Journal: International Journal of Biological Sciences

Article Title: Ebastine exerts antitumor activity and induces autophagy by activating AMPK/ULK1 signaling in an IPMK-dependent manner in osteosarcoma

doi: 10.7150/ijbs.69541

Figure Lengend Snippet: High-throughput screening generates candidate anti-inflammatory agents for osteosarcoma therapy. ( a ) The composition of this immunology inflammation screening library (L4100). ( b-d ) The inhibition rate of every compound in all three osteosarcoma cell lines. ( e ) The inhibition rate > 95% in three osteosarcoma cell lines was achieved by ebastine.

Article Snippet: The immunology inflammation screening library (L4100), dorsomorphin (S7840) and 3-methyladenine (3-MA, S2767) were purchased from Selleck (Shanghai, China).

Techniques: High Throughput Screening Assay, Inhibition

Journal: Nature Communications

Article Title: A phenopushing platform to identify compounds that alleviate acute hypoxic stress by fast-tracking cellular adaptation

doi: 10.1038/s41467-025-57754-1

Figure Lengend Snippet: a Overview of AH-to-CH phenopushing screening and hit-calling framework. N: Normoxia. AH: Acute hypoxia (1 d). CH: Chronic hypoxia (6 d). D: Distance from the phenotypic profile of a perturbation (a given compound at a given dose) to the centroid of the phenotypic profiles of CH DMSO controls. θ : Angle of deviation from the phenotypic profile of a perturbation relative to the centroid of the phenotypic profiles of CH DMSO controls. The cell icons: Created in BioRender. Wu, A. (2025) https://BioRender.com/q85j552 . b Summary of geometric hit calls in the primary screen. Scatter plot and density map for distance (x-axis) and direction (y-axis) of bioactive compounds. Red solid line and dotted line show cutoffs for distance and angle, respectively. Vehicle (DMSO) controls in N, AH, and CH are shown for reference. Bioactive non-hit compounds (black dots), hits (red dots), and four hit examples (large red dots) are shown. c Screening funnel for AH-to-CH phenopushing hits. d Representative images for DMSO under N, AH, or CH compared to hit examples (highlighted in b) in AH (Temsirolimus, Deforolimus, KU-0063794, WYE-354). The experiment was independently repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Screened compounds included (1) Selleck FDA-approved & Passed Phase I Drug Library (L3800), (2) Selleck Kinase Inhibitor Library (L1200), (3) Selleck Apoptosis Compound Library (L3300), (4) Selleck Epigenetics Compound Library (L1900), (5) Selleck Bioactive Library (L1700), (6) manually curated FDA-approved & Passed Phase I drugs from other vendors, and (7) manually curated bioactive compounds from other vendors.

Techniques:

Journal: Nature Communications

Article Title: A phenopushing platform to identify compounds that alleviate acute hypoxic stress by fast-tracking cellular adaptation

doi: 10.1038/s41467-025-57754-1

Figure Lengend Snippet: a Overview of the ischemia rescue assay. The cell icons: Created in BioRender. Wu, A. (2025) https://BioRender.com/q85j552 . b Scatter plot of hit compound effects on HepG2 survival in ischemia-like stress. Cell survival: normalized to DMSO-treated cells, averaged across all tested well replicates and doses for each drug, and ranked based on their survival rate. Dashed lines: survival of DMSO-treated hypoxia naïve cells (bottom) or DMSO-treated cells pre-exposed to hypoxia for 6 days (top). mTOR/PI3Ki: AH-to-CH phenopushing hits targeting mTOR and/or PI3Ks. BETi: AH-to-CH phenopushing hits targeting BET proteins. Other: AH-to-CH phenopushing hits other than mTOR/PI3Ki and BET proteins. c Normalized cell survival of each tested dose for the top 25 hits shown in b . d Ischemia rescue effect comparison between the most effective tested dose of phenopushing (red) and non-phenopushing, bioactive (blue) compounds. Kernel density estimation used to smooth histograms. Dashed lines: (grey) mean survival of DMSO-treated cells or (red) 3x DMSO survival. Source data are provided as a Source Data file.

