Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Chemical structure of ( a ) ORG25543 and ( b ) RPIGLYT2-82. c Glycine dose-responses in the presence of different concentrations of RPI-GLYT2-82 showing non-competitive inhibition. A Brown-Forsythe ANOVA test with Dunnett’s T3 multiple comparisons determined there is no statistically significant difference in the glycine EC 50 across all four conditions ( p = 0.49, n = 5 per condition). There is a significant reduction in I/I 300 between 0 nM RPI-GLYT2-82 and 600 nM ( p < 0.0001, n = 5) and 1.8 µM RPI-GLYT2−82 ( p < 0.0001, n = 5), determined by an ordinary one-way ANOVA with Dunnett’s multiple comparisons. There is no significant reduction in I/I 300 between 0 nM and 200 nM RPI-GLYT2-82 ( p = 0.07, n = 5). d RPI-GLYT2-82 concentration-dependent inhibition of glycine transport. The difference between hGlyT2 WT ( n = 6) and hGlyT2 Δ185 ( n = 5) is not significant ( p = 0.45), determined by a two-tailed unpaired T-test. e ORG25543 concentration-dependent inhibition of glycine transport. The difference between hGlyT2 WT ( n = 6) and hGlyT2 Δ185 ( n = 5) is not statistically significantly ( p = 0.60), determined by a two-tailed unpaired T-test. f RPI-GLYT2-82 washes out within 5-min (WT not fitted due to full reversal before the first time point) ( n = 5 for both conditions). g ORG25543 has prolon g ed inhibitory effects with minimal restoration of glycine-induced currents ( n = 5 for both conditions). h RPI-GLYT2-82 inhibits GlyT2 ( n = 5) with minimal effect on GlyT1 ( n = 5) ( p < 0.0001) determined by a two-tailed unpaired T-test. i The RPI-GLYT2-82 IC 50 at 20 mM Na + (IC 50 = 159 nM (95% CI: 81 to 238 nM), n = 5) is significantly lower than 100 mM Na + (IC 50 = 279 nM (95% CI: 182 to 376 nM), n = 5) ( p = 0.04). j The ORG25543 IC 50 at 20 mM Na + (IC 50 = 18 nM (95% CI: 6.8 to 29 nM), n = 5) is not significantly different to 100 mM Na + (IC 50 = 17.6 nM (95% CI: 5.5 to 30 nM), n = 5) ( p > 0.9999). Data in i , j analysed by a two-way ANOVA with Tukey’s multiple comparisons. k Normalised glycine-dependent transport currents showing comparable transport by hGlyT2 WT ( n = 5) and hGlyT2 Δ185 ( n = 5). ( p = 0.78), determined by a two-tailed unpaired T-test. n indicates biological replicates. Error bars represent ± SD from the mean. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Inhibition, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Cryo-EM density maps of hGlyT2 Δ185 in ( a ) substrate-free state (purple map contour level = 0.04 in ChimeraX), bound to b substrate glycine (pink map contour level = 0.046), c ORG25543 (green map contour level = 0.055 in ChimeraX), and d RPI-GLYT2-82 (blue map contour level = 0.038 in ChimeraX). Lipid densities shown in light yellow, densities corresponding to sterols built into the models shown in dark yellow. e-h Surface representation of substrate-free, substrate-bound, and inhibitor-bound structures of hGlyT2 Δ185 viewed parallel to the membrane. e Slice view of hGlyT2 Δ185 in substrate-free form. Closed extracellular vestibule around W215 (blue) and open intracellular pathway are displayed. f Slice view of hGlyT2 Δ185 showing glycine binding pocket (cyan, compound density shown at map contour level = 0.046 in ChimeraX) g Slice view of hGlyT2 Δ185 showing ORG25543 binding pocket (orange, compound density shown at map contour level = 0.055 in ChimeraX). h Slice view of hGlyT2 Δ185 showing RPI-GLYT2-82 binding pocket (dark purple, compound density shown at map contour level = 0.038 in ChimeraX). Residues W215 (TM1, blue), R216 (TM1, blue), P543 (EL4, yellow), and D633 (TM10, orange) are shown as sticks in e-h ; TM6 shown in green in f .

