Journal: eLife

Article Title: An NAD + -dependent novel transcription factor controls stage conversion in Entamoeba

doi: 10.7554/eLife.37912

Figure Lengend Snippet: ( A ) EMSA results with purified GST tagged ERM-BP and radiolabeled ERM probe. Unlabeled ERM oligonucleotides in 100X excess were used as a specific competitor and GST as control protein. The red arrow indicates the major shifted band that exhibits specific binding. ( B ) EMSA results with radiolabeled ERM and nuclear extracts from both cysts/trophozoites from control cells and nuclear extracts from myc-tagged ERM-BP_OX trophozoites. Unlabeled ERM oligonucleotide in excess at 100X was used as a specific competitor. The red arrow indicates the major band that exhibits specific binding. 10.7554/eLife.37912.012 Figure 2—source data 1. All proteins identified from three independent mass-spec experiments. Mass spectrometry was performed on three paired samples (ERM-WT and ERM-core). In one experiment, ERM-WT with trophozoite nuclear extract was used as a second control. The cut-off criteria: minimum protein, 95%; minimum number of peptides, 1; minimum peptide, 95% were used for analysis. All the proteins identified are listed with Gene ID, spectral count and fold change (Excel file).

Article Snippet: GST-fusion proteins were expressed in Escherichia coli (BL21) and purified by using glutathione beads (GE-healthcare).

Techniques: Purification, Binding Assay, Mass Spectrometry

Journal: eLife

Article Title: An NAD + -dependent novel transcription factor controls stage conversion in Entamoeba

doi: 10.7554/eLife.37912

Figure Lengend Snippet: Representative EMSA results by using recombinant ERM-BP-WT and mutant proteins (ERM-BP-DBM, C198A, K150A and D12A) with radiolabeled ERM in presence of different concentrations of NAD + (0, 1, 2, 3 and 4 mM). EMSA shifted bands are shown by red arrows and free probes at the bottom. GST was used as negative control.

Article Snippet: GST-fusion proteins were expressed in Escherichia coli (BL21) and purified by using glutathione beads (GE-healthcare).

Techniques: Recombinant, Mutagenesis, Negative Control

Journal: eLife

Article Title: An NAD + -dependent novel transcription factor controls stage conversion in Entamoeba

doi: 10.7554/eLife.37912

Figure Lengend Snippet: The fluorescence intensity (FI) at varying temperatures (upper panel) and derivative melt curves calculated by differences in FI at each temperature (lower panel) are shown. Peak temperature in the lower panel of graph dFl/dT is considered as Tm. The recombinant proteins are ( A ) GST, ( B ) ERM-BP-WT, ( C ) D12A, ( D ) ERM-BP-DBM, ( E ) K12A and ( F ) C198A. Different concentrations of NAD + were used as indicated as 0 mM (blue), 1 mM (red), 2 mM (green) and 4 mM (pink). Experiments were done in quadruplicate for each NAD + concentration for each protein and in three independent experiments. Representative thermal shift assay is shown from one experiment. ( G ) Thermal stability assay of ERM-BP-WT with different amounts of NADP as indicated 0 mM (green), 1 mM (blue), 2 mM (pink) and 4 mM (orange) in quadruplicate for each NADP concentration.

Article Snippet: GST-fusion proteins were expressed in Escherichia coli (BL21) and purified by using glutathione beads (GE-healthcare).

Techniques: Fluorescence, Recombinant, Concentration Assay, Thermal Shift Assay, Stability Assay

Journal: eLife

Article Title: An NAD + -dependent novel transcription factor controls stage conversion in Entamoeba

doi: 10.7554/eLife.37912

Figure Lengend Snippet: The graph represents percentage of nicotinamide turn over into nicotinic acid by ERM-BP-WT and mutant recombinant proteins. Bacterial PncA was used as positive control and GST as a negative control. Data are mean ±s.d. (n = 3). The catalytic activities of the wild type (WT) ERM-BP and mutant recombinant proteins were determined by HPLC analysis. Bacterial nicotinamidase (PncA) was used as positive control with 100% conversion; GST was a negative control with 0% conversion. LC-traces are shown as a representative example from one experiment out of three independent experiments.

Article Snippet: GST-fusion proteins were expressed in Escherichia coli (BL21) and purified by using glutathione beads (GE-healthcare).

Techniques: Mutagenesis, Recombinant, Positive Control, Negative Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Affinity-based protein profiling to reveal targets of puerarin involved in its protective effect on cardiomyocytes.

doi: 10.1016/j.biopha.2020.111160

Figure Lengend Snippet: Fig. 1. Cardiomyocyte protection by puerarin from oxidative stress and high glucose stress and synthesis of the puerarin probe. A) The extent of apoptosis of AC16 cells analyzed by flow cytometry. AC16 cells were pretreated with DMSO or puerarin (Pue, 100 μM) for 12 h, followed by addition of H2O2 (400 μM) or a high glucose (4.5 g/L) stimulations. The cells were then stained with Annexin V-FITC for 30 min and propidine iodide (PI) for 5 min, and determined by flow cytometry. B) Synthesis of the puerarin probe (Pue-DA) with a photosensitive crosslinker for profiling of Pue targets. C) The protective of Pue-DA on cardiomyocytes was assessed by flow cytometry.

Article Snippet: Human cardiomyocytes (AC16 cell line) were purchased from American Type Culture Collection (ATCC, MD, USA).

Techniques: Flow Cytometry, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Affinity-based protein profiling to reveal targets of puerarin involved in its protective effect on cardiomyocytes.

doi: 10.1016/j.biopha.2020.111160

Figure Lengend Snippet: Fig. 3. Verification of puerarin-binding proteins responsible for the protection of human AC16 cardiomyocytes. A) Workflow to identify puerarin targets by using the strategy of affinity-based protein profiling (AfBPP). B) in situ fluorescence labeling of human AC16 cells treated with Pue-DA using UV irradiation or not. C) in situ fluorescence labeling of human AC16 cells treated with Pue-DA at different concentrations. For the competition assay, AC16 cells were pretreated with excessive Pue (20X) for 30 min, and then co-treated with Pue-DA (50 μM) for 4 h.