Article Snippet: Screened compounds included (1) Selleck FDA-approved & Passed Phase I Drug Library (L3800), (2) Selleck Kinase Inhibitor Library (L1200), (3) Selleck Apoptosis Compound Library (L3300), (4) Selleck Epigenetics Compound Library (L1900), (5) Selleck Bioactive Library (L1700), (6) manually curated FDA-approved & Passed Phase I drugs from other vendors, and (7) manually curated bioactive compounds from other vendors.

Techniques: Rescue Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Activation of ERK and p38 Reduces AZD8055-Mediated Inhibition of Protein Synthesis in Hepatocellular Carcinoma HepG2 Cell Line

doi: 10.3390/ijms222111824

Figure Lengend Snippet: Screening anticancer drugs for identification of translation inhibitors. ( a , b ) The level of translation in mouse embryonic fibroblasts (MEFs) was measured using puromycin incorporation assay. Polypeptide chains in wild-type MEFs treated with 10 μM ( a ) or the indicated concentrations ( b ) of Selleck Anti-cancer Compound Library for 24 h were labeled with puromycin for 10 min. The number of cells (left column) and Alexa Fluor 488 intensities of each area (right column) were assessed using Operetta High-Content analysis system. Operetta images were analyzed using Harmony software. Red dots in the red dotted circle in B indicate MEFs treated with various concentrations of AZD8055. To recognize the shape of cells, cells were stained with Hoechst 33342 prior to the measurement of cytoplasmic Alexa Fluor 488 intensities. The number of compounds include DMSO- and Tg-treated samples. ( c – e ) Wild-type (S/S) or eIF2α mutant (A/A) MEFs were treated with the indicated concentrations of AZD8055 for 24 h; two representative confocal images are shown ( c ). The intensity of Alexa Fluor 488 was visualized ( c ) and MEFs treated with various concentrations of AZD8055 were magnified ( c ). Scale bar = 200 μM. The obtained images were measured using Harmony Software ( d ). Immunoblot analyses were performed to determine the relative amount of puromycin and phosphorylated form of eIF2α using specific antibodies for puromycin, eIF2α, phosphorylated form of eIF2α (p-eIF2α), and GAPDH ( e ). Treatment with thapsigargin (Tg), an ER stress inducer, was used as a positive control. Fold changes in d were calculated by comparing DMSO control group of each cell line with AZD8055 treatment groups. Statistically significant differences were determined by Student’s t -test and were indicated as *** p < 0.001.

Article Snippet: The cells were treated with dimethyl sulfoxide (DMSO; Biosesang, Korea) or Selleck Anti-Cancer Compound Library for 24 h. Cells treated with 1 µM Tg for 30 min served as positive control.

Techniques: Labeling, High Content Screening, Software, Staining, Mutagenesis, Western Blot, Positive Control

Journal: Stem cell reports

Article Title: DOT1L regulates lung developmental epithelial cell fate and adult alveolar stem cell differentiation after acute injury.

doi: 10.1016/j.stemcr.2023.07.006

Figure Lengend Snippet: Figure 1. Alveolar organoid screen to identify epigenetic pathways regulating alveolar regeneration (A) Schematic of how AT2 cells isolated from SftpcEGFP mice were used in the screen. (B) An example of fluorescent reporter detection from SftpcEGFP AT2 cells in a 96-well plate. Arrows indicate negative (water or DMSO) or positive (FGF7) controls used for the screen. (C) Results from the epigenetic small molecular inhibitor library using a Z score of 2.0 as a cutoff for a positive increase in alveolar organoid total fluorescence. Data are representative of the screening assay performed in duplicate.

Article Snippet: Forty-eight hours after seeding cells, medium was changed to include 10 mM of one small molecule from the epigenetic library (Selleckchem, L1900) or an equal volume of H2O or DMSO as negative controls and 50 ng/mL of Fgf7 as a positive control.