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a Cartoon representation of substrate-free hGlyT2 Δ185 , glycine-bound hGlyT2 Δ185 , ORG25543 -bound hGlyT2 Δ185 , and RPI-GLYT2-82-bound hGlyT2 Δ185 . b Upper panel is a magnified view of collapsed Na1 site, usual Na1 site-forming residues shown with corresponding densities (contour level = 0.030 in ChimeraX). Lower panel is of Cl – (green) modelled in substrate-free hGlyT2 Δ185 with coordinating residues (mean coordination distance of 3.1 Å ± 0.4) and densities shown (contour level = 0.030 in ChimeraX). c Upper panel is a magnified view of Na + (purple) modelled into Na2 with coordinating residues (mean coordination distance of 2.9 Å ± 0.4) with densities (contour level = 0.046 in ChimeraX). Lower panel is a magnified view of Cl – (green) modelled in glycine-bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 3.2 Å ± 0.5) and densities (contour level = 0.046 in ChimeraX). d Upper panel is a magnified view of Na + (purple) modelled into the density at Na1 with coordination residues (contour level = 0.050 in ChimeraX). ORG25543 shown in orange and water molecules shown in red (with corresponding densities). Lower panel is a magnified view of Cl – (green) modelled in ORG25543 -bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 2.8 Å ± 0.3) and densities shown (contour level = 0.050 in ChimeraX). e Cartoon representation of RPI-GLYT2-82-bound hGlyT2 Δ185 . Upper panel is a magnified view of Na + (purple) modelled into the density at Na1 with coordination residues (contour level = 0.045 in ChimeraX). RPI-GLYT2-82 is in magenta and water molecules in red (with corresponding densities). Lower panel shows Cl – (green) modelled in RPI-GLYT2-82-bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 2.9 Å ± 0.5) and densities (contour level = 0.045 in ChimeraX).

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques:

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a Cryo-EM density of glycine (contour level = 0.042 in ChimeraX). b Glycine (cyan) bound to the central site of hGlyT2. Interacting residues shown in sticks, H-bonds and ionic interactions shown as dashed grey lines. Na + modelled into Na2 shown as a green circle, modelled Cl – shown as a purple circle, and modelled water molecule shown as a red circle. c Glycine binds in distinct poses in GlyT2 and GlyT1. Glycine-bound hGlyT2 (pink) overlayed with glycine-bound GlyT1 (PDB ID 8WFI, grey). Interacting residues shown as sticks (hGlyT2 = pink, hGlyT1 = black). d Cryo-EM density of ORG25543 (contour level = 0.055 in ChimeraX). e Cryo-EM density of RPI-GLYT2-82 (contour level = 0.04 in ChimeraX). f ORG25543 (orange) bound to hGlyT2 Δ185 (green). Interacting residues shown as sticks and H-bonds and ionic interactions shown as grey dashed lines. N213 is likely involved in a weak hydrogen bond interaction with the polarised C–H of the methyl group attached to positively charged ammonium nitrogen of R4 substituent. g RPI-GLYT2-82 (purple) bound to hGlyT2 Δ185 (blue). Interacting residues shown as sticks, and H-bonds and ionic interactions shown as grey dashed lines. h , i Chemical structure of h ORG25543 and i RPI-GLYT2-82 with key chemical substituents R1 (blue), R2 (orange), R3 (green), and R4 (yellow) outlined. j ORG25543 concentration-dependant inhibition of glycine transport by hGlyT2 WT (black) ( n = 6), hGlyT2 W215F (red) ( n = 5) and hGlyT2 D633E (blue) ( n = 5). k Reversibility of ORG25543 inhibition of glycine transport currents mediated by hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 5) and hGlyT2 D633E ( n = 5). l RPI-GLYT2-82 dose-response curves of hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 6) and hGlyT2 D633E ( n = 5). The IC 50 values for hGlyT2 W215F and hGlyT2 D633E are greater than the maximal tested dose ( > 10 µM). m RPI-GLYT2-82 is a reversible inhibitor of glycine transport by hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 5), and hGlyT2 D633E ( n = 5). n refers to biological replicates with error bars representing ± SD from the mean. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Concentration Assay, Inhibition

