gluc-on promoter reporter clones  (Genecopoeia)

Journal: Frontiers in Transplantation

Article Title: Mechanisms inducing differentiation of adult islet progenitor-like cells into functional islet-like organoids

doi: 10.3389/frtra.2026.1740314

Figure Lengend Snippet: Requirement of CN/NFAT signaling to recruit p300 to RFX6 and NEUROD1 promoters during ISX9-induced IPC islet organoid differentiation (A) induction of NFAT family isoform genes in IPC clusters treated with ISX9 for 24 h in the presence of CN inhibitor FK506. (B) RFX6 and NEUROD1 promoter activation by ISX9 in IPCs in the presence of FK506 or transfected with gene vectors overexpressing dominant negative NFAT (dnNFAT) and mutated control (dnNFATm). (C) ChIP assay of association of NFATC2, p300, HDAC1, HDAC2, and HDAC3 with RFX6, NEUROD1, and NEUROG3 promoters upon 6 h treatment of IPCs with ISX9 with and without 24 h pretreatment with ITF2357. Graphed values are expressed as mean ± SD. Asterisks above bars indicate statistically significant differences (* p < 0.05, *** p < 0.001) in mean values for treatments based on a two-way ANOVA and Sidak's multiple comparison test. Data shown are results from at least three independent experiments using IPCs derived from three individual donors.

Article Snippet: Gluc-ON reporters for RFX6 (HPRM53326-PG04), NEUROD1 (HPRM69533-PG04), and INS (HPRM30189-PG04) promoters were obtained from GeneCopoeia.

Techniques: Activation Assay, Transfection, Dominant Negative Mutation, Control, Comparison, Derivative Assay

Journal: Frontiers in Transplantation

Article Title: Mechanisms inducing differentiation of adult islet progenitor-like cells into functional islet-like organoids

doi: 10.3389/frtra.2026.1740314

Figure Lengend Snippet: Schematic overview of IPC isolation, expansion, and differentiation. Adult human pancreatic tissue obtained from islet cell isolation fractions was expanded in vitro to generate a CD9 + PROCR + IPC-enriched population. IPCs were identified using transcriptomic and phenotypic analyses (scRNA-seq, flow cytometry, and immunofluorescence). An RGS16 + organoid-forming subset was characterized within the expanded population. IPC clusters were subsequently subjected to ISX9-mediated differentiation. ISX9 treatment stimulated calcineurin (CN)/NFAT signaling, promoted NFATC2–p300 association and binding at RFX6 and NEUROD1 promoters, and induced downstream endocrine transcriptional programs (NGN3, RFX6, NEUROD1, NKX2.2, NKX6.1, MAFA), resulting in functional islet organoids.

Article Snippet: Gluc-ON reporters for RFX6 (HPRM53326-PG04), NEUROD1 (HPRM69533-PG04), and INS (HPRM30189-PG04) promoters were obtained from GeneCopoeia.

Techniques: Isolation, Cell Isolation, In Vitro, Flow Cytometry, Immunofluorescence, Binding Assay, Functional Assay

Journal: Molecular Cancer Therapeutics

Article Title: PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER + Breast Cancer

doi: 10.1158/1535-7163.MCT-23-0564

Figure Lengend Snippet: PKMYT1 mRNA and protein levels are E2-regulated in ER + breast cancer PDXs and cell lines. A, Volcano plot showing the effect of E2-deprivation on kinase levels in 22 ER + PDXs whose tumor growth was either E2-dependent or E2 independent. All mice were ovariectomized before exogenous E2 was supplied or not. Levels were determined by KIPA. Differentially expressed kinases with P -values < 0.05 are labeled. B, Absolute PKMYT1 protein levels in individual PDX lines were measured by KIPA-SureQuant. C, PKMYT1 mRNA levels in individual PDX lines were determined by RNA-seq. D, RT-qPCR of PKMYT1 mRNA in hormone-deprived MCF7 and T47D cells treated with vehicle, 10 nmol/L E2, and E2 with 100 nmol/L fulvestrant for 2 days. E, Immunoblotting of protein lysates from hormone-deprived MCF7 and T47D cells treated with vehicle, 10 nmol/L E2, and E2 with 100 nmol/L fulvestrant for 2 days. ERα was decreased by fulvestrant as expected. GAPDH serves as a loading control. The figure is a representative image of three independent biological replicates. In ( A ), P -values were calculated by paired t test. In ( B ) and ( C ), Wilcoxon signed-rank test P -values were shown after the median difference; paired t test P -values were shown after the mean difference. Wilcoxon rank sum test and t test were used to compare E2-deprived samples. In ( D ), bars show the means of individual biological repeats indicated by the dots. P -values were calculated with one-way ANOVA and Tukey HSD.