Techniques: Isolation, Screening Assay

Journal: Stem cell reports

Article Title: DOT1L regulates lung developmental epithelial cell fate and adult alveolar stem cell differentiation after acute injury.

doi: 10.1016/j.stemcr.2023.07.006

Figure Lengend Snippet: Figure 7. Model of how Dot1l regulation of AT2-AT1 differentiation through regula- tion of OxPhos and a subset of transcrip- tion and epigenetic factors

Article Snippet: Forty-eight hours after seeding cells, medium was changed to include 10 mM of one small molecule from the epigenetic library (Selleckchem, L1900) or an equal volume of H2O or DMSO as negative controls and 50 ng/mL of Fgf7 as a positive control.

Techniques:

Journal: Journal of Cancer

Article Title: CC-115 Mediates GSDME-Dependent Pyroptosis in Lung Adenocarcinoma Through the Akt/Bax Pathway

doi: 10.7150/jca.83175

Figure Lengend Snippet: CC-115 induces the pyroptosis of LUAD cells. ( A ) Representative images of LUAD cells treated with CC-115 (scale bar, 50 μm). ( B, C ) Results of the ELISA assay used to quantify LDH release and IL-1β secretion in LUAD cells. ( D ) Flow cytometry plots showing the percentage of Annexin V PE and 7-AAD double-positive LUAD cells. ( E ) Western blotting of pyroptosis markers in LUAD cells treated with CC-115. ** P < 0.01, *** P < 0.001. Ctl: control.

Article Snippet: The Pyroptosis Compound Library (L7400), CC-115 (S7891), and the Akt activator SC79 (S7863) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot, Control

Journal: Journal of Cancer

Article Title: CC-115 Mediates GSDME-Dependent Pyroptosis in Lung Adenocarcinoma Through the Akt/Bax Pathway

doi: 10.7150/jca.83175

Figure Lengend Snippet: GSDME knockdown abrogates the pyroptosis-inducing effect of CC-115 in LUAD cells. ( A ) Knockdown efficiency of GSDME determined via qRT-PCR and western blotting. ( B ) Representative images of LUAD cells in the indicated groups (scale bar, 50 μm). ( C, D ) ELISA determination of the release of LDH and secretion of IL-1β in the indicated groups. ( E ) Flow cytometry plots showing the percentage of Annexin V PE and 7-AAD double-positive cells in the indicated groups. ( F ) Western blotting of pyroptosis markers in the indicated groups. ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: The Pyroptosis Compound Library (L7400), CC-115 (S7891), and the Akt activator SC79 (S7863) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Journal of Cancer

Article Title: CC-115 Mediates GSDME-Dependent Pyroptosis in Lung Adenocarcinoma Through the Akt/Bax Pathway

doi: 10.7150/jca.83175

Figure Lengend Snippet: CC-115 regulates GSDME-dependent pyroptosis through Akt/Bax signaling. ( A ) Western blotting of Akt, Bax, and Cyt-c in A549 cells treated with or without CC-115 (5 μM) and/or SC79 (5 μM). ( B ) Representative images of the mitochondrial membrane potential signal in A549 cells determined using the JC-10 assay in the indicated groups (scale bar, 50 μm). ( C ) Representative bright field images of A549 cells in the indicated groups (scale bar, 50 μm). ( D, E ) Release of LDH and secretion of IL-1β in A549 cells as determined using ELISA. ( F ) Analysis of caspase-3 and GSDME expression using western blotting in A549 cells in the indicated groups. *** P < 0.001.

Article Snippet: The Pyroptosis Compound Library (L7400), CC-115 (S7891), and the Akt activator SC79 (S7863) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Western Blot, Membrane, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Cancer

Article Title: CC-115 Mediates GSDME-Dependent Pyroptosis in Lung Adenocarcinoma Through the Akt/Bax Pathway

doi: 10.7150/jca.83175

Figure Lengend Snippet: CC-115-induced GSDME-dependent pyroptosis is downstream of the Bax-mitochondrial intrinsic pathway. ( A ) Knockout efficiency of Bax determined via qRT-PCR and western blotting. ( B ) Representative images of the mitochondrial membrane potential of LUAD cells determined using the JC-10 assay in the indicated groups (scale bar, 50 μm). ( C, D ) Release of LDH and secretion of IL-1β in LUAD cells in the indicated groups. ( E ) Western blotting of GSDME-dependent pyroptosis markers in LUAD cells in the indicated groups. *** P < 0.001, ns: not significant.