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a RPI-GLYT2-82 reduced response rate to von Frey stimulus in chronic constriction injury (CCI) mice. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test comparing vehicle and every other group showed significant effects of time ( p < 0.001; F (5, 149) = 27.3) and treatment ( p < 0.001; F (3, 28) = 48.3), and an interaction effect ( p < 0.001; F (21, 196) = 6.30). A 50 mg/kg (i.p.) dose of RPI-GLYT2-82 demonstrated efficacy against mechanical allodynia, significant at 2 and 3 hours post-injection compared to vehicle ( p = 0.003, p < 0.001, respectively) A 100 mg/kg dose of RPI-GLYT2-82 showed a similar trend at 2 and 3 hours ( p = 0.001, p < 0.001, respectively). b RPI-GLYT2-82 reduced response rate to acetone stimulus in CCI mice. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test was conducted between CCI model mice administered vehicle and every other group showing significant main effects of time ( p < 0.0001; F (4, 93) = 25.8) and treatment ( p < 0.0001; F (3, 26) = 21.8, and an interaction effect ( p < 0.0001; F (11, 93) = 4.4). Both 50 and 100 mg/kg (i.p.) significantly reduced cold allodynia between 0.5 and 6-hours postinjection compared to vehicle ( p < 0.05-0.001). Gabapentin was also significant at 1-6 hours ( p < 0.05-0.001). c In PSNL mice, RPI-GLYT2-82 reduced von Frey response rates. Two-way ANOVA showed significant main effects of time ( p < 0.001; F (4, 46) = 12.7) and treatment ( p < 0.001; F (2, 13) = 20.8), and an interaction effect ( p < 0.001; F (14, 91) = 5.86). A 50 mg/kg (i.p.) dose reduced mechanical allodynia, significant at 90 minutes and 2-hours post-injection compared to vehicle ( p = 0.02, p = 0.03, respectively). d – f No deficits were observed at 50 mg/kg RPI-GLYT2-82. At 250 mg/kg, RPI-GLYT2-82 caused transient reductions in d rotarod performance, e grip strength (at 30 minutes but fully recovered by 60 minutes), and f sedation. g – i RPI-GLYT2−82 (50 mg/kg and 250 mg/kg) examined against positive morphine control (10 mg/kg) and vehicle (saline). Activity of compounds using whole-body plethysmography, measuring ( g ) respiratory frequency, h min volume, and i tidal volume. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test conducted between mice administered saline and every other group. j RPI-GLYT2-82 did not increase preference in a conditioned place preference paradigm. One-way ANOVA with Dunnett’s multiple comparison post-hoc test conducted between mice administered vehicle and every other group (F (3, 25) = 3.03; p = 0.0482). RPI-GLYT2-82 50 mg/kg ( n = 8, p = 0.78) and 150 mg/kg ( n = 8, p = 0.15) compared to positive 10 mg/kg morphine control ( n = 6, p = 0.0287) and vehicle (5% solutol, 95% PBS) ( n = 7). a – i Data shown as mean ± SEM. J Individual replicates shown with error bars representing ± SEM from the mean. Significance is denoted as: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. n = biological replicates. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Comparison, Injection, Control, Saline, Activity Assay, Conditioned Place Preference

