μ m ), dasatinib (Selleck; 20 μ m ), bisindolylmaleimide II (BisII; MCE; 10 μ m ), Go6976 (Selleck; 5 μ m ), Enzastaurin (Selleck; 5 μ m ), and Rottlerin (Selleck; 5 μ m ).

Techniques: Inhibition, Western Blot, Expressing, Binding Assay, Comparison, Phospho-proteomics, Over Expression, Mutagenesis, Biomarker Discovery, Luciferase, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Phosphorylation of the rod-tail hinge region of cingulin regulates its interaction with nonmuscle myosin-2B

doi: 10.64898/2026.04.02.716052

Figure Lengend Snippet: Inhibition of either CK1 or CK2 promotes the rescue of junctional NM2B in cingulin-KO cells expressing the depho-6 mutant of cingulin. (A) Top: scheme of GFP-tagged canine CGN (cCGN-FL), with the GFP tag (green), globular head (gray), coiled-coil rod (white) and globular tail (blue) domains (amino-acid residue boundaries are indicated above the scheme). Bottom: C-terminal sequences of WT canine CGN (cCGN-FL, sequence 1140–1190 with specific residues indicated in the region) and corresponding sequences of the cCGN-dephosphomimetic-6 mutant (depho-6), which does not rescue junctional NM2B (Fig. S1K’). (B-G) IF microscopy analysis and localization (left) and quantification of junctional labeling (relative fluorescence intensity) (right) of NM2B in CGN-KO MDCK cells rescued with GFP-cCGN-FL (B, D and F), or with GFP-cCGN-depho-6 mutant (C, E and G) treated either with DMSO (B and C) or with CK1 inhibitor (D and E: 25 μM, 8h) or with CK2 inhibitor (F and G: 25 μM, 14h). Arrows and arrowheads show increased, normal and decreased/undetected junctional labeling, respectively. Quantifications of relative fluorescent intensity (RFI) shows the ratio between the junctional staining of NM2B versus the junctional marker PLEKHA6 (n=70 junctions) from three independent experiments. Data in quantifications are represented as mean±SD. Statistical significance was determined by unpaired Mann-Whitney’s test. ***p≤0.001. Scale bar (G)= 10 μm.

Article Snippet: Drugs treatments were as follows (final concentration, duration, catalog number and source): CK1 inhibitor (25 μM, 8 h, D4476 MedChemExpress) and CK2 inhibitor (5 μM, 14 h, CX4945 Selleckchem).

Techniques: Inhibition, Expressing, Mutagenesis, Residue, Sequencing, Microscopy, Labeling, Fluorescence, Staining, Marker

Journal: Cell Death & Disease

Article Title: TRIM24 promotes proliferation and metastasis of gastric cancer via mediating NRBP1 ubiquitination

doi: 10.1038/s41419-025-08346-w

Figure Lengend Snippet: A The numbers of distinct proteins found by mass spectrometry in MKN74 and NUGC3 cells transfected with TRIM24 shRNA lentivirus compared with vector lentivirus. B Common differentially expressed proteins identified by proteomic screening of MKN74 and NUGC3 cells. C Volcano plot showing differentially expressed proteins in NUGC3 cells following TRIM24 knockdown. D TRIM24 and NRBP1 protein amounts evaluated in the vector, TRIM24 shRNA (1# and 2#), and overexpression TRIM24 lentiviruses groups by WB. E TRIM24 and NRBP1 protein amounts evaluated in orthotopic tumors of MKN74 and NUGC3 transfected vector, TRIM24 shRNA (1# and 2#), and overexpression TRIM24 lentivirus by WB. Colony forming ( F ), wound healing ( G , H ), migration and invasion ( I , J ) assays were performed in MKN74 and NUGC3 cells transfected with TRIM24 shRNA-1# lentivirus alone or TRIM24 shRNA-1# lentivirus and NRBP1 siRNA-1# simultaneously. Data represent mean ± SD (n = 3). Data are shown as mean ± SD. ns no statistical difference, *P < 0.05, * *P < 0.01, ** *P < 0.001.