Article Snippet: The Pyroptosis Compound Library (L7400), CC-115 (S7891), and the Akt activator SC79 (S7863) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Knock-Out, Quantitative RT-PCR, Western Blot, Membrane

Journal: Journal of Cancer

Article Title: CC-115 Mediates GSDME-Dependent Pyroptosis in Lung Adenocarcinoma Through the Akt/Bax Pathway

doi: 10.7150/jca.83175

Figure Lengend Snippet: CC-115 shows the antitumor effect through GSDME-dependent pyroptosis in vivo . ( A ) Images of nude mice with subcutaneous xenografts in the indicated groups. ( B ) Xenograft tumors isolated from sacrificed mice (28 days) with or without CC-115 treatment (2.5 mg/kg per day). ( C ) Average weights of xenograft tumors in the indicated groups. ( D ) Kinetics of tumor growth by volume of subcutaneous xenograft tumors. ( E ) Immunohistochemistry of p-Akt, Bax, cleaved caspase 3, and GSDME in subcutaneous mouse tumors from the NC vs. NC+CC-115 groups (scale bar, 100 μm). ( F, G ) Release of serum LDH and secretion of IL-1β in nude mice. ** P < 0.01, *** P < 0.001.

Article Snippet: The Pyroptosis Compound Library (L7400), CC-115 (S7891), and the Akt activator SC79 (S7863) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: In Vivo, Isolation, Immunohistochemistry

Journal: Journal of Cancer

Article Title: CC-115 Mediates GSDME-Dependent Pyroptosis in Lung Adenocarcinoma Through the Akt/Bax Pathway

doi: 10.7150/jca.83175

Figure Lengend Snippet: Diagram of the proposed mechanism and function of CC-115 in LUAD. CC-115 exerts its function as a dual DNA-PK/mTOR inhibitor, suppressing the activity of Akt. This induces significant mitochondrial damage and triggers caspase-3 activation, which induces GSDME-dependent pyroptosis, ultimately affecting cell survival in lung adenocarcinoma.

Article Snippet: The Pyroptosis Compound Library (L7400), CC-115 (S7891), and the Akt activator SC79 (S7863) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activity Assay, Activation Assay

Journal: Journal of Biomedical Science

Article Title: USP9X-mediated REV1 deubiquitination promotes lung cancer radioresistance via the action of REV1 as a Rad18 molecular scaffold for cystathionine γ-lyase

doi: 10.1186/s12929-024-01044-3

Figure Lengend Snippet: REV1 downregulates CTH expression by promoting Rad18 binding and ubiquitinating CTH for degradation. A Heatmap generated from the results of enrichment of Gly/Ser/Thr signaling pathways in RNA sequencing. B REV1 was knocked down in A549 and H1299 cells, and the relative mRNA levels of CTH were detected by RT-PCR. *** P < 0.001, ( n = 3). C A549, H1299, and H1975 cells were transfected with siREV1, and the expression of the corresponding antibody was detected by Western blotting after 48 h ( n = 3). D DMSO and JH-RE-06-treated A549, H1299, and H1975 cells were harvested and CTH expression was detected by Western blotting ( n = 3). E-F Endogenous interaction of CTH with REV1 and Rad18 in A549 cells and H1299 cells. G Proteins of HEK293T cells transfected with Flag-Rad18 were collected and added to GST-only and GST-CTH fusion proteins, respectively. The expression of the corresponding molecules was detected by Western blotting after overnight enrichment and elution ( n = 3). H A549, H1299, and H1975 cells were transfected with siRad18, and the expression of the corresponding proteins was detected by Western blotting after 48 h ( n = 3). I Scrambled and Rad18-deleted A549 cells were treated with DMSO or MG132 for 4 h before protein collection, and the expression of the indicating molecules was examined by Western blotting ( n = 3). J Cells were transfected with the corresponding plasmids and siRNA, MG132 was added 4 h before protein collection treatment, and the in vitro CTH ubiquitination level was detected using the immunoprecipitation technique ( n = 3). K Left panel: Scrambled and Rad18-deleted A549 cells were treated with CHX (20 µg/mL). Cellular proteins were collected at the corresponding times, and the protein levels of CTH were detected by Western blotting ( n = 3). Right panel: quantitative statistical analysis of CTH protein ( n = 3). L Proteins of HEK-293T cells transfected with the corresponding plasmids were harvested and incubated with GST-only or GST-CTH fusion proteins overnight and analyzed by Western blotting ( n = 3). M-N A549 and H1975 cells were transfected with the corresponding siRNA or plasmid and analyzed by Western blotting using the corresponding antibodies ( n = 3)