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Schematic showing conformational differences between inhibitor-bound outward-open, glycine-bound inward-occluded and substrate-free inward-open states of GlyT2. ORG25543 (orange) and RPI-GLYT2-82 (magenta) lock GlyT2 in an outward-open conformation. Key binding site residues stabilizing the compounds, L211, W215, R216, P543, and D633, shown as sticks. The slow dissociation rate of ORG25543 prohibits the transporter cycling to an inward-open state. RPI-GLYT2-82 binding to GlyT2 is reversible allowing the transporter to sample other conformational states after release. Glycine is bound to GlyT2 in the inward-occluded state, S479 forming a key interaction to stabilize glycine in the central pocket. In the substrate-free inward-open state, the extracellular gate formed by R216 and D633 closes access to the extracellular side and W215 occupies the allosteric site. Cl – (green circle) is bound in all states, Na + (purple circle) in Na1 is bound only in the inhibitor-bound states, and Na + in Na2 is observed only in the substrate-bound state. Transmembrane helices TM1, TM6, and TM10 shown in blue, green, and orange, respectively, and EL4 is shown in yellow.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Time-dependent expression of CDK4 and cyclin D1 in G1/S transition. A – D , dynamic changes in the expression or phosphorylation of individual proteins in MCF-7 cells stably expressing FUCCI, detected by western blotting up to 36 h after treatment with 10 nM HRG. A and B , ErbB2 pathway proteins. C and D , proteins regulating G1/S transition. Protein and phosphorylation levels were normalized relative to those of GAPDH and divided by that of the 0 h sample (0 h = 1); n = 3. The data are displayed as the means ± SEM. FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Expressing, Phospho-proteomics, Stable Transfection, Western Blot, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: High ErbB2 expression induces cell cycle arrest by repressing cyclin D1 expression via c-Myc under CDK4 inhibition. A and B , WT MCF-7 and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and harvested 24 h after HRG stimulation. Western blotting of ( A ) p-ErbB2, p-RB (Ser807/811), p-RB (Thr373), RB, p-AKT (Thr308), AKT, p-ERK (Thr202/Thr204), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, with DMSO treatment) after normalization with GAPDH expression ( B ). The data are displayed as the means ± SD. C - F , ErbB2-overexpressing cells (moderate) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). Hsp90 and ErbB2 were coimmunostained. The scale bar represents 25 μm ( C ). Colocalization rate of Hsp90 and ErbB2 based on Mander’s tM1 and Pearson’s R values determined using Coloc 2 plugin of Image J; n = 10 ( D ). Hsp90 and PY100 were coimmunostained. The scale bar represents 25 μm ( E ). Colocalization rate of Hsp90 and PY100 based on Mander’s tM2 and Pearson’s R values; n = 10 ( F ). G and H , PLA signals detected in WT MCF-7 cells. Cells were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). PLA was performed using ErbB2 and Hsp90 antibodies. G , PLA signals detected in the presence of HER2 and Hsp90 antibodies ( left ). No signals detected after omitting Hsp90 antibody ( right ). The scale bar represents 100 μm. H , mean intensity of PLA signals per image was measured under each condition in ( G ) ( Left ). Number of nuclei per image was counted ( Right ). n = 59. I and J , FUCCI stably expressing MCF-7 cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG and collected 8 h later. I , ErbB2 and GAPDH expression determined by western blotting. ( J ) ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SD. K and L , ErbB2 cycloheximide pulse-chase assay was performed with FUCCI stably expressing MCF-7 cells. Cells were treated with CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, then 100 μg/ml cycloheximide was added 16 h after stimulation with 10 nM HRG. Cells were harvested at 0, 2, and 4 h after cycloheximide treatment; n = 3. K , ErbB2 and GAPDH expression determined by western blotting. L , ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SDs. M , coimmunoprecipitation assay in ErbB2-overexpressing MCF-7 cells (moderate). Cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG, and collected 8 h later. Immunoprecipitates and whole cell lysates were immunoblotted with anti-ubiquitin or anti-ErbB2, or GAPDH antibodies. N , schematic model: In CDK4i-unresponsive WT MCF-7 cells, the loss of ErbB2 protein stability impairs the regulation of c-Myc-mediated G1/S transition, leading to the delayed G1/S transition. O , WT MCF-7 and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after stimulation. The scale bar represents 100 μm. P , Images in ( J ) were analyzed to determine the proportion (%) of each cell cycle phase in the entire set of images; n = 3. In ( B ) and ( J ), ∗ p < 0.05 (Tukey’s test). In ( D ), ∗ p < 0.05 (with respect to control and CDK4i; Welch’s t test). In ( F ) and ( H ), ∗ p < 0.05 (with respect to control and CDK4i; two-tailed Student’s t test). In ( L ), the two-way ANOVA revealed significant main effects of both time ( p < 0.0001) and CDK4i treatment (∗ p = 0.02). FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin; PLA, proximity ligation assay; DMSO, dimethyl sulfoxide; RB, retinoblastoma protein.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Expressing, Inhibition, Western Blot, Control, Stable Transfection, Pulse Chase, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Two Tailed Test, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Reversible cell cycle arrest is maintained under c-Myc transcriptional activity. A - G , MCF-7 cells overexpressing ErbB2 (high levels). A and B , cells were treated with 250 nM CDK4i 12 h after HRG stimulation, subjected to a wash/no-wash treatment 4 h later, and harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, p-c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. The data are displayed as the means ± SD. C , washing operations were performed under the same conditions as in ( A and B ); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3859. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. D and E , cells were pretreated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation, D ). E , percentage distributions of G1 and S/G2/M phases based on the images in ( D ); n = 4. The scale bar represents 100 μm. The data are displayed as the means ± SD. F , cells were treated with 250 nM CDK4i or DMSO (control) 12 h after HRG stimulation and CDKN1B mRNA levels up to 20 h after HRG stimulation were examined by qPCR; n = 3. G , after the cells were treated with or without EN4 under the same conditions as in ( D - E ), RNA was collected 8 h after treatment with inhibitors, and CDKN1B mRNA levels were examined by qPCR; n = 3. The data are displayed as the means ± SD. H , WT MCF-7, WT T47D, and ErbB2 ( high ) OE MCF-7 cells were pretreated for 30 min with/without 4 μM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation, incubated with 250 nM CDK4i for 6 days, and SA-β-Gal staining was assessed. I , percentage SA-β-Gal-stained cells based on the images in ( H ); n = 12. J , ErbB2 levels modulate the irreversibility of the G1/S transition under CDK4i treatment. In ( B ), ( E ), ( F ), and ( G ), ∗ p < 0.05 (with respect to control and each inhibitor; two-tailed Student’s t test). In ( I ), ∗ p < 0.05 (with respect to control and each inhibitor; Welch’s t test). DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; SA-β-Gal, senescence-associated β-galactosidase; qPCR, quantitative PCR; RB, retinoblastoma protein.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Activity Assay, Western Blot, Expressing, Incubation, Control, Staining, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Proposed model the ErbB2 receptor regulation during G1/S transition via cyclin D1 and c-Myc in CDK4i nonresponsive cells. A , in low ErbB2-expressing cells, CDK4 inhibition leads to reduced pathway activity due to ErbB2 degradation via Hsp90 dis-colocalization. This attenuates the transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells unresponsive to CDK4i exhibits a delayed G1/S transition. B , in high ErbB2-expressing cells, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in cell cycle arrest after CDK4 inhibition. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Expressing, Inhibition, Activity Assay