Article Snippet: T47D, T47D Palbo-R, MCF7 EDR, and MCF7 EDR Palbo-R cell lines expressing Gaussia luciferase (GLuc) under the control of the human PKMYT1 promoter were created by lentiviral transduction using a PKMYT1 promoter-GLuc reporter obtained from GeneCopoeia (cat. # HPRM40000-LvPG04).

Techniques: Labeling, RNA Sequencing, Quantitative RT-PCR, Western Blot, Control

Journal: Molecular Cancer Therapeutics

Article Title: PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER + Breast Cancer

doi: 10.1158/1535-7163.MCT-23-0564

Figure Lengend Snippet: PKMYT1 mRNA levels are significantly associated with patient outcome and endocrine therapy response. A, Kaplan–Meier survival curves of disease-specific survival of patients with Luminal A and B (ER + ) breast cancer in the METABRIC cohort, stratified by the median of PKMYT1 mRNA. Numbers of patients with high or low PKMYT1 mRNA are shown at the bottom. B, PKMYT1 mRNA level of pre- and post- AI treatment tumors of the patients from the ACOSOG Z1031B trial, grouped by AI clinical response (AI sensitive or AI resistant). C, PKMYT1 mRNA level of pre- and post-anastrozole treatment tumors of the patients from the NeoPalAna trial, grouped by anastrozole clinical response. In ( A ), the P -value and hazard ratio were calculated by the Cox Proportional-Hazards model. In ( B and C ), Wilcoxon signed-rank test P -values were shown after the median difference; paired t test P -values were shown after the mean difference. Wilcoxon rank sum test and t test were used to compare E2 post-treatment samples.

Article Snippet: T47D, T47D Palbo-R, MCF7 EDR, and MCF7 EDR Palbo-R cell lines expressing Gaussia luciferase (GLuc) under the control of the human PKMYT1 promoter were created by lentiviral transduction using a PKMYT1 promoter-GLuc reporter obtained from GeneCopoeia (cat. # HPRM40000-LvPG04).

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER + Breast Cancer

doi: 10.1158/1535-7163.MCT-23-0564

Figure Lengend Snippet: Expression of PKMYT1 is negatively regulated by palbociclib treatment in sensitive, but not resistant, ER + breast cancer cell lines and patients. A, Volcano plot of the correlation of PKMYT1 protein levels and Hallmark ssGSEA scores in 22 ER + breast cancer in ovariectomized PDX mice given exogenous E2. Hallmark pathways with P -values < 0.05 are labeled. B, RT-qPCR of relative PKMYT1 mRNA level after 1 μmol/L palbociclib treatment for 2 days, adjusted by GAPDH mRNA and normalized by the vehicle-treated cells. C, Luciferase assays were performed to determine PKMYT1 promoter activities in cells transduced with a lentivirus-expressing Gaussia luciferase (GLuc) under the control of a ∼1.3 kb PKMYT1 promoter (see “Methods”). Cells were treated with 1 μmol/L palbociclib for 2 days, and GLuc values were normalized by the vehicle-treated cells. D, Immunoblotting of protein lysates made from cells treated with 1 μmol/L palbociclib or vehicle for 2 days. GAPDH serves as a loading control. The figure is a representative image of three independent biological replicates. E, Scatterplots of PKMYT1 mRNA and Hallmark pathway “E2F Targets” score of NeoPalAna patient samples collected at three stages of treatments [baseline, cycle 1 day 1 (4 weeks treatment with anastrozole) and cycle 1 day 15 (2 weeks treatment with anastrozole plus palbociclib)]. F, PKMYT1 mRNA level of pre- and post-anastrozole and palbociclib (A+P) treatment tumors of the patients from the NeoPalAna trial who did not initially respond to anastrozole alone, grouped by A+P response. In (B and C ), bars show the means of three individual biological repeats. P -values were calculated by t test. In ( E ), the trend line was calculated by a linear regression model and P -values were calculated by Pearson and Spearman correlation. In ( F ), Wilcoxon signed-rank test P -values are shown after the median difference; paired t test P -values are shown after the mean difference. Wilcoxon rank sum test and t test were used to compare E2 post-treatment samples.

