Journal: Nucleic Acids Research

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

doi: 10.1093/nar/gkt410

Figure Lengend Snippet: IGF2BP1 knockdown promotes epithelial-like cell properties in HEK293 cells. ( A and B ) HEK293 cells were transfected with control (siC) or IGF2BP1-directed (siI1-2 or siI1-3) siRNAs for 72 h. Cell morphology was monitored by light microscopy (A). The size of adherent cells was analyzed on immunostaining for CTNNB1 as well as F-actin labeling by phalloidin and is depicted as box plots (B). Images were acquired by LSM microscopy. Adherent cells were traced by manual labeling using CTNNB1-defined cell borders to determine the cell area (µm 2 ) using the Leica-SP5× software (also see Supplementary Figure S1A ). ( C ) HEK293 cells were transfected with control (siC) or three distinct IGF2BP1-directed siRNAs (siI1-1, siI1-2 or siI1-3) for 72 h. IGF2BP1 paralogue-specific knockdown was analyzed by western blotting using IGF2BP1-, IGF2BP2- or IGF2BP3-directed monoclonal antibodies. VCL served as a loading control. ( D and E ) HEK293 cells were transfected with indicated siRNAs as in (A). The F-actin cytoskeleton and cell–cell contact formation was analyzed by phalloidin labeling and immunostaining for CTNNB1 (D) or CDH1 (E). Where indicated nuclei were stained by DAPI. Enlargements of boxed regions (left panels) are shown in the right panels (enlargement). Note the enrichment of CTNNB1 and CDH1 at adherens junctions and a knockdown-induced enhancement of cortical F-Actin (also see Supplementary Figure S1F ). Representative images were acquired by LSM microscopy; bars, 10 µm. ( F ) HEK293 cells were transfected with indicated siRNAs as in (A). CDH1, CTNNB1 and IGF2BP1 protein abundance was analyzed by western blotting with indicated antibodies. Protein levels on IGF2BP1 knockdown were determined relative to controls (siC) by normalization to VCL, as indicated above panels. Representative western blots of three independent analyses are shown. ( G ) Soluble FN1 levels were analyzed by ELISA in HEK293 cells transfected with indicated siRNAs for 72 h. Statistical significance was validated by Student’s t -test: ** P < 0.005. Error bars indicate standard deviation (SD) of at least three independent analyses.

Article Snippet: Soluble FN1 protein levels secreted by HEK293 cells were determined using a human FN1 enzyme-linked immunosorbent assay (ELISA) (Boster Biological Technology).

Techniques: Knockdown, Transfection, Control, Light Microscopy, Immunostaining, Labeling, Microscopy, Software, Western Blot, Bioprocessing, Staining, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

doi: 10.1093/nar/gkt410

Figure Lengend Snippet: IGF2BP1 promotes LEF1 expression by preventing LEF1 mRNA degradation. ( A and B ) HEK293 cells were transfected with control (siC) or indicated IGF2BP1-directed (siI1-1, siI1-2) siRNAs for 72 h. Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) by western blotting using VCL and TUBA4A for cross-normalization, as indicated above panels. Representative western blots of three independent analyses are shown. ACTB and LEF1 mRNA levels were analyzed by qRT-PCR. Changes in RNA abundance on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) by the ΔΔC t -method using PPIA for normalization. ( C ) RNA decay was monitored in HEK293 cells transfected with indicated siRNAs for 72 h by blocking mRNA synthesis using ActD (5 µM) for indicated times. RNA levels were determined by qRT-PCR using normalization to PPIA by the ΔΔC t -method. RPLP0 served as a control. RNA decay is depicted in semi-logarithmic scale. Statistical significance determined over three independent analyses was analyzed by Student’s t -test, as shown in panels ( P -values). ( D and E ) The association of indicated mRNAs with IGF2BP1 in HEK293 cells was analyzed by RIP using formaldehyde fixation to stabilize mRNPs prior purification. Endogenous IGF2BP1 was immunopurified (I1) by a monoclonal antibody, as indicated by western blotting in the lower panel (IB). Co-purification of indicated mRNAs was analyzed relative to the input fraction (I, 10% of cell lysates) by semi-quantitative (D) as well as qRT-PCR (E). IgG-agarose served as a control (C) for unspecific mRNA binding. The enrichment of mRNAs by immunopurification of IGF2BP1 (I1) was determined relative to the input fraction by using the ΔC t -method (E). ( F ) Upper panel: Scheme of used Firefly reporters comprising the two alternative LEF1 3′-UTRs (A: Acc.No., NM_016269 /001130713/ 001166119; B: Acc.No., NM_001130714) or the vector-encoded BGH-3′UTR (C). Lower panel: HEK293 cells were transfected with control or indicated IGF2BP1-directed siRNAs for 48 h before the co-transfection of Firefly luciferase reporters (A–C: see scheme in upper panel) and Renilla luciferase control reporters for 24 h. Changes in Firefly luciferase reporter activities on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) on normalization by Renilla activities. Statistical significance was validated by Student’s t -test: * P < 0.05; ** P < 0.005; *** P < 0.0005. Error bars indicate SD of at least three independent analyses.