Article Snippet: For the validation of the kinase responsible for the phosphorylation at the S42 site of NRBP1, HEK293T cells were treated with the CK1 inhibitor D4476 (MCE, Cat. No# HY-10324) for 6 h, followed by immunoblotting analysis.

Techniques: Mass Spectrometry, Transfection, shRNA, Plasmid Preparation, Knockdown, Over Expression, Migration

Journal: Cell Death & Disease

Article Title: TRIM24 promotes proliferation and metastasis of gastric cancer via mediating NRBP1 ubiquitination

doi: 10.1038/s41419-025-08346-w

Figure Lengend Snippet: A NRBP1 mRNA expression in the vector and shTRIM24 (1# and 2#) groups detected by qPCR. B , C GC cells infected with vector, overexpression TRIM24, and TRIM24 shRNA lentiviruses were treated with 100 µg/ml cycloheximide, and protein lysates were collected at the indicated times for WB. D Vector and overexpression TRIM24 GC cells were treated with 10 µg/mL MG-132 for 6 h and analyzed by WB. The numbers shown below the bands represent the relative densitometry values. E MKN74 and NUGC3 cell lysates were immunoprecipitated with control IgG or anti-TRIM24 antibody, followed by WB. F MKN74 and NUGC3 cell lysates were immunoprecipitated with control IgG or anti-NRBP1 antibody, followed by WB. G – I Transfect His-NRBP1 truncation fragments into HEK293T cells, followed by immunoprecipitation and WB analysis. J HEK293T cells (vector or overexpression flag-TRIM24) transfected with His-NRBP1 plasmid, and followed by WB. K HEK293T cells expressing His-NRBP1 and HA-ub were treated with 10 µg/mL MG-132 for 6 h and subjected to sequential immunoprecipitation and WB. L HEK293T cells expressing His-NRBP1, HA-ub, and Flag-TRIM24 were treated with 10 µg/mL MG-132 for 6 h and subjected to sequential immunoprecipitation-WB. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the validation of the kinase responsible for the phosphorylation at the S42 site of NRBP1, HEK293T cells were treated with the CK1 inhibitor D4476 (MCE, Cat. No# HY-10324) for 6 h, followed by immunoblotting analysis.

Techniques: Expressing, Plasmid Preparation, Infection, Over Expression, shRNA, Immunoprecipitation, Control, Transfection

Journal: Cell Death & Disease

Article Title: TRIM24 promotes proliferation and metastasis of gastric cancer via mediating NRBP1 ubiquitination

doi: 10.1038/s41419-025-08346-w

Figure Lengend Snippet: A Effects of the mutations of five phosphorylation sites on TRIM24-mediated NRBP1 expression in HEK293T cells, detected by WB. B Effects of NRBP1 S42 site mutation on TRIM24-mediated NRBP1 ubiquitination in HEK293T cells. Colony forming ( C ), wound healing ( D ), migration and invasion ( E ) assays were performed in MKN74 (vector or overexpression TRIM24) transfected with NRBP1 S42 site mutation plasmid. F Effects of the mutations of six ubiquitination sites on TRIM24-mediated NRBP1 expression in HEK293T cells, detected by WB. G Effect of NRBP1 K430 site mutation on TRIM24-mediated NRBP1 ubiquitination in HEK293T cells. Colony forming ( H ), wound healing ( I ), migration and invasion ( J ) assays were performed in MKN74 (vector or overexpression TRIM24) transfected with NRBP1 K430 site mutation plasmid. Data are shown as mean ± SD. ns no statistical difference, *P < 0.05, * *P < 0.01, ** *P < 0.001.

Article Snippet: For the validation of the kinase responsible for the phosphorylation at the S42 site of NRBP1, HEK293T cells were treated with the CK1 inhibitor D4476 (MCE, Cat. No# HY-10324) for 6 h, followed by immunoblotting analysis.

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Ubiquitin Proteomics, Migration, Plasmid Preparation, Over Expression, Transfection