Article Snippet: T47D, T47D Palbo-R, MCF7 EDR, and MCF7 EDR Palbo-R cell lines expressing Gaussia luciferase (GLuc) under the control of the human PKMYT1 promoter were created by lentiviral transduction using a PKMYT1 promoter-GLuc reporter obtained from GeneCopoeia (cat. # HPRM40000-LvPG04).

Techniques: Expressing, Labeling, Quantitative RT-PCR, Luciferase, Transduction, Control, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER + Breast Cancer

doi: 10.1158/1535-7163.MCT-23-0564

Figure Lengend Snippet: A clinical-grade PKMYT1 inhibitor (RP-6036) and nucleoside analog (gemcitabine) synergistically and significantly reduce the viability of palbociclib-resistant ER + breast cancer cells that lack functional p53 protein. A, Dose–response curves of RP-6306 effect on the viability of T47D parental and Palbo-R cells, with and without 1 nmol/L gemcitabine cotreatment. B and C, Loewe synergy scores ( B ) and combination viability scores ( C ) of T47D parental and Palbo-R cells treated with different concentrations of RP-6306 and gemcitabine. D and E, Loewe synergy scores ( D ) and combination viability scores ( E ) of MCF7 EDR and MCF7 EDR Palbo-R cells stably transduced with lentiviruses expressing nontargeting shRNA (shNC) or two different TP53 -targeting shRNAs, treated with different concentrations of RP-6306 and gemcitabine. In ( A ), curves and IC 50 s were derived by the three-parameter log-logistic model. Vertical error bars show the standard errors of the mean of the viabilities. Horizontal error bars show the standard errors of the IC 50 s. In ( B and D ), P -values were calculated by Wilcoxon signed-rank test, adjusted by the Holms method if more than one comparison was performed. The (arrows) display whether the value is higher or lower than the baseline. In ( C and E ), error bars show the SEM, the approximate P -values were calculated by Z -test using the SEM of the model, adjusted by the Holms method if more than one comparison was performed. In all experiments, data were analyzed from three independent biological replicates.

Article Snippet: T47D, T47D Palbo-R, MCF7 EDR, and MCF7 EDR Palbo-R cell lines expressing Gaussia luciferase (GLuc) under the control of the human PKMYT1 promoter were created by lentiviral transduction using a PKMYT1 promoter-GLuc reporter obtained from GeneCopoeia (cat. # HPRM40000-LvPG04).

Techniques: Functional Assay, Stable Transfection, Transduction, Expressing, shRNA, Derivative Assay, Comparison

Journal: Molecular Cancer Therapeutics

Article Title: PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER + Breast Cancer

doi: 10.1158/1535-7163.MCT-23-0564

Figure Lengend Snippet: The combination of RP-6306 and gemcitabine increases apoptosis and activates DNA damage signaling in palbociclib-resistant T47D cells. A, The relative abundance of dead cells after 3 days drug treatment, measured by a CellTox Green assay, is shown relative to the vehicle treatment. Bars show the means of individual biological repeats indicated by the dots. B, Quantification of immunoblotting of protein lysates made from cells treated with 100 nmol/L RP-6306 and/or 2 nmol/L gemcitabine for 3 days. Cleaved PARP1 and caspase 3 are known markers of apoptosis. GAPDH serves as a loading control. The figure is a representative image of three independent biological replicates. C, Quantification of immunoblotting of protein lysates made from cells treated with 100 nmol/L RP-6306 and/or 2 nmol/L gemcitabine for 1 day. PKMYT1 phosphorylation of CDK1 at pT14 was assayed. DNA damage was assayed by induction of phosphorylation of ATR (pY1989) and histone variant H2AX pS139 (also known as γH2AX). Total CDK1 and ATR serve as normalizing controls of the corresponding phosphorylated proteins, while GAPDH serves as a loading control. The figure is a representative image of four independent biological replicates. Supplementary Fig. S18 shows representative immunoblots for quantified data in ( B and C ). In all data panels, P -values were calculated by Dunnett’s test (using the vehicle as the baseline) within each cell line.