Article Snippet: The main antibodies and reagents used in this study are as follows: mouse anti-REV1 (Santa Cruz, sc-393022, 1:100), rabbit anti-USP9X (Proteintech, 55054-1-AP, 1:1000), rabbit anti-CTH (Abcam. ab151769, 1:1000), mouse anti-GAPDH (ABclonal, AC033, 1:3000), rabbit anti-Rad51 (Abcam, ab133534, 1:1000), rabbit anti-USP24 (Proteintech, 13126 -1-AP, 1:1000), rabbit anti-USP5 (Proteintech, 10473-1-AP, 1:1000), rabbit anti-USP1 (Proteintech, 14346-1-AP, 1:1000), rabbit anti-USP14 ( Proteintech, 14517-1-AP, 1:1000), rabbit anti-UCHL5 (Proteintech, 11527-1-AP, 1:1000), mouse anti-Flag (Sigma-Aldrich, F1804, 1:3000), rabbit anti-GST (Abcolonal, AE006, 1:1000), rabbit anti-HA (Cell Signaling Technology, #3724, 1:3000), Ubiquitination Compound Library (Selleck, L6000), JH-RE-06 (Topscience, T15611), MG132 (Calbiochem, 474,790) and cycloheximide (Sigma-Aldrich, A8244).

Techniques: Expressing, Binding Assay, Generated, Protein-Protein interactions, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, In Vitro, Ubiquitin Proteomics, Immunoprecipitation, Incubation, Plasmid Preparation

Journal: Journal of Biomedical Science

Article Title: USP9X-mediated REV1 deubiquitination promotes lung cancer radioresistance via the action of REV1 as a Rad18 molecular scaffold for cystathionine γ-lyase

doi: 10.1186/s12929-024-01044-3

Figure Lengend Snippet: USP9X positively regulates REV1 expression through the ubiquitin-proteasome system pathway. A Protein extracted from A549 and H1299 lung cancer cells after 4 h of MG132 treatment and protein expression of REV1 detected by Western blotting. B Flow chart of the ubiquitination compound library screening system. C The four drugs obtained from the screening that inhibited REV1 protein expression most significantly were used to treat A549 cells for 24 h. The proteins were extracted, and the expression of REV1 protein was detected by Western blotting. D The targets of the four small-molecule drugs obtained from the screening. Interaction between USP9X and REV1 ( n = 3). E Endogenous interaction between REV1 and small molecule drug targets. F A549 cells were transfected with SFB-REV1 plasmid, and the interaction between USP9X and REV1 was detected by co-IP assay after 24 h ( n = 3). G siUSP9X was transfected into A549 and H1299 lung cancer cells, and REV1 expression was detected by Western blotting after 48 h ( n = 3). H A549 cells were transfected with the corresponding plasmids for 24 h, treated with MG132 for 4 h before protein extraction, and treated with S beads for immunoprecipitation. I A549 cells were transfected with Scramble or siUSP9X for 48 h, DMSO or MG132 was added 4 h before protein extraction, and the expression of the corresponding proteins was detected by Western blotting ( n = 3). J Cells were transfected with the corresponding siRNA or plasmid, MG132 was added 4 h before protein extraction, and immunoprecipitation experiments were performed with S beads for immunoprecipitation experiments ( n = 3). K Left panel: A549 lung cancer cells transfected with scramble or siUSP9X for 48 h before CHX (100 µg/mL) treatment. Cellular proteins were collected at the corresponding times, and the protein levels of REV1 were detected by Western blotting ( n = 3). Right panel: quantitative statistical analysis of REV1 protein ( n = 3)