Article Snippet: Soluble FN1 protein levels secreted by HEK293 cells were determined using a human FN1 enzyme-linked immunosorbent assay (ELISA) (Boster Biological Technology).

Techniques: Expressing, Transfection, Control, Quantitative Proteomics, Knockdown, Western Blot, Quantitative RT-PCR, Blocking Assay, Purification, Copurification, Binding Assay, Immu-Puri, Plasmid Preparation, Cotransfection, Luciferase

Journal: Nucleic Acids Research

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

doi: 10.1093/nar/gkt410

Figure Lengend Snippet: IGF2BP1 modulates FN1 and SNAI2 (SLUG) transcription via LEF1. ( A ) Schematic of luciferase reporters comprising the full-length in silico predicted (FN-839) or 5′-truncated fragments of the human FN1 promoter. The proposed transcription start is indicated by +1 with a reported 5′-UTR of 266 nt. Putative LEF1-binding sites predicted by ‘PROMO’ are depicted as white boxes with labels ‘1-5’ in 5′-to-3′ direction. ( B ) The Firefly luciferase activity of indicated promoter fragments or empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. Firefly activities were normalized by Renilla activities [relative luciferase units (RLU)], serving as internal controls. All reporters comprising the putative LEF1-binding site four showed promoter activity and were activated by LEF1. ( C and D ) Binding of endogenous LEF1 protein to the human FN1 promoter in HEK293 cells was assessed by ChIP. The association of endogenous LEF1 or histone H3 to the FN1 promoter was monitored by semi-quantitative (C) as well as quantitative PCR (D) using to FN1 promoter specific amplicons (P1 and P2, indicated in lower panel). An intergenic probe served as positive control. IgG-agarose was used to monitor unspecific binding (C, negative control). In (D), the enrichment of indicated genomic DNA fragments (P1 and P2) or the intergenic control (intergenic) was determined relative to the diluted input fraction (I) normalized by IgG-controls using the ΔC t -method. ( E ) HEK293 cells were co-transfected with FN-839 luciferase reporter and IGF2BP1-directed (shI1-1), LEF1-directed (shL1-1) or control shRNA encoding vectors for 48 h. RLUs were determined as described in (B). ( F ) HEK293 cells were transfected with IGF2BP1-directed (siI1-2) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to IGF2BP1 knockdown was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. ACTB served as control. ( G ) HEK293 cells transfected as in (F) were treated with ActD (5 µM) to block transcription for indicated times. SNAI2 mRNA turnover was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RNA decay is depicted in semi-logarithmic scale revealing no significant difference in mRNA turnover ( P -value not shown). ( H ) HEK293 cells were transfected with LEF1-directed (siL1-1) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to LEF1 depletion was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RPLP0 served as control. ( I ) Schematic of Firefly luciferase reporters comprising the SNAI1 or SNAI2 promoter sequences, as previously reported ( , ). Indicated putative LEF1-binding sites within the SNAI1 or SNAI2 promoter were predicted [white boxes; as described in (A)] or as previously reported [gray boxes, only for SNAI2; ]. ( J ) The Firefly activity of SNAI1 or SNAI2 promoter fragments cloned in pGL4 as well as the activity of empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. RLUs were determined as described in (B). LEF1 only enhanced the activity of the SNAI2 promoter. ( K ) HEK293 cells were co-transfected with SNAI1 or SNAI2 promoter reporters and indicated shRNA-encoding vectors for 48 h. RLUs were determined as described in (B). SNAI2 promoter activity was reduced by IGF2BP1 as well as LEF1 knockdown, whereas the SNAI1 reporter activity remained largely unaffected and was barely elevated compared with the empty control reporter. Statistical significance was validated by Student’s t -testing: * P < 0.05; *** P < 0.0005. Error bars indicate SD of at least three independent analyses.

