am580  (Tocris)

Journal: Advanced Science

Article Title: Retinoic Acid Reprograms Mast Cells Toward a Proinflammatory State to Enhance Antitumor Immunity

doi: 10.1002/advs.202509340

Figure Lengend Snippet: RA–RARα signaling induces CIITA‐dependent antigen‐presenting programming in mast cells. A) Flow cytometry analysis showing HLA‐DR expression after IFN‐γ +TTNPB and IFN‐γ +Bexarotene treatment for 48 h ( n = 3). B) Flow cytometry analysis showing CD40 expression after IFN‐γ +TTNPB and IFN‐γ +Bexarotene treatment for 48 h ( n = 3). C) Flow cytometry analysis showing HLA‐DR expression after IFN‐γ +AM580, IFN‐γ +Adapalene, and IFN‐γ +Palovarotene treatment for 48 h ( n = 3). D) Flow cytometry analysis showing CD40 expression after IFN‐γ +AM580, IFN‐γ +Adapalene, and IFN‐γ +Palovarotene treatment for 48 h ( n = 3). E) Flow cytometry analysis showing HLA‐DR expression after IFN‐γ +RA, IFN‐γ + RA+ Ro 41‐5253 treatment for 48 h ( n = 3). F) Flow cytometry analysis showing CD40 expression after IFN‐γ +RA, IFN‐γ + RA+ Ro 41‐5253 treatment for 48 h ( n = 3). G) Flow cytometry analysis showing CMV antigen presentation by mast cells to CD4⁺ and CD8⁺ T cells under different treatments, with T cell activation assessed by 4‐1BB and TNF‐α expression. H) Volcano plot showing significantly differentially expressed genes between the IFN‐γ + RA treatment group and the IFN‐γ treatment alone group. I) The mRNA expression of CIITA after IFN‐γ, RA, IFN‐γ +RA, IFN‐γ + RA+ Ro 41‐5253 treatment for 48 h ( n = 3).

Article Snippet: The cells were then treated with 100 U mL −1 IFN‐γ (Propretech, #300‐02) (IFN‐γ group) or 100 U mL −1 IFN‐γ plus 50 nM RA (MeilunBio, #MB1302) (IFN‐γ + RA group) for 48 or 72 h. TTNPB (#HY‐15682), Bexarotene (#HY‐14171), AM580 (#HY‐10475), Adapalene (#HY‐B0091), Palovarotene (#HY‐14799), Ro 41‐5253 (#HY‐116248) were purchased from MCE.

Techniques: Flow Cytometry, Expressing, Immunopeptidomics, Activation Assay

Journal: mBio

Article Title: AUP1 and UBE2G2 complex targets STING signaling and regulates virus-induced innate immunity

doi: 10.1128/mbio.00602-25

Figure Lengend Snippet: VSV infection induces AUP1 protein accumulation, and knockdown of AUP1 alters lipid metabolism genes. ( A ) Immunoblot analysis of the indicated proteins at different time intervals upon VSV (MOI = 1) infection in HT1080 cells (left) and densitometric analyses to quantitate increased expression of AUP1 (right). GAPDH was used as an internal control. ( B and C ) LDs (stained with Nile Red) visualized using confocal imaging. Scale bar represents 50 µm. The fluorescence intensity of lipid was analyzed with ImageJ. ( D ) qRT-PCR analysis of mRNA expression levels of various lipid metabolism-associated factors in wild-type and AUP1 knockdown HeLa cells. ( E and F ) HeLa cells were transfected with vector or Flag-CPT1A for 24 h, followed by infection with VSV (MOI = 0.1) for indicated time. The mRNA and protein levels of VSV were determined by qRT-PCR and Western blot. ( G and H ) HeLa cells were transfected with vector or Flag-MGLL for 24 h, followed by infection with VSV (MOI = 0.1) for indicated times. The mRNA and protein levels of VSV were determined by qRT-PCR and Western blot. ( I and J ) HeLa cells were infected with VSV (MOI = 1) and then treated with DMSO or AM580 (20 µM) for indicated times. The mRNA and protein levels of VSV were determined by qRT-PCR and Western blot. Data are representative of three experiments with similar results. Bar graphs show the means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following reagents were also used: HT-DNA (Sigma-Aldrich, D6898), poly(I:C) (InvivoGen, tlrl-pic), Lipofectamine 2000 reagent (Invitrogen, 11668019), 2′,3′-cGAMP (InvivoGen, tlrl-nacga23-02), diABZI (Selleck, S8796), brefeldin A (Selleck, S7046), cycloheximide (Selleck, S7418), 4μ8c (MedChemExpress, HY-19707), AM580 (TargetMOI, T5854), and Nile Red (HY-D0718, MedChemExpress).