Article Snippet: The main antibodies and reagents used in this study are as follows: mouse anti-REV1 (Santa Cruz, sc-393022, 1:100), rabbit anti-USP9X (Proteintech, 55054-1-AP, 1:1000), rabbit anti-CTH (Abcam. ab151769, 1:1000), mouse anti-GAPDH (ABclonal, AC033, 1:3000), rabbit anti-Rad51 (Abcam, ab133534, 1:1000), rabbit anti-USP24 (Proteintech, 13126 -1-AP, 1:1000), rabbit anti-USP5 (Proteintech, 10473-1-AP, 1:1000), rabbit anti-USP1 (Proteintech, 14346-1-AP, 1:1000), rabbit anti-USP14 ( Proteintech, 14517-1-AP, 1:1000), rabbit anti-UCHL5 (Proteintech, 11527-1-AP, 1:1000), mouse anti-Flag (Sigma-Aldrich, F1804, 1:3000), rabbit anti-GST (Abcolonal, AE006, 1:1000), rabbit anti-HA (Cell Signaling Technology, #3724, 1:3000), Ubiquitination Compound Library (Selleck, L6000), JH-RE-06 (Topscience, T15611), MG132 (Calbiochem, 474,790) and cycloheximide (Sigma-Aldrich, A8244).

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Drug discovery, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Protein Extraction, Immunoprecipitation

Journal: Journal of Biomedical Science

Article Title: USP9X-mediated REV1 deubiquitination promotes lung cancer radioresistance via the action of REV1 as a Rad18 molecular scaffold for cystathionine γ-lyase

doi: 10.1186/s12929-024-01044-3

Figure Lengend Snippet: Schematic. Left: REV1 induces metabolic abnormalities involved in the development of radioresistance in lung cancer. USP9X mediates REV1 deubiquitination to stabilize its expression in lung cancer. Aberrantly expressed REV1 acts as a scaffolding protein to assist the E3 ubiquitin ligase Rad18 in interacting with CTH, promoting CTH ubiquitination degradation and upregulating intra- and extracellular Gly/Ser/Thr levels, leading to lung cancer radiotherapy resistance. Right: Therapeutic potential of targeting REV1 in lung cancer. JH-RE-06 promotes the formation of loss-of-function dimers of REV1 and induces remodeling of intra- and extracellular amino acid metabolism, thereby enhancing the radiosensitivity of lung cancer cells

Article Snippet: The main antibodies and reagents used in this study are as follows: mouse anti-REV1 (Santa Cruz, sc-393022, 1:100), rabbit anti-USP9X (Proteintech, 55054-1-AP, 1:1000), rabbit anti-CTH (Abcam. ab151769, 1:1000), mouse anti-GAPDH (ABclonal, AC033, 1:3000), rabbit anti-Rad51 (Abcam, ab133534, 1:1000), rabbit anti-USP24 (Proteintech, 13126 -1-AP, 1:1000), rabbit anti-USP5 (Proteintech, 10473-1-AP, 1:1000), rabbit anti-USP1 (Proteintech, 14346-1-AP, 1:1000), rabbit anti-USP14 ( Proteintech, 14517-1-AP, 1:1000), rabbit anti-UCHL5 (Proteintech, 11527-1-AP, 1:1000), mouse anti-Flag (Sigma-Aldrich, F1804, 1:3000), rabbit anti-GST (Abcolonal, AE006, 1:1000), rabbit anti-HA (Cell Signaling Technology, #3724, 1:3000), Ubiquitination Compound Library (Selleck, L6000), JH-RE-06 (Topscience, T15611), MG132 (Calbiochem, 474,790) and cycloheximide (Sigma-Aldrich, A8244).

Techniques: Expressing, Scaffolding, Ubiquitin Proteomics