Article Snippet: Human cardiomyocytes (AC16 cell line) were purchased from American Type Culture Collection (ATCC, MD, USA).

Techniques: Binding Assay, In Situ, Fluorescence, Labeling, Irradiation, Competitive Binding Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Affinity-based protein profiling to reveal targets of puerarin involved in its protective effect on cardiomyocytes.

doi: 10.1016/j.biopha.2020.111160

Figure Lengend Snippet: Fig. 4. Mass spectrometry results of the Pue-DA probe-binding proteins in cardiomyocytes. A) Venn diagram representation of the number of proteins for all three repeats determined by AfBPP in which the fold change is greater than 2 and p-value is less than 0.05. These experi ments were performed in triplicate for each group. B) Volcano plot showing the targets identified by LC–MS/MS analysis. Red blots indicated significant targets with fold change greater than 2 and p value less than 0.05. These experiments were performed in triplicate for each group. Blue plots labeled with blue lettering indicate the significant targets enriched both in H2O2 (400 μM, left) or high glucose (4.5 g/L, right) damage groups. C) Protein hits were enriched both in H2O2 (400 μM) or high glucose (4.5 g/L) damage groups. These experiments were performed in triplicate for each group.

Article Snippet: Human cardiomyocytes (AC16 cell line) were purchased from American Type Culture Collection (ATCC, MD, USA).

Techniques: Mass Spectrometry, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Labeling

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Affinity-based protein profiling to reveal targets of puerarin involved in its protective effect on cardiomyocytes.

doi: 10.1016/j.biopha.2020.111160

Figure Lengend Snippet: Fig. 5. Verification of the binding tar gets of Pue. A) The three enrichment targets of Pue- DA in the damaged cardiomyocytes were verified by western blotting. These experiments were performed in triplicate for each group, and data are represented as mean ± SEM, *p < 0.05 versus DMSO group. B) Cellular thermal shift assay was then used to confirm the stabilizing effect of Pue on the enrichment targets. Three independent experiments were performed for each group. C) Three siRNA strands of chaf1b were assessed by western-blotting assay. Three indepen dent experiments were performed for each group. Data are represented as mean ± SD, **p < 0.01 and ***p < 0.001 versus negative control group. D) AC16 cells were transfected with NC/CHAF1B- siRNA-2 (20 nM) and co-treated with equivalent volume DMSO or Pue (100 μM) for 48 h, and the cell survival was detected using CCK-8 kit. These experi ments were performed in triplicate for each group. Data are represented as mean ± SD, ***p < 0.001 versus nega tive control with DMSO group and ##p < 0.01 versus negative control with Pue group. E) AC16 cells were transfected with NC/ CHAF1B-siRNA-2 (20 nM) and co-treated with equivalent volume DMSO or Pue (100 μM) for 48 h, and the percent of apoptotic cells was determined by flow cytometry.

Article Snippet: Human cardiomyocytes (AC16 cell line) were purchased from American Type Culture Collection (ATCC, MD, USA).

Techniques: Binding Assay, Western Blot, Thermal Shift Assay, Negative Control, Transfection, CCK-8 Assay, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Small molecule activation of the tumor suppressor kinase LKB1

doi: 10.1101/2024.12.17.628051

Figure Lengend Snippet: A. Model of hypothesized enhancement of LKB1 activation through the use of SMALS to stabilize the STRAD pseudokinase “active” conformation, thereby enhancing trimeric complex formation and LKB1 kinase activity. B. TNP-ATP screen of advanced kinase inhibitor library, measuring ability of small molecules to bind STRAD’s ATP-binding pocket. Screen was performed on recombinant STRAD/MO25. Significant hits are colored blue, the top five hits are labeled. ATP is shown and labeled in red. C. TNP-ATP assay validating NVP-AEW541 displacement of ATP from recombinant STRAD/MO25. D. NVP-AEW541 activates LKB1 in an in vitro kinase assay using the LKB1/STRAD/MO25 trimer. Kinase activity was read out with a Caliper device. E. Schematic of the calculated size of wild-type of STRAD and its conjugation with the SMALS-c3, anticipated to occur at STRAD Cysteine 160 (C160); LC-MS analysis shows that the majority of wild type of STRAD is conjugated with SMALS-c3 after 6 hours’ incubation. F. C160S LC-MS of STRAD incubated with SMALS-c3 demonstrating loss of covalent binding.

Article Snippet: Samples were eluted with 5 μ L of 5x loading dye and analyzed by western blotting. anti-LKB1 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 3050S), anti-STRAD (1:1000 in 5% BSA in TBS-T; Abcam; ab192879), anti-MO25 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 2716S)

Techniques: Activation Assay, Activity Assay, Binding Assay, Recombinant, Labeling, ATP Assay, In Vitro, Kinase Assay, Conjugation Assay, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: bioRxiv

Article Title: Small molecule activation of the tumor suppressor kinase LKB1

doi: 10.1101/2024.12.17.628051

Figure Lengend Snippet: A. Selected examples of aminopyrazinamide (APA) and pyrrolopyrimidine (PYP) analogue structure-activity relationship. B. Summary graph of compound potency vs. enhancement of LKB1 activity in vitro for APA and PYP scaffolds. PYP compounds had lower EC 50 values, APA compounds had a greater enhancement of LKB1 kinase activity. C. Dose-response curve for SMAL-323 (PYP) and SMALS-329 (APA), demonstrating differences in ceiling of activation and inhibition of LKB1 at higher concentrations by SMALS-323. D. Competition assay between reversible compound SMALS-329 with irreversible covalent SMALS-c3: Left panel shows SMALS-c3 conjugation to STRAD, which is reduced when STRAD/MO25 is preloaded with 20 µM SMALS-329 for 30 minutes.