Techniques: Infection, Knockdown, Western Blot, Expressing, Control, Staining, Imaging, Fluorescence, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) CMA activity in 5 human NSCLC cell lines and lung epithelial BEAS-2B cells. CMA activity was measured using the KFERQ-PS-Dendra reporter assay (left) and quantified as fluorescent puncta per cell (right). Representative images (center) of cells expressing KFERQ-PS-Dendra in red and nuclei highlighted with DAPI. Inserts show higher magnification of the red channel. n ≥ 105 cells in three independent experiments. (**** P ≤ 0.0001). Created in BioRender. https://BioRender.com/a8dxxz3 . ( B ) Heatmap of the transcriptional differences of CMA-related genes (left) and calculated CMA score (right) in the same cell lines as ( A ). n = 3 independent experiments. (** P = 0.0013, ** P = 0.0051, * P = 0.0283, ** P = 0.0019, *** P = 0.0001). ( C ) Immunofluorescence staining for NCoR1 and RARα in the indicated cell lines. Top: representative images of single or merged (overlay) channels with nuclei highlighted with DAPI. Full field images are shown in Appendix Fig. ). Bottom: quantification of nuclear intensity for each protein. n ≥ 158 cells in three independent experiments (*** P = 0.0004, **** P ≤ 0.0001). ( D ) CMA activity in A549 cells expressing KFERQ-PS-Dendra upon transfection with control (Ctrl) siRNA or siRNA targeting RARα. Representative images (left) as in ( A ) and quantification of fluorescent puncta per cell (right). n ≥ 212 cells from six independent experiments (* P = 0.0317). ( E ) CMA activity in A549 cells expressing KFERQ-PS-Dendra and maintained in serum-free media supplemented with 10 µM ATRA or equal volume vehicle (DMSO). n ≥ 87 cells from three independent experiments. Representative images are shown in Fig. . (**** P ≤ 0.0001). ( F ) CMA activity in A549 cells expressing KFERQ-PS-Dendra and maintained in media supplemented with 5 µM AM580 or equal volume vehicle (DMSO). n ≥ 72 cells from two independent experiments. Representative images are shown in Fig. . (*** P = 0.0007). ( G ) CMA activity in A549 cells expressing KFERQ-PS-Dendra upon transfection with control (Ctrl) siRNA or siRNA targeting NCoR1. Representative images (left) as in ( A ) and quantification of fluorescent puncta per cell (right). n ≥ 124 cells from four independent experiments. (**** P ≤ 0.0001). ( H ) CMA activity in A549 cells expressing the KFERQ-PS-Dendra upon overexpression of either wild-type (WT) RARα or the AHT RARα mutant. Representative images (left) as in ( A ) and quantification of number of fluorescent puncta per cell (right) n ≥ 45 cells from three independent replicates (* P = 0.0492). Data information: All values are mean + SEM, with individual data points when n < 10. Ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons post-hoc test ( A–C ) or unpaired two-tailed t test ( D–H ) were used. .

Article Snippet: AM580 , Millipore Sigma , Cat# A8843.