Article Snippet: T47D, T47D Palbo-R, MCF7 EDR, and MCF7 EDR Palbo-R cell lines expressing Gaussia luciferase (GLuc) under the control of the human PKMYT1 promoter were created by lentiviral transduction using a PKMYT1 promoter-GLuc reporter obtained from GeneCopoeia (cat. # HPRM40000-LvPG04).

Techniques: CellTox Assay, Western Blot, Control, Phospho-proteomics, Variant Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

doi: 10.1007/s00018-023-05095-x

Figure Lengend Snippet: SND1 bound with SRF-CArG complex to promote VSMC proliferation- and migration-related gene transcription. A EMSA assays were performed using purified SRF1(1–266 aa, 0.1 μg), or SND1 (1–660 aa, 0.1 μg) protein and Biotin-labeled probes targeting the CArG motif (–CCATATTAGG–, 20 fmoL) of the Fos gene. Unlabeled- or unspecific probes were used for competition experiments. B , C 100 μg of cell nuclear lysate were collected from 1.5 × 10 7 primary Snd1-F/F and Snd1-F/F Tagln-Cre VSMCs. SRF enrichments at Cdk2 , Fos , Cdk6 , Rock1 , Rock2 , and Iqgap1 promoter were analyzed by ChIP assay. D , G The Snd1-F/F and Snd1-F/F Tagln-Cre VSMCs were allowed to grow until the cells were 70% confluent (2 × 10 5 per well in 6-well plates) and infected with the GLuc- Cdk2 , GLuc- Fos , GLuc- Cdk6 , GLuc- Rock1 , GLuc- Rock2 , and GLuc- Iqgap1 promoter plasmids, empty GLuc-Vector as the negative control (NC). Relative luciferase activity was evaluated by the ratio of GLuc / SEAP activity. 2 × 10 6 primary VSMCs were obtained in a 6 cm dish. E , H The mRNA levels of Cdk2 , Fos , Cdk6, Rock1 , Rock2 , and Iqgap1 were detected by RT-qPCR. F , I The protein levels of SND1, CDK2, FOS, CDK6, ROCK1, ROCK2, IQGAP1 and ACTB were detected by western blotting. Western blotting results were analyzed by ImageJ (2 ×) software. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed by unpaired two-tailed Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns no significance

Article Snippet: The wild type and ELK1 binding sites (–CCGGAAGT–) mutant promoter sequences of Snd1 were cloned into the Gluc-ON™ promoter-reporter vector (GeneCopoeia, EZX-LvPG04).

Techniques: Migration, Purification, Labeling, Infection, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Software, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

doi: 10.1007/s00018-023-05095-x

Figure Lengend Snippet: ELK1 promoted transcriptional activation of Snd1 in VSMC proliferation. A Venn diagram showed the upstream transcription factors of Snd1 predicted by JASPAR, EPD, GeneCard, GTRD, and PROMO bioinformatics software. B ELK1 binding motif (–CCGGAAGT–) in Snd1 promoter region across vertebrates was analyzed by the JASPAR database. C EMSA assays were performed using purified ELK1 (0.1 μg) protein and Biotin-labeled probes targeting motif (–CCGGAAGT–, 20 fmoL) of the Snd1 , unlabeled- or unspecific probes were used for competition experiments. D Primers were designed for ChIP based on the promoter regions containing the ELK1 binding motif (Region a), the region located outside the promoter region (Region b) as the negative control. E The mice femoral arteries were obtained after the injury ( n = 8 /group), ChIP analysis of ELK1 enrichment at different regions of Snd1 promoter. F 100 μg of cell nuclear lysate were collected by 1.5 × 10 7 primary cells for ChIP assay. ChIP analysis of ELK1 enrichment at different regions of Snd1 promoter in VSMCs that infected with pLVX-IRES-ELK1 or pLVX-IRES-Vector and stimulated with or without PDGF (40 ng/mL) for 24 h. G , J The primary VSMCs were allowed to grow until the cells were 70% confluent (2 × 10 5 per well in 6-well plates). Then primary VSMCs were infected with the GLuc plasmids carrying wild-type Snd1 promoter (− 2500 to + 500 bp) or Snd1 promoter with ELK1 binding site mutant ( Snd1 -mut), empty GLuc-Vector as the negative control (NC), together with pLVX-IRES-ELK1, or pLVX-IRES-Vector plasmids, or TRC2-pLKO-shElk1-1, TRC2-pLKO-shElk1-2, or TRC2-pLKO-Vector, with or without PDGF treatment for 24 h. Relative luciferase activity was evaluated by the ratio of GLuc / SEAP activity. H , I The primary VSMCs were infected with pLVX-IRES-ELK1 or pLVX-IRES-Vector and then were stimulated with or without PDGF (40 ng/mL) for 24 h, 2 × 10 6 primary VSMCs were obtained in a 6 cm dish. The mRNA levels of Snd1 were detected by RT-qPCR. The protein levels of SND1, ELK1, pELK1 and ACTB in were detected by western blotting. K – L The primary VSMCs were infected with TRC2-pLKO-Vector, shElk1-1 or shElk1-2 and then stimulated with or without PDGF (40 ng/mL) for 24 h, 2 × 10 6 primary VSMCs were obtained in a 6 cm dish. The mRNA levels of Snd1 were detected by RT-qPCR. The protein levels of SND1, ELK1, pELK1 and ACTB were detected by western blotting. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed by one-way ANOVA (from E, F, H, I, K, and L), or three-way ANOVA (G, J) followed by Bonferroni post-hoc correction. ** P < 0.01; *** P < 0.001; ns no significance