Article Snippet: Samples were eluted with 5 μ L of 5x loading dye and analyzed by western blotting. anti-LKB1 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 3050S), anti-STRAD (1:1000 in 5% BSA in TBS-T; Abcam; ab192879), anti-MO25 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 2716S)

Techniques: Activity Assay, In Vitro, Activation Assay, Inhibition, Competitive Binding Assay, Conjugation Assay

Journal: bioRxiv

Article Title: Small molecule activation of the tumor suppressor kinase LKB1

doi: 10.1101/2024.12.17.628051

Figure Lengend Snippet: A. Immunoprecipitation of doxycycline (dox)-inducible 3xFLAG-LKB1 WT in A549 cells, a lung cancer line with an early nonsense mutation in LKB1 . An increase in the association of complex components is correlated with a SMALS-329 dose. B. Quantified results from cellular thermal shift assay in A549 cells. Greater 3xFLAG-LKB1 WT and STRAD stabilization is observed following 1 hour treatment with 20 µM SMALS-329 when LKB1 WT is stably transduced in A549 cells. C. Left: Western blot analysis of proximal downstream signaling in H358 cells after a time course of 8 µM treatment with SMALS-329. LKB1 activation results in increased SIK phosphorylation. Right: Cells were treated with a range of doses (DMSO, 310 nM, 930 nM, 2.8 µM, 8.33 µM, 25 µM) of SMALS-329 and harvested at 4 hours’ time point. D. Effect of 120 hours of SMALS-329 on cell viability in H358 cells stably transduced with dCas9-KRAB and LKB1 targeting sgRNA or non-targeting control sgRNA, measured by CellTiter-Glo. Inset: western blot confirming knockdown.

Article Snippet: Samples were eluted with 5 μ L of 5x loading dye and analyzed by western blotting. anti-LKB1 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 3050S), anti-STRAD (1:1000 in 5% BSA in TBS-T; Abcam; ab192879), anti-MO25 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 2716S)

Techniques: Immunoprecipitation, Mutagenesis, Thermal Shift Assay, Stable Transfection, Western Blot, Activation Assay, Transduction, Control, Knockdown

Journal: bioRxiv

Article Title: Small molecule activation of the tumor suppressor kinase LKB1

doi: 10.1101/2024.12.17.628051

Figure Lengend Snippet: A. Cell screen to evaluate the growth inhibition of cancer cells by SMALS-329 and SMALS-357 treatment by Cell Titer Glo. Neither tissue of origin nor tumor type predicted sensitivity to LKB1 activation. SMALS-329 GI 50 vs. SMALS-357 GI 50 : Pearson r (62) = .37, p** = .0026, GI 50 cut off: 50µM. Four of the most sensitive cells include: ACHN (kidney), BICR56 (tongue), NCI-H358 (lung), and RL95-2 (endometrium). Red dots represent cell lines with LKB1 inactivation. B. Summary gene set enrichment analysis (GSEA) of RNA-seq datasets derived from the Cancer Cell Line Encyclopedia (CCLE) to assess gene expression-based SMALS-329 sensitivity profile. Significant ‘Hallmark’ gene sets are shown. Cell lines sensitive to LKB1 stimulation had downregulation of inflammation-relevant Hallmark Gene Sets. C. Western blot analysis of proximal downstream signaling in ACHN cells with CRISPR deletions of LKB1 , STRAD or T Cell Receptor Alpha Constant ( TRAC , a CRISPR control). Cells were treated with a range of doses of SMALS-329 (DMSO, 310 nM, 930 nM, 2.8 µM, 8. 3 µM, 25 µM). Treatment with SMALS-329 in control cells resulted in increased phosphorylation of SIKs and AMPK. This effect is lost in cells with CRISPR deletions of LKB1 and STRAD . D. Western blot analysis of proximal downstream signaling in ACHN cells treated with a range of doses of SMALS-329 and a range of doses of a negative control compound lacking the suspected hinge binding motif, SMALS-357. Treatment with SMALS-329 results in increased phosphorylation of LKB1 and increased phosphorylation of AMPK. Treatment with SMALS-537 results in no change in LKB1 phosphorylation and decreased phosphorylation of AMPK.

Article Snippet: Samples were eluted with 5 μ L of 5x loading dye and analyzed by western blotting. anti-LKB1 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 3050S), anti-STRAD (1:1000 in 5% BSA in TBS-T; Abcam; ab192879), anti-MO25 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 2716S)

Techniques: Inhibition, Activation Assay, RNA Sequencing Assay, Derivative Assay, Expressing, Western Blot, CRISPR, Control, Negative Control, Binding Assay

Journal: bioRxiv

Article Title: Small molecule activation of the tumor suppressor kinase LKB1

doi: 10.1101/2024.12.17.628051

Figure Lengend Snippet: A. Western blot analysis of proximal downstream signaling in ACHN and RL95-2 after a time course of 8 µM treatment with SMALS-329. LKB1 activation results in increased SIKs and AMPK phosphorylation, albeit with distinct kinetics. B. RNAseq analysis in ACHN after 24h treatment with 8 µM SMALS-329. Hits regulating cell polarity (all on downregulated side), cell adhesion/motility (all on the downregulated side), inflammation and unfolded protein response (UPR) are highlighted.