Article Snippet: Soluble FN1 protein levels secreted by HEK293 cells were determined using a human FN1 enzyme-linked immunosorbent assay (ELISA) (Boster Biological Technology).

Techniques: Luciferase, In Silico, Binding Assay, Activity Assay, Plasmid Preparation, Cotransfection, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Control, Transfection, shRNA, Knockdown, Quantitative RT-PCR, Blocking Assay, Clone Assay

Journal: Nature Communications

Article Title: Functionally deficient UBOX5 variants and primary angle-closure glaucoma

doi: 10.1038/s41467-025-62775-x

Figure Lengend Snippet: a A UBOX5 chimeric construct (UBOX5-UBD) where the UBOX5 open reading frame was fused by a flexible linker to a FLAG-tagged ubiquitin binding domain was generated. FLAG-tagged UBOX5 (3 lanes on the right) or FLAG-tagged UBOX5-UBD construct (4 lanes on the left) and HA-tagged ubiquitin was co-transfected into HEK293 cells. After the first immunoprecipitation of lysates by anti-FLAG antibody, 20% of eluates was kept for analysis. Eluate from the UBOX5-UBD was further immunoprecipitated with anti-HA antibody to enrich for ubiquitinated proteins. To serve as antibody specificity control (negative controls), lysates were mock immunoprecipitated with mouse immunoglobulin. Immunoblotting of BIP was performed on inputs and eluates as indicated. Bands corresponding to ubiquitinated BIP and ubiquitinated UBOX5-UBD chimeric protein are indicated by vertical lines, while the unmodified proteins are indicated by arrows on the right. Positions of the molecular weight markers are indicated by arrows on the left. IB: FLAG and IB:HA are shown in Supplementary Fig. . The experiment was repeated independently 3 times. Source data are provided as a Source data file. b MYC-tagged BIP, empty vector, UBOX5, or HA-tagged ubiquitin was co-transfected into HEK293 cells in the indicated combinations. 24 h later, cells were treated with 0.7 uM Tharpsigargin for 16 h, and then further treated with MG132 (a proteasomal inhibitor) for 6 h as indicated. Cells were then harvested and a MYC immunoprecipitation was performed on the input lysates. Eluates were immunoblotted with antibodies against HA to assess the extent of BIP ubiquitination. The membrane was then stripped and a MYC immunoblot was performed to assess immunoprecipitation efficiency. Ubiquitination of BIP was only observed when UBOX5 was expressed. The degree of ubiquitination of BIP did not appear to differ with the addition of MG132, a proteasomal inhibitor, suggesting that the ubiquitinated BIP was not degraded by the proteasome pathway. The experiment was repeated independently 3 times. Source data are provided as a Source data file. (c) UBOX5 or its empty vector contains a GFP open reading frame, which allows for assessment of transfection efficiency by assessing GFP abundance in input lysates. Human UBOX5 immunoblots were used to verify expression of UBOX5. GAPDH was used as loading control. The experiment was repeated independently 3 times. Source data are provided as a Source data file.

Article Snippet: HEK293 cells were transiently transfected with 1.8 μg wild-type UBOX5 expression plasmid per 10 cm dish and treated with 0.7 μM Thapsigargin (#sc-24017, Santa Cruz) for 16 h. Cells were then collected and lysed in Subcellular (SF) Buffer (250 mM Sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, PPI cocktail), and left to rotate on a rotary shaker for 30 min at 4 °C.

Techniques: Construct, Ubiquitin Proteomics, Binding Assay, Generated, Transfection, Immunoprecipitation, Control, Western Blot, Molecular Weight, Plasmid Preparation, Membrane, Expressing