Techniques: Activity Assay, Reporter Assay, Expressing, Immunofluorescence, Staining, Transfection, Control, Over Expression, Mutagenesis, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) RARα protein levels and RNA expression in A549 cells transfected with control (ctrl) or RARα siRNA. Representative immunoblot (left), protein level quantification (center), and RNA expression normalized to control siRNA (right) are shown. n = 4 independent experiments. (* P = 0.0107, ** P = 0.0081). ( B ) Representative fluorescence images of A549 cells expressing KFERQ-PS-Dendra treated with 10 µM ATRA or equal volume DMSO in serum-free media for 24 h. Quantification is shown in Fig. . ( C ) Representative fluorescence images of A549 cells expressing KFERQ-PS-Dendra treated with 5 µM AM580 or equal volume DMSO for 24 h. Quantification is shown in Fig. . ( D ) NCoR1 protein levels and RNA expression in A549 cells transfected with control (ctrl) or NCoR1 siRNA. Representative immunoblot (left), protein level quantification (center), and RNA expression normalized to control siRNA (right) are shown. n = 3 independent experiments. (** P = 0.0028, * P = 0.0400). ( E ) Representative immunoblot (left) of A549 cells expressing KFERQ-PS-Dendra transfected with equal amounts of DNA of WT RARα or AHT RARα. ( F ) Schematic of mCherry-GFP-LC3 reporter (Kimura et al, ). When the reporter is associated to autophagosomes, both mCherry and GFP fluoresce and autophagosomes are visualized as yellow puncta. Once the autophagosome fuses with a lysosome GFP fluorescence is quenched and autophagolysosomes appear as red fluorescent puncta. Created in BioRender. https://BioRender.com/oqvzi51 . ( G , H ) Macroautophagy activity in A549 cells expressing mCherry-GFP-LC3 treated with 10 µM ATRA ( G ) or 5 µM AM580 ( H ) or equal volume DMSO (No treatment; NT) for 24 h. Representative images (left) and quantification of number of the indicated autophagic compartments (right). AV: autophagic vacuoles (total number of fluorescent red puncta), APG: autophagosomes (total number of green fluorescent puncta) and AUT: autolysosomes (total number of red – green fluorescent puncta). n = 35–45 cells from 2 independent experiments. ( G : ** P = 0.0086, ns = not significant, ** P = 0.0098, H : **** P ≤ 0.0001, * P = 0.0448, ** P = 0.0015). Data Information: All values are mean + SEM. Unpaired two-tailed t test ( A , D ) and two-way ANOVA ( G , H ) were used. Insets show higher magnification and nuclei are highlighted with DAPI. are available online for this figure.

Article Snippet: AM580 , Millipore Sigma , Cat# A8843.

Techniques: RNA Expression, Transfection, Control, Western Blot, Fluorescence, Expressing, Activity Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) Cell viability of various NSCLC cell lines (blue circles), compared to BEAS-2B non-tumorigenic cells (grey squares) after exposure to increasing concentrations of CIM7 for 72 h. n = 4–6 independent experiments. (* P = 0.0147, ** P = 0.0073, ** P = 0.0019, * P = 0.0264, ** P = 0.0083, **** P ≤ 0.0001). ( B ) Cell viability of A549 cells exposed to increasing concentrations of CIM7 or its dead derivative CIM7.2 for 72 h. n = 3 independent experiments. (**** P ≤ 0.0001). ( C ) Cell viability of A549 cells exposed to increasing concentrations of CIM7, ATRA, or AM580 for five days, with treatment refreshed every 24 h. n = 4 technical replicates. ( D ) Cell viability of various cell lines exposed to 10 µM CIM7 for five days, with treatment refreshed every 24 h. n = 4 technical replicates. ( E ) Colony formation of A549 cells exposed to increasing concentrations of CIM7 over 1 week. Representative images (left) and quantification of crystal violet dye based on optical density at 580 nm (right). n = 13–19 wells from three independent experiments. (** P = 0.0066, **** P ≤ 0.0001). ( F ) Schematic of in vivo experimental design. Created in BioRender. https://BioRender.com/tcyxxdd . ( G ) Changes in tumor growth over time, measured every 3 days during the 30-day course of daily administration to mice of vehicle or 25 mg/kg of CIM7. n = 9–11 mice per treatment group. (* P = 0.0248, **** P ≤ 0.0001). ( H ) Heatmap of transcriptional changes in CMA-related genes and calculated CMA scores in tumors from vehicle- or 25 mg/kg CIM7-treated mice. n = 9–11 mice per treatment group. ( I ) TUNEL staining in representative tumors from vehicle- or 25 mg/kg CIM7-treated mice (left) and quantification of percentage of cells showing positive staining (right) in the same tumors as ( H ). ( J ) Changes in tumor growth over time, measured every 3 days during the 30-day course of daily administration to mice of vehicle or 25 mg/kg of CIM7 of the same mice in g-i with treated mice separated by responsive or unresponsive. n = 4–9 mice per group. (* P = 0.0415, ** P = 0.0049, **** P ≤ 0.0001 responsive versus vehicle). Data information: All values are mean + SEM, with individual data points in bar graphs when n < 10. Ordinary two-way ANOVA ( A , B , G , J ) or one-way ANOVA ( E ) followed by Bonferroni’s multiple comparisons post-hoc test and non-linear regression ( A , E ) were used. .

Article Snippet: AM580 , Millipore Sigma , Cat# A8843.

Techniques: In Vivo, TUNEL Assay, Staining