Article Snippet: Samples were eluted with 5 μ L of 5x loading dye and analyzed by western blotting. anti-LKB1 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 3050S), anti-STRAD (1:1000 in 5% BSA in TBS-T; Abcam; ab192879), anti-MO25 (1:1000 in 5% BSA in TBS-T; Cell Signaling Technology; 2716S)

Techniques: Western Blot, Activation Assay

Journal: RSC Chemical Biology

Article Title: Covalent targeting of PSMD14 by Eupalinolide B induces oncoprotein degradation and apoptosis in acute promyelocytic leukemia cells

doi: 10.1039/d5cb00197h

Figure Lengend Snippet: (A) Co-localization of PSMD14 (green) and EB-P (red) in HL-60 cells confirmed by immunofluorescence staining (scale bar = 10 μm). (B) Pull-down assay using EB-P, followed by Western blotting, confirming that EB binds to PSMD14 in situ . (C and D) Cellular thermal shift assay (CETSA)-WB indicating the direct interaction between EB and PSMD14. (E, Left) Structural model of the predicted PSMD14–EB complex. The protein is shown as a gray cartoon, and EB is depicted in orange sticks. Residues forming the predicted binding pocket are highlighted. (E, Right) Detailed view of the predicted binding interface. The key residue His183 of PSMD14 is represented as sticks and is labeled. (F) Mapping of the EB binding site on recombinant human PSMD14 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (G) Western blot showing PSMD14 protein levels in HL-60 cells incubated with or without EB (15 μM). (H) In vitro activity assay showing that EB (100 μM or 200 μM) significantly inhibits PSMD14 enzymatic activity. (I) Both EB (15 μM) and CZM (40 μM, a known PSMD14 inhibitor) markedly inhibited HL-60 cell proliferation. (J) Cell viability was assessed by CCK-8 assay following PSMD14 knockdown. (K) HL-60 cells transfected with siPSMD14-2 or siNC were treated with different concentrations of EB, and cell viability was measured by CCK-8 assay. (L–O) Cell cycle distribution of HL-60 cells treated with CZM. (P–S) PSMD14 knockdown significantly altered cell cycle progression in HL-60 cells. ** P < 0.01, *** P < 0.001, ns = not significant.

Article Snippet: To identify the direct binding site of EB on PSMD14, 40 μg of recombinant human PSMD14 protein (Signalchem Lifesciences) was incubated with 40 μM EB for 2 h at 37 °C.

Techniques: Immunofluorescence, Staining, Pull Down Assay, Western Blot, In Situ, Thermal Shift Assay, Binding Assay, Residue, Labeling, Recombinant, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Incubation, In Vitro, Activity Assay, CCK-8 Assay, Knockdown, Transfection

Journal: RSC Chemical Biology

Article Title: Covalent targeting of PSMD14 by Eupalinolide B induces oncoprotein degradation and apoptosis in acute promyelocytic leukemia cells

doi: 10.1039/d5cb00197h

Figure Lengend Snippet: (A and B) WB analysis of AKT1 and CDK4 protein expression following PSMD14 knockdown in HL-60 cells. (C and D) WB analysis of PSMD14, AKT1, and CDK4 in HL-60 cells treated with the PSMD14 inhibitor CZM. (E and F) Expression of PSMD14, AKT1, and CDK4 after treatment with cycloheximide (CHX, 20 μM), analyzed by Western blot. (G and H) WB analysis of PSMD14, AKT1, and CDK4 in HL-60 cells transfected with siPSMD14-2 and subsequently treated with EB; siNC was used as the negative control. (I–K) Quantification of PSMD14 (I), AKT1 (J), and CDK4 (K) protein levels with or without EB treatment. * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant. Identical letters indicate no statistically significant difference, while different letters indicate P < 0.05.

Article Snippet: To identify the direct binding site of EB on PSMD14, 40 μg of recombinant human PSMD14 protein (Signalchem Lifesciences) was incubated with 40 μM EB for 2 h at 37 °C.

Techniques: Expressing, Knockdown, Western Blot, Transfection, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic di-GMP suppresses cancer metastasis by targeting proteasome 26S subunit non-ATPase 3 independently of STING

doi: 10.1038/s41392-025-02553-9

Figure Lengend Snippet: c-di-GMP inhibits human cancer metastasis in nude mice. a Chemical structure of c-di-GMP (cdG, 3′,5′-cyclic diguanylic acid or 3′,5′-cyclic dimeric guanosine monophosphate). b Representative images of the wound-healing assay (upper) and transwell migration assay (lower) performed on MDA-MB-231 cells treated with the indicated doses of c-di-GMP for 15–17 h. Migrated cells were quantified manually. *** P < 0.001 compared with the control group ( n = 6–8 random fields from 3 separate wells or inserts per condition). The data are presented as the means ± SDs. Scale bar, 200 μm. c Representative bioluminescence images of breast cancer cell metastases. MDA-MB-231-luc cells were injected intravenously into female nude mice. PBS or c-di-GMP was administered intraperitoneally on day 0 and subsequently every other day. Lung metastases were monitored weekly via an in vivo imaging system. d Quantification of bioluminescence (luciferase flux; p/s = photons/second) for lung metastases. n = 8 mice per group. e Mean bioluminescence at the end of the indicated treatments. Each dot represents an individual mouse ( n = 8 per group). The data are presented as the means ± SEMs. * P < 0.05; *** P < 0.001 (one-way ANOVA followed by Bonferroni’s multiple comparison test). f H&E staining of lung sections from the indicated groups harvested on day 42. Scale bar = 50 μm as indicated

Article Snippet: Various human cancer cell lines, such as MDA-MB-231 (RRID:CVCL_0062), MDA-MB-231-Luc2 (RRID:CVCL_D582), BT-549 (RRID:CVCL_1092), MCF-7 (RRID:CVCL_0031), Hs578T (RRID:CVCL_0332), PANC-1 (RRID:CVCL_0480), LoVo (RRID:CVCL_0399), H1299 (RRID:CVCL_0060), Calu-1 (RRID:CVCL_0608), B16-F10-Luc (JCRB Cat# NIHS0699, RRID:CVCL_4Y00) and HEK293T (ATCC Cat# CRL-3216, RRID:CVCL_0063), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO), 1% penicillin and streptomycin (GIBCO).