Article Snippet: The wild type and ELK1 binding sites (–CCGGAAGT–) mutant promoter sequences of Snd1 were cloned into the Gluc-ON™ promoter-reporter vector (GeneCopoeia, EZX-LvPG04).

Techniques: Activation Assay, Software, Binding Assay, Purification, Labeling, Negative Control, Infection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

doi: 10.1007/s00018-023-05095-x

Figure Lengend Snippet: Inhibition of the phosphorylation of ELK1 by U0126 retards injury-induced SND1 upregulation and neointima hyperplasia. A Wire-induced mouse femoral artery injury models were constructed, and U0126 was injected intraperitoneally every 3 days (1 mg/kg) after wire injury. The femoral arteries were collected ( n = 4 /group). Representative HE staining of sham-operated and wire-injured femoral arteries from DMSO and U0126 treated mice. ( N neointima; M media) B Averaged data of the neointima area, neointima/media ratio, lumen area, media area, peripheral vessel circumference, lumen vessel circumference of sham-operated or wire-injured femoral arteries ( n = 4 /group). C 100 μg of cell nuclear lysate were collected by 1.5 × 10 7 primary cells for ChIP assay. ChIP analysis of ELK1 enrichment at different regions of Snd1 promoter in primary VSMCs with PDGF in the presence or absence of U0126 (10 mM). D The primary VSMCs were allowed to grow until the cells were 70% confluent (2 × 10 5 per well in 6-well plates). Then cells were infected with the GLuc plasmids carrying wild-type Snd1 promoter (− 2500 to + 500 bp) or Snd1 promoter with ELK1 binding site mutant ( Snd1 -mut), empty GLuc-Vector as the negative control (NC), together with PDGF in the presence or absence of U0126 (10 mM). Relative luciferase activity was evaluated by the ratio of GLuc/SEAP activity. E , F The primary VSMCs were allowed to grow until the cells were 70% confluent and were stimulated with or without PDGF for 24 h in the presence or absence of U0126 (10 mM) for 24 h. The mRNA levels of Snd1 were detected by RT-qPCR. The protein levels of SND1, ELK1, pELK1 and ACTB were detected by western blotting. All western blotting results were analyzed by ImageJ (2 ×) software. Scale bar, 100 μm. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed by unpaired two-tailed Student’s t -test (A), one-way ANOVA (C, E, and F) or three-way ANOVA (A and D) followed by Bonferroni post-hoc correction. * P < 0.05; ** P < 0.01; *** P < 0.001; ns no significance

Article Snippet: The wild type and ELK1 binding sites (–CCGGAAGT–) mutant promoter sequences of Snd1 were cloned into the Gluc-ON™ promoter-reporter vector (GeneCopoeia, EZX-LvPG04).

Techniques: Inhibition, Construct, Injection, Staining, Infection, Binding Assay, Mutagenesis, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Software, Two Tailed Test