Journal: Nature Communications

Article Title: Functionally deficient UBOX5 variants and primary angle-closure glaucoma

doi: 10.1038/s41467-025-62775-x

Figure Lengend Snippet: a , b UBOX5 and BIP are both induced by ER stress. a NIH3T3 cells were treated with the indicated ER stress inducers tunicamycin (Tu) and thapsigargin (Tg). (Upper) Endogenous UBOX5 mRNA abundance was quantified by qPCR in biological triplicates, normalised against mouse beta-actin transcript. Relative fold change of transcript is reported against control DMSO treatment. Error bars represent standard deviation. ( b , Top) Protein abundance of endogenous mouse UBOX5 in ER-stressed NIH3T3 cells. Beta-actin was used as loading control. Of note, UBOX5 mRNA and protein is induced in response to ER stress. ( b , Bottom): Densitometric quantitation of UBOX5 bands. UBOX5 band intensity of each sample is normalised to corresponding beta-actin intensity. The amount of TG or TU used is indicated on the x -axis. P -values were generated from two-sided Welch’s t -test. Error bars represent standard deviation. Source data are provided as a Source data file. c Cellular Localization of UBOX5: Immunoblots of HEK293 cells transiently transfected with UBOX5 expression plasmid and treated with 0.7 uM Thapsigargin for 16 h. Cellular fractions are indicated above. Whole cell lysates were fractionated into cytoplasmic, endoplastic reticulum (ER) fractions and nuclear fractions by stepwise centrifugation. Indicated antibodies are shown. Positions of molecular weight markers are indicated on the left with arrows. GFP is used as transfection control, Calnexin is used as fractionation control for ER and nuclear fraction; Histone H2B is used as fractionation control for nuclear fraction. GAPDH is used as fractionation control for cytoplasmic and nuclear fractions. The experiment was repeated independently 3 times. Source data are provided as a Source data file. d , e The ability of UBOX5 to ubiquitinate BIP is not dependent on cellular stress. d MYC-tagged BIP, empty vector, UBOX5, or HA-tagged ubiquitin was co-transfected into HEK293 cells in the indicated combinations. 24 hours later, cells were treated with 0.7 μM Thapsigargin (TG) or DMSO for 16 h. TG is a known inducer of endoplasmic reticulum stress. Cells were then harvested and a MYC immunoprecipitation was performed on the input lysates. Eluates were immunoblotted with antibodies against HA to assess the extent of BIP ubiquitination. e UBOX5 or its empty vector contains a GFP open reading frame, which allows for assessment of transfection efficiency by assessing GFP abundance in input lysates. GAPDH was used as loading control.

Article Snippet: HEK293 cells were transiently transfected with 1.8 μg wild-type UBOX5 expression plasmid per 10 cm dish and treated with 0.7 μM Thapsigargin (#sc-24017, Santa Cruz) for 16 h. Cells were then collected and lysed in Subcellular (SF) Buffer (250 mM Sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, PPI cocktail), and left to rotate on a rotary shaker for 30 min at 4 °C.

Techniques: Control, Standard Deviation, Quantitative Proteomics, Quantitation Assay, Generated, Western Blot, Transfection, Expressing, Plasmid Preparation, Centrifugation, Molecular Weight, Fractionation, Ubiquitin Proteomics, Immunoprecipitation

Journal: Nature Communications

Article Title: Functionally deficient UBOX5 variants and primary angle-closure glaucoma

doi: 10.1038/s41467-025-62775-x

Figure Lengend Snippet: a Wild-type UBOX5 increases the half-life of BIP in the presence of thapsigargin-induced ER stress. Top left panel: Pulse chase of BIP with or without expression of UBOX5 in HEK293 cells. HEK 293 cells were transfected with UBOX5 or empty vector. 24 h later, cells were treated with a pulse of 0.25 μM Tharpsigargin for 2 hours, and cycloheximde (150 mM) was added. Cells were collected at indicated time points for immunoblotting. GAPDH was used as loading control. Positions of molecular weight standards are indicated on the left. Top right panel: The same experiment repeated with Tharpsigargin substituted with DMSO carrier control. Bottom panels: Quantitation of BIP band intensities normalized to GAPDH intensity for every indicated time point. Separate graphs were shown for cells treated with Tharpsigargin (Left) or DMSO control (Right). Intensities are shown as fold changes compared to normalized BIP intensity at t = 0 of pulse chase. Data are presented as mean values +/- standard deviation. Error bars indicate standard deviation; 3 biological replicates were used for quantitation. Source data are provided as a Source data file. b Variant UBOX5 and their effects on the half-life of BIP. Top: Pulse chase of BIP in the presence of wildtype UBOX5 or variant UBOX5 (D33N, K291R, R301Q, and S465C) in HEK293 cells. HEK 293 cells were transfected with wildtype UBOX or indicated variants. 24 h later, cells were treated with a pulse of 0.25 μM Tharpsigargin for 2 h, and cycloheximde (150 mM) was added at time = 0. Cells were then subsequently collected at indicated time points (in hours) for immunoblotting. GAPDH was used as loading control. UBOX5 expression was verified as indicated. GFP, expressed from a separate locus in the vector used, was also used to verify success of transfection. Bottom: Densitometric quantitation of BIP band intensities in cells expressing indicated UBOX5 variants, normalized to GAPDH intensity for every time point on the same blot. Intensities are shown as fold changes compared to normalized BIP intensity at t = 0 of pulse chase. Three biological replicates were analyzed. Error bars represent standard deviation. Source data are provided as a Source data file.