Techniques: Wound Healing Assay, Transwell Migration Assay, Control, Injection, In Vivo Imaging, Luciferase, Comparison, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic di-GMP suppresses cancer metastasis by targeting proteasome 26S subunit non-ATPase 3 independently of STING

doi: 10.1038/s41392-025-02553-9

Figure Lengend Snippet: The antimetastatic effect of c-di-GMP is independent of STING. a MDA-MB-231 cells were treated with the indicated doses of c-di-GMP for 24 h, and total STING levels were assessed by WB analysis. The numbers underneath the bands represent the normalized density quantified by densitometry via ImageJ. b MDA-MB-231 cells were treated with 5 μM c-di-GMP for the indicated time points (3–24 h), and total STING levels and downstream protein expression were assessed via WB analysis. c Genetic knockout of STING in MDA-MB-231 cells was achieved via CRISPR-Cas9. Several MDA-MB-231 clones stably expressing Cas9 and either control sgRNA (sg-Ctrl) or three different sgRNAs targeting STING were isolated. Total STING levels and downstream protein expression were assessed via WB analysis. d Metastasis was analyzed via a transwell migration assay with or without 5 μM c-di-GMP for 16 h in STING KO clones. Representative images are shown (upper), and migrated cells were manually quantified (lower). n = 6‒11 random fields from 3 separate inserts per condition. The data are presented as the means ± SDs. *** P < 0.001 compared with the control group; ns not significant. Scale bar, 100 μm e The migration of MDA-MB-231 cells following treatment with different concentrations of STING agonists (c-di-AMP, 2′3′-cGAMP or c-di-GMP) was evaluated via a wound-healing scratch assay at the indicated time points. An IncuCyte WoundMaker kit was used, and images were captured via the IncuCyte Zoom system. Representative images showing the 5 μM treatment at 20 h. Wound width was analyzed via IncuCyte software ( n = 6). Scale bar, 400 μm. The results were quantified and are presented as the means ± SDs; *** P < 0.001; ** P < 0.01; ns not significant

Article Snippet: Various human cancer cell lines, such as MDA-MB-231 (RRID:CVCL_0062), MDA-MB-231-Luc2 (RRID:CVCL_D582), BT-549 (RRID:CVCL_1092), MCF-7 (RRID:CVCL_0031), Hs578T (RRID:CVCL_0332), PANC-1 (RRID:CVCL_0480), LoVo (RRID:CVCL_0399), H1299 (RRID:CVCL_0060), Calu-1 (RRID:CVCL_0608), B16-F10-Luc (JCRB Cat# NIHS0699, RRID:CVCL_4Y00) and HEK293T (ATCC Cat# CRL-3216, RRID:CVCL_0063), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO), 1% penicillin and streptomycin (GIBCO).

Techniques: Expressing, Knock-Out, CRISPR, Clone Assay, Stable Transfection, Control, Isolation, Transwell Migration Assay, Migration, Wound Healing Assay, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic di-GMP suppresses cancer metastasis by targeting proteasome 26S subunit non-ATPase 3 independently of STING

doi: 10.1038/s41392-025-02553-9

Figure Lengend Snippet: c-di-GMP suppresses the NF-κB signaling pathway. a Volcano plot of overall differentially expressed genes (DEGs) between the c-di-GMP treatment and control groups identified via RNA-seq. p value < 0.05; |log2FoldChange| > 0. b KEGG enrichment analysis of genes downregulated in MDA-MB-231 cells following c-di-GMP treatment. The top 20 signaling pathways are shown. c Gene set enrichment analysis (GSEA) of the NF-κB signaling pathway between the c-di-GMP treatment group and the control group (NES = −1.416; p value = 0.028). d MDA-MB-231 cells were treated with increasing concentrations of c-di-GMP for 24 h, and whole-cell lysates were analyzed by Western blotting. e c-di-GMP inhibits the nuclear localization of phosphorylated NF-κB (p-NF-κB Ser536). MDA-MB-231 cells were treated with different concentrations of c-di-GMP for 24 h, stimulated with TNFα (1 nM) for 1 h, and then subjected to cell fractionation. GAPDH was used as a cytoplasmic marker, and PARP was used as a nuclear marker. f The mRNA expression levels of NF-κB target genes were assessed via RT‒qPCR following c-di-GMP treatment for 24 h. Data were quantified and are presented as a graph with the means ± SDs, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 by two-tailed t test

Article Snippet: Various human cancer cell lines, such as MDA-MB-231 (RRID:CVCL_0062), MDA-MB-231-Luc2 (RRID:CVCL_D582), BT-549 (RRID:CVCL_1092), MCF-7 (RRID:CVCL_0031), Hs578T (RRID:CVCL_0332), PANC-1 (RRID:CVCL_0480), LoVo (RRID:CVCL_0399), H1299 (RRID:CVCL_0060), Calu-1 (RRID:CVCL_0608), B16-F10-Luc (JCRB Cat# NIHS0699, RRID:CVCL_4Y00) and HEK293T (ATCC Cat# CRL-3216, RRID:CVCL_0063), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO), 1% penicillin and streptomycin (GIBCO).

Techniques: Control, RNA Sequencing, Protein-Protein interactions, Western Blot, Cell Fractionation, Marker, Expressing, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic di-GMP suppresses cancer metastasis by targeting proteasome 26S subunit non-ATPase 3 independently of STING

doi: 10.1038/s41392-025-02553-9

Figure Lengend Snippet: c-di-GMP directly binds to PSMD3 in breast cancer cells and in vitro. a MDA-MB-231 cell lysates were incubated with biotin-c-di-GMP or biotin alone in the absence or presence of a 20-fold excess of unlabeled c-di-GMP, followed by pull-down using streptavidin-agarose. The precipitates were resolved via SDS‒PAGE and visualized via silver staining. The arrows indicate bands excised for LC–MS-MS analysis (W-2, the band specifically pulled down by biotin-c-di-GMP; W-1, the control band; binding candidates competed out by excess unlabeled c-di-GMP). b Table listing protein candidates directly targeted by c-di-GMP identified in the W-2 group with at least two unique peptides via LC‒MS/MS. c Cellular thermal shift assay (CETSA). MDA-MB-231 cell lysates were incubated with or without c-di-GMP, followed by exposure to different temperatures. PSMD3 protein levels were analyzed by Western blotting (left). The expression ratio of PSMD3 (normalized to its expression at the lowest temperature) was quantified via ImageJ (right). d Chemical structure of c-di-GMP (upper), which is composed of two guanine nucleotides linked via 3′–5′ phosphodiester bonds. SPR sensorgrams showing the concentration-dependent binding of c-di-GMP to immobilized recombinant PSMD3 E61–S460. A six-point, twofold dilution series (3.9–125 μM) was used. The colored traces represent experimental data, and the black lines indicate global fits to a 1:1 binding model, yielding an apparent equilibrium dissociation constant (K d = 8.5 ± 1.5 μM). e Chemical structure of 2′3′-cGAMP (upper), composed of guanine and adenine nucleotides linked via mixed 2′–5′ and 3′–5′ phosphodiester bonds. SPR sensorgrams showing no detectable binding between PSMD3 and 2′3′-cGAMP under identical experimental conditions to those for panel ( d ). No measurable association or dissociation was observed across a seven-point, twofold dilution series of analyte concentrations (3.9–250 μM), indicating the absence of a detectable interaction. f ITC profile of PSMD3 titrated with c-di-GMP at 25 °C. The raw thermogram (upper) and integrated heat (lower) show a saturating exothermic isotherm fit to a one-site model (K d = 4.2 ± 0.5 μM; n = 0.91 ± 0.05; Δ H ≈ –1.3 kcal·mol⁻¹; TΔS ≈ +8.7 kcal·mol⁻¹). g ITC analysis of PSMD3 titrated with 2′3′-cGAMP under the same buffer and temperature conditions as ( f ) showing no significant heat exchange, which is consistent with a lack of binding under saturating conditions

Article Snippet: Various human cancer cell lines, such as MDA-MB-231 (RRID:CVCL_0062), MDA-MB-231-Luc2 (RRID:CVCL_D582), BT-549 (RRID:CVCL_1092), MCF-7 (RRID:CVCL_0031), Hs578T (RRID:CVCL_0332), PANC-1 (RRID:CVCL_0480), LoVo (RRID:CVCL_0399), H1299 (RRID:CVCL_0060), Calu-1 (RRID:CVCL_0608), B16-F10-Luc (JCRB Cat# NIHS0699, RRID:CVCL_4Y00) and HEK293T (ATCC Cat# CRL-3216, RRID:CVCL_0063), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO), 1% penicillin and streptomycin (GIBCO).

Techniques: In Vitro, Incubation, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Control, Binding Assay, Thermal Shift Assay, Western Blot, Expressing, Concentration Assay, Recombinant

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic di-GMP suppresses cancer metastasis by targeting proteasome 26S subunit non-ATPase 3 independently of STING

doi: 10.1038/s41392-025-02553-9

Figure Lengend Snippet: PSMD3 and TBK1 bind to each other via their coiled coil domains. a HEK293T cells were transfected with HA-TBK1 and Flag-PSMD3 plasmids as indicated. Anti-Flag beads were used for the co-IP assay. b HEK293T cells were transfected with Flag-PSMD3 and HA-TBK1 plasmids as indicated, followed by co-IP using anti-HA beads. c Endogenous PSMD3-TBK1 complexes in MDA-MB-231 cells were detected by co-IP using a PSMD3 antibody with IgG as a negative control. Treatment with c-di-GMP (5 μM, 24 h) disrupted the endogenous interaction between PSMD3 and TBK1 in MDA-MB-231 cells. d His-tagged TBK1 fragments and full-length fusion proteins were expressed in E. coli and purified via HisPur™ Ni-NTA Resin. Equal amounts of lysates from Flag-PSMD3-transfected HEK293T cells were incubated with these beads. Binding between TBK1 and PSMD3 was analyzed by WB using an anti-FLAG antibody. Equal loading of His-tagged proteins was confirmed by WB analysis with an anti-His antibody. e HA-tagged TBK1 and Flag-tagged PSMD3 full-length and truncated plasmids were cotransfected into HEK293T cells as indicated, followed by a co-IP assay using anti-Flag beads. f Left, schematic representation of the PSMD3 binding region on TBK1. FL full length, KD kinase domain, ULD ubiquitin-like domain, CC coiled-coil domain, Right, schematic representation of the TBK1-binding region within the PSMD3 domain structure. FL full length, N,N-terminal fragment (unstructured region containing potential regulatory motifs), CC connector region (coiled-coil–like helical segment), PCI proteasome component domain (core functional domain), C-ter C-terminus

Article Snippet: Various human cancer cell lines, such as MDA-MB-231 (RRID:CVCL_0062), MDA-MB-231-Luc2 (RRID:CVCL_D582), BT-549 (RRID:CVCL_1092), MCF-7 (RRID:CVCL_0031), Hs578T (RRID:CVCL_0332), PANC-1 (RRID:CVCL_0480), LoVo (RRID:CVCL_0399), H1299 (RRID:CVCL_0060), Calu-1 (RRID:CVCL_0608), B16-F10-Luc (JCRB Cat# NIHS0699, RRID:CVCL_4Y00) and HEK293T (ATCC Cat# CRL-3216, RRID:CVCL_0063), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO), 1% penicillin and streptomycin (GIBCO).