Article Snippet: HEK293 cells were transiently transfected with 1.8 μg wild-type UBOX5 expression plasmid per 10 cm dish and treated with 0.7 μM Thapsigargin (#sc-24017, Santa Cruz) for 16 h. Cells were then collected and lysed in Subcellular (SF) Buffer (250 mM Sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, PPI cocktail), and left to rotate on a rotary shaker for 30 min at 4 °C.

Techniques: Pulse Chase, Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Molecular Weight, Quantitation Assay, Standard Deviation, Variant Assay

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: δ-opioid receptor (DOR)-expressing HEK293 cells and HEK293 cells stained with anti-DOR antibody and followed with FITC-conjugated anti-mouse IgG ( A ). Volcano plot of identified differentially expressed genes (DEGs) in DOR-HEK293 cells after 1 h treatment with CM-10 ( B ). Red boxes show changed gene expression over 1.4-time higher and less than 0.71 with p -value predicted by EdgeR of less than 0.05.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Expressing, Staining, Gene Expression

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: Predicted DOR agonistic signaling networks in DOR-HEK293 cells after 1 h treatment with CM-10. Fourteen genes with suggested involvement in cAMP-dependent protein kinases, transcriptional regulators in response to cAMP, circadian rhythm, stress and depression are shown in and were applied for network analysis by STRING. Red: circadian rhythm, Green: regulation of transcription of Notch receptor target, Yellow: PKA activation in glucagon signalling.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Activation Assay

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Immunofluorescence analysis of PD-1 binding to PD-L1 on RKO cells treated with TER. RKO cells were incubated with recombinant human PD-1 Alexa 647 protein and treated with the indicated concentrations of TER. Red fluorescence represents PD-1 binding; the nuclei were stained with Hoechst (blue). B Competitive ELISA assay measuring the inhibition of PD-1/PD-L1 interaction by TER. The binding between PD-1 and PD-L1 in the presence of increasing concentrations of TER was assessed. C The 3D structure of TER (upper panel) and its predicted binding pose within the active site of PD-1 (PDB ID: 3RRQ, lower panel). The key interacting residues include PHE56, SER57, ASN58, THR59, SER62, PHE63, PHE82, PRO83, GLN99, ASN102, GLY103, and ARG104. D Protein–ligand complex showing TER docked to PD-1, with the binding pocket highlighted. E Surface plasmon resonance (SPR) analysis showing the binding of TER to PD-1. αPD-L1 was used as a positive control. *** p < 0.001 and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant Jurkat-T cells expressing human PD-1 and an NFAT reporter gene (hPD-1/NFAT Jurkat-T cells, #60535) and recombinant CHO-K1 cells expressing human PD-L1 and a T-cell receptor (TCR) activator (hPD-L1/TCR CHO-K1 cells, #60536) were obtained from BPS Bioscience.

Techniques: Immunofluorescence, Binding Assay, Incubation, Recombinant, Fluorescence, Staining, Competitive ELISA, Inhibition, SPR Assay, Positive Control, Control

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-1 Jurkat-T cells and hPD-L1 CHO cells following treatment with the indicated concentrations of TER for 24 h. B Luciferase activity measured using a PD-1/PD-L1 blockade bioassay. hPD-1 Jurkat-T cells (effector cells) were co-cultured with hPD-L1-expressing aAPC/CHO-K1 cells (target cells) in the presence of indicated concentrations of TER. The luminescence signal indicates the level of TCR signaling activation. αPD-L1 was used as a positive control. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant Jurkat-T cells expressing human PD-1 and an NFAT reporter gene (hPD-1/NFAT Jurkat-T cells, #60535) and recombinant CHO-K1 cells expressing human PD-L1 and a T-cell receptor (TCR) activator (hPD-L1/TCR CHO-K1 cells, #60536) were obtained from BPS Bioscience.

Techniques: Luciferase, Activity Assay, Bioassay, Cell Culture, Expressing, Activation Assay, Positive Control, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of small molecules and a druggable groove that regulate DNA binding and release of the AP-1 transcription factor ΔFOSB

doi: 10.1016/j.jbc.2025.111080

Figure Lengend Snippet: Cellular activity of JPC0661 . A , cell viability of JPC0661 comm and HTS10307 assessed in the range of 0 to 20 μM after 24 h ( top ) and 72 h ( bottom ) in neural progenitor cells (NPCs) using the CellTiter-Glo viability assay. Cell viability measures are normalized to the negative control containing no compound but 0.6% DMSO. Dotted lines indicate 100% cell viability. The mean of n replicates is shown for 24 h (n = 3) and 72 h (n = 2) with error bars indicating the SEM. B , stability of JPC0661 comm and HTS10307 in mouse liver microsomes. C and D , effects of JPC0661 comm (0–100 μM; ( C ) and JPC0661 (0–100 μM; ( D ) on AP-1-driven luciferase activity in AP-1-luc HEK293 cells. The dose-dependent activation of the AP-1-driven luciferase reporter was measured based on changes in the luciferase signal and expressed as relative fluorescence units (RFUs). Each compound was tested twice (n = 4 wells per experiment; total n = 6–8 wells for JPC0661 comm and n = 7–8 wells for JPC0661 per concentration), with results normalized to the luciferase signals of blank wells from corresponding experiments (n = 8 wells). Nonlinear regression using a three-parameter model was applied to fit the luciferase signal and calculate IC 50 values, which are reported with a 95% confidence interval (CI), and data points are presented as the mean ± SEM. E , effect of serially diluted JPC0661 (0.003–100 μM) on the viability of AP-1-luc HEK293 cells using the Celltiter-Glo viability assay (2 h). Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 7 wells). Data points are presented as the mean ± SEM, with a total n = 10 to 12 wells per concentration. F , effect of JPC0661 (at doses 12.5, 25, and 50 μM) on the viability of Neuro 2A cells (72 h) using the Celltiter-Glo viability assay to assess potential toxicity in mouse neuronal cells. Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 8 wells). Data points are presented as the mean ± SEM, with a total n = 12 wells per concentration. AP-1, activator protein 1; DMSO, dimethyl sulfoxide.

Article Snippet: The AP-1-luciferase reporter HEK293 cell line was obtained from BPS Bioscience and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Activity Assay, Viability Assay, Negative Control, Luciferase, Activation Assay, Fluorescence, Concentration Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of small molecules and a druggable groove that regulate DNA binding and release of the AP-1 transcription factor ΔFOSB

doi: 10.1016/j.jbc.2025.111080

Figure Lengend Snippet: Validation of JPC0661 as an inhibitor of ΔFOSB . A , a panel of JPC0661 analogs was designed to probe the role of the sulfonic acid group and the amino-pyrazolone group. B , the table summarizing the JPC0661 analogs tested in AP-1-reporter assays using AP-1-luc HEK293 cells, yielding cell-based IC 50 values and FP-DRC assays. Lower IC 50 values are marked with more plus signs (+) and indicate higher activity. The plots from the FP-DRC and cell-based DRC assays for these compounds are shown in . C , ΔFOSB–JUND bZIP incubated with compounds (0.5 mM) with 100 μM diamide (“ ox ,” oxidized) or without diamide (“ red ,” reduced) and assessed by SDS-PAGE (with or without reducing agent in the loading buffer). D , ΔFOSB–JUND bZIP protein incubated with compounds (0.5 mM) with no diamide (“ red ”) or protein alone (no compound) incubated with diamide (“ ox ”) as a control and then assessed by SDS-PAGE (with or without reducing agent in the loading buffer). In ( C ) and ( D ), “cntrl” denotes the ΔFOSB–JUND bZIP protein in the absence of compound. AP-1, activator protein 1; bZIP, basic leucine zipper; FP-DRC, fluorescence polarization–dose–response curve; HEK293, human embryonic kidney 293 cell line.

Article Snippet: The AP-1-luciferase reporter HEK293 cell line was obtained from BPS Bioscience and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Biomarker Discovery, Activity Assay, Incubation, SDS Page, Control, Fluorescence