Techniques: Transfection, Co-Immunoprecipitation Assay, Negative Control, Purification, Incubation, Binding Assay, Ubiquitin Proteomics, Functional Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic di-GMP suppresses cancer metastasis by targeting proteasome 26S subunit non-ATPase 3 independently of STING

doi: 10.1038/s41392-025-02553-9

Figure Lengend Snippet: PSMD3 is required for cancer cell migration and is highly expressed in aggressive and late-stage breast cancers. a WB analysis of the effect of PSMD3 knockdown on TBK1-NF-κB activation in MDA-MB-231 cells. b PSMD3 knockdown in MDA-MB-231 cells was achieved via siRNA. Cell migration was analyzed via a transwell migration assay with or without 5 μM c-di-GMP treatment for 16 h. Representative images are shown. Scale bar, 100 μm. c Migrated cells were quantified via ImageJ software and normalized to the control. *** P < 0.001, ** P < 0.01 compared with the control group; ns not significant ( n = 3‒4 separate inserts per condition). The data are presented as the means ± SDs. d The efficiency of PSMD3 knockdown was confirmed by immunoblotting. e MDA-MB-231 cells were transfected with the indicated siRNAs and/or Flag-PSMD3 for 72 h, followed by a transwell migration assay. Representative images are shown. Scale bar, 100 μm. f Migrated cells were quantified by ImageJ software and normalized to the control. *** P < 0.001, ** P < 0.01 compared with the control group ( n = 3 separate inserts per condition). The data are presented as the means ± SDs. g The efficiency of PSMD3 knockdown and overexpression in the same batch of transwell assays ( e ) was confirmed by immunoblotting, and the effects on TBK1 activation were analyzed. h Schematic illustration of the proposed mechanism by which c-di-GMP suppresses the PSMD3-TBK1-NF-κB pathway. i Relative mRNA expression of PSMD3 in normal ( n = 114) and breast cancer ( n = 1097) tissues from the UALCAN Breast Invasive Carcinoma Cohort ( p = 1.62E−12). j Kaplan–Meier analysis of PSMD3 expression and overall survival in an untreated breast cancer cohort ( n = 313) from the Kaplan‒Meier plotter database. The P value was calculated via the log-rank test

Article Snippet: Various human cancer cell lines, such as MDA-MB-231 (RRID:CVCL_0062), MDA-MB-231-Luc2 (RRID:CVCL_D582), BT-549 (RRID:CVCL_1092), MCF-7 (RRID:CVCL_0031), Hs578T (RRID:CVCL_0332), PANC-1 (RRID:CVCL_0480), LoVo (RRID:CVCL_0399), H1299 (RRID:CVCL_0060), Calu-1 (RRID:CVCL_0608), B16-F10-Luc (JCRB Cat# NIHS0699, RRID:CVCL_4Y00) and HEK293T (ATCC Cat# CRL-3216, RRID:CVCL_0063), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO), 1% penicillin and streptomycin (GIBCO).

Techniques: Migration, Knockdown, Activation Assay, Transwell Migration Assay, Software, Control, Western Blot, Transfection, Over Expression, Expressing

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: Identification of Prdx6-Ser32 as a direct target of 5-MTP in PMVECs after hypoxia. A . The workflow of LiP-SMap showed the process to identify the potential targets of 5-MTP by limited proteolysis and LC-MS; B . The match graph showed Ser32 in differential peptide segments of Prdx6 identified by limited proteolysis. Green square, the differential peptide segments in Prdx6. Red square, the S32 site of Prdx6; C . The immunoblotting and quantitative data of CETSA calculated relative to 37 o C group. The curve is fitted using non-linear methods; D . The graphic result of molecular docking showed 5-MTP bonded to Prdx6 directly. The blue line indicates hydrogen bonds, the gray dotted line indicates hydrophobically interaction and the red dotted circle indicates the hydrogen bond between Ser32-Prdx6 and the carbonyl group of 5-MTP; E. The 2-dimensional chemical structure of 5-MTP. F. The RMSD, RMSF, Rg and SASA of wt (the protein monomer) and the protein-ligand complex, assessed by MD simulation; G. The free energy landscape generated from the last 10ns of RMSD and Rg trajectories identified the lowest free energy state (blue region in the 2D plot), corresponding to the most stable conformation of the protein–ligand complex during the simulation (black arrow); H. The fitting curve of fraction bound and 5-MTP concentration generated by MST results, indicating the binding affinity. The Values were mean ± SEM for n = 3. ∗∗∗∗ p < 0.0001, compared with the 1 ‰ DMSO group. LiP-SMap, limited proteolysis combined with mass spectrometry; 5-MTP, 5-methoxytryptophan; DMSO, dimethyl sulfoxide; PMVECs, mouse pulmonary microvascular endothelial cells; LC-MS, liquid chromatography-tandem mass spectrometry; CETSA, cellular thermal shift assay; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation analysis; Rg, radius of gyration; SASA, solvent accessible surface area; MD, molecular dynamics; MST, microscale thermophoresis. Statistical significance was evaluated using one-way ANOVA.

Article Snippet: Primary antibodies against NF-κB p50 (Proteintech, 14220-1-AP, 1:500), Prdx6 (Proteintech, 13585-1-AP, 1:1000) and Lamp2 (Abcam, ab13524, 1:500) were diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) and incubated overnight at 4 o C. PBS was used for negative control.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Generated, Concentration Assay, Binding Assay, Mass Spectrometry, Liquid Chromatography, Thermal Shift Assay, Solvent, Microscale Thermophoresis

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: 5-MTP prevented the decline of Prdx6 and mitigated lipid peroxidation in ALI. A . Immunoblotting and quantitative data of Prdx6 in PMVECs; B-D. The SOD, MDA and GSH level in PMVECs; E. The representative fluorescent images and relative fluorescent intensity of Liperfluo probe in living PMVECs. Scale bar, 200 μm; F . DPPP-oxide level in PMVECs; G-I. The SOD, MDA and GSH level in mice lung; J. DPPP-oxide level in mice lung. A-F. Values were mean ± SEM for n = 3. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, compared with the Control + Vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with Hypoxia + Vehicle group. G-J. Values were mean ± SEM for n = 6. ∗∗∗∗ p < 0.0001, compared with the Control + Vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with Hypoxia + Vehicle group. B-D, F-J. The data were normalized by the total protein content measured by BCA. 5-MTP, 5-methoxytryptophan; ALI, acute lung injury; PMVECs, mouse pulmonary microvascular endothelial cells; DMSO, dimethyl sulfoxide; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, glutathione; DPPP, diphenyl-1-pyrenylphosphine; BCA bicinchoninic acid. Statistical significance was evaluated using one-way ANOVA.

Article Snippet: Primary antibodies against NF-κB p50 (Proteintech, 14220-1-AP, 1:500), Prdx6 (Proteintech, 13585-1-AP, 1:1000) and Lamp2 (Abcam, ab13524, 1:500) were diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) and incubated overnight at 4 o C. PBS was used for negative control.

Techniques: Western Blot, Control

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: 5-MTP reduced the lysosomes localization and degradation of Prdx6 induced by hypoxia. A. qPCR assessed the relative mRNA level of Prdx6 in PMVECs; B. The immunoblotting and quantitative data of p62 and Prdx6 in PMVECs; C, D. The immunoblotting and quantitative data of relative lysosomes/cytoplasm localization of Prdx6 in PMVECs; E. Immunofluorescence staining and quantitative data of colocalization of Prdx6 and Lamp2 in PMVECs. Scale bar, 25 μm (5 μm in the enlarged view). Values were mean ± SEM for n = 3. B, C. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, compared with the Hypoxia + DMSO group; D, E. ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the Control + DMSO group. # p < 0.05, ## p < 0.01, compared with Hypoxia + DMSO group. 5-MTP, 5-methoxytryptophan; qPCR, quantitative polymerase chain reaction; PMVECs, mouse pulmonary microvascular endothelial cells; DMSO, dimethyl sulfoxide; MG132, Z-Leu-Leu-Leu-al; BafA1, bafilomycin A1. Statistical significance was evaluated using one-way ANOVA.

Article Snippet: Primary antibodies against NF-κB p50 (Proteintech, 14220-1-AP, 1:500), Prdx6 (Proteintech, 13585-1-AP, 1:1000) and Lamp2 (Abcam, ab13524, 1:500) were diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) and incubated overnight at 4 o C. PBS was used for negative control.

Techniques: Western Blot, Immunofluorescence, Staining, Control, Real-time Polymerase Chain Reaction

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: Prdx6-S32A blocked the protective effects of 5-MTP on lipid peroxidation and reduced lysosomal localization of Prdx6. A. Immunoblotting and quantitative data of VE-cadherin and Prdx6 in PMVECs transfected with EV, Prdx6-WT and Prdx6-S32A plasmids; B. The representative fluorescent images and relative intensity of Liperfluo probe in living PMVECs transfected with EV, Prdx6-WT and Prdx6-S32A plasmids. Scale bar, 200 μm; C. The POD activity of PMVECs transfected with EV, Prdx6-WT and Prdx6-S32A plasmids; D. The representative fluorescent images and relative intensity of Liperfluo probe in living PMVECs transfected with EV, Prdx6-WT and Prdx6-S32A plasmids without 5-MTP treatment. Scale bar, 200 μm; E. The immunoblotting and quantitative data of relative lysosomes/cytoplasm localization of Prdx6 in PMVECs transfected with EV, Prdx6-WT and Prdx6-S32A. plasmids; F. Immunofluorescence staining and quantitative data of colocalization of Prdx6 and Lamp2 in PMVECs transfected with EV, Prdx6-WT and Prdx6-S32A. plasmids. Scale bar, 25 μm (5 μm in the enlarged view). Values were mean ± SEM for n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, compared with EV group. # p < 0.05, ## p < 0.01, ### p < 0.001, compared with Prdx6-WT group. 5-MTP, 5-methoxytryptophan; PMVECs, mouse pulmonary microvascular endothelial cells; EV, empty vector. Statistical significance was evaluated using one-way ANOVA.

Article Snippet: Primary antibodies against NF-κB p50 (Proteintech, 14220-1-AP, 1:500), Prdx6 (Proteintech, 13585-1-AP, 1:1000) and Lamp2 (Abcam, ab13524, 1:500) were diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) and incubated overnight at 4 o C. PBS was used for negative control.

Techniques: Western Blot, Transfection, Activity Assay, Immunofluorescence, Staining, Plasmid Preparation

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: Prdx6-S32A attenuated the protective effects of 5-MTP on ALI AAV-EV, AAV-Prdx6-WT and AAV-Prdx6-S32A were injected intratracheally into mice 3 weeks before the mice were induced ALI and treated with 100 mg/kg 5-MTP. A. Representative images of mice lung hematoxylin-eosin staining. Scale bar, 100 μm; B, C . Representative image and quantitative data of Evans Blue assays. Scale bar, 1 cm; D . The lung wet to dry ratio indicated the degree of pulmonary edema; E, F . The total protein and cell density in BALF; G . Immunoblotting and quantitative data of VE-cadherin and Prdx6 in mice lungs; H-J. The SOD, MDA and GSH level in mice lung homogenates, normalized by the content of total protein measured by BCA; K. DPPP-oxide content in mice lungs, normalized by the content of total protein measured by BCA. Values were mean ± SEM for n = 6. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, compared with EV group. # p < 0.05, ### p < 0.001, #### p < 0.0001, compared with Prdx6-WT group. 5-MTP, 5-methoxytryptophan; AAV, adeno-associated virus; ALI, acute lung injury; EV, empty vector; BALF, bronchoalveolar lavage fluid; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, glutathione; DPPP, diphenyl-1-pyrenylphosphine; BCA bicinchoninic acid. Statistical significance was evaluated using one-way ANOVA.

Article Snippet: Primary antibodies against NF-κB p50 (Proteintech, 14220-1-AP, 1:500), Prdx6 (Proteintech, 13585-1-AP, 1:1000) and Lamp2 (Abcam, ab13524, 1:500) were diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) and incubated overnight at 4 o C. PBS was used for negative control.

Techniques: Injection, Staining, Western Blot, Virus, Plasmid Preparation