Journal: Oncology Letters

Article Title: Stimulator of interferon genes signaling network-driven prognostic signature for pancreatic cancer: Hub gene discovery with multimodal validation

doi: 10.3892/ol.2025.15348

Figure Lengend Snippet: Molecular characterization of STING signaling pathway-related genes in PAAD. (A) Protein-protein interaction (PPI) network of core STING signaling components identified through STRING database analysis (minimum interaction score: 0.4). (B) Expanded PPI network showing secondary interactors of STING pathway genes (minimum interaction score: 0.7). (C) Consensus matrix heatmap classifying patients with PAAD into two molecular subtypes (C1 and C2) based on STING-related gene expression patterns. (D) Heatmap comparing expression profiles of STING-related genes across PAAD subtypes (C1 vs. C2) with expression gradient key (red, high expression; blue, low expression). (E) Kaplan-Meier survival analysis showing significantly reduced overall survival in the STING-enriched subtype (C1) compared with the STING-depleted subtype (C2). STING, stimulator of interferon genes; PAAD, pancreatic ductal adenocarcinoma; CGAS, cyclic GMP-AMP synthase; TBK1, TANK-binding kinase 1; MAVS, mitochondrial antiviral-signaling protein; TRIM56, tripartite motif-containing 56; TREX1, three prime repair exonuclease 1; TRIM32, tripartite motif-containing 32; IFI16, interferon γ inducible protein 16; IFIT1, interferon-induced protein with tetratricopeptide repeats 1; DDX41, DEAD-box helicase 41; NLRP4, NLR family pyrin domain containing 4; DTX4, deltex E3 ubiquitin ligase 4; ZDHHC1, zinc finger DHHC-type containing 1; PRKDC, protein kinase DNA-activated catalytic subunit; XRCC, X-ray repair cross complementing; ELF4, E74-like ETS transcription factor 4; NLRC3, NLR family CARD domain containing 3; ARF1, ARF GTPase 1; DDX56, RNA sensor RIG-I; IKBKE, inhibitor of nuclear factor κB kinase subunit ε; IRF3, interferon regulatory factor 3; RNF5, ring finger protein 5.

Article Snippet: For overexpression, full-length zinc finger DHHC-type containing 1 (ZDHHC1) complementary DNA (OriGene Technologies, Inc.) was cloned into the pCDH-CMV-MCS-EF1-Puro vector (cat. no. CD510B-1; System Biosciences, LLC).

Techniques: Gene Expression, Expressing, Binding Assay, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Stimulator of interferon genes signaling network-driven prognostic signature for pancreatic cancer: Hub gene discovery with multimodal validation

doi: 10.3892/ol.2025.15348

Figure Lengend Snippet: Development and validation of a STING1 signaling-related prognostic signature for PAAD. (A) Univariate Cox regression analysis of STING1 pathway-related genes in PAAD. Genes with significant prognostic value (P<0.05) are included. (B) LASSO regression 10-fold cross-validation curve showing optimal λ selection (minimum criteria). (C) LASSO coefficient profiles of STING1-related genes across log(λ) sequence. (D) Kaplan-Meier survival analysis for the prognostic signature in TCGA-PAAD cohort (high-risk vs. low-risk groups; cutoff: median risk score). (E) Validation of prognostic signature in GSE224564 cohort. (F) Risk score distribution plot with corresponding survival status and expression heatmap for 6 signature genes in TCGA-PAAD. (G) Validation in GSE224564 cohort showing identical configuration to (F). STING, stimulator of interferon genes; PAAD, pancreatic ductal adenocarcinoma; LASSO, least absolute shrinkage and selection operator; TCGA, The Cancer Genome Atlas; MAVS, mitochondrial antiviral-signaling protein; IFI16, interferon γ inducible protein 16; IFIT2, interferon-induced protein with tetratricopeptide repeats 2; DTX4, deltex E3 ubiquitin ligase 4; ZDHHC1, zinc finger DHHC-type containing 1; PRKDC, protein kinase DNA-activated catalytic subunit; STAT6, signal transducer and activator of transcription 6; XRCC5, X-ray repair cross complementing: ELF4, E74-like ETS transcription factor 4; DDX58, RNA sensor RIG-I.

Article Snippet: For overexpression, full-length zinc finger DHHC-type containing 1 (ZDHHC1) complementary DNA (OriGene Technologies, Inc.) was cloned into the pCDH-CMV-MCS-EF1-Puro vector (cat. no. CD510B-1; System Biosciences, LLC).

Techniques: Biomarker Discovery, Selection, Sequencing, Expressing, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Stimulator of interferon genes signaling network-driven prognostic signature for pancreatic cancer: Hub gene discovery with multimodal validation

doi: 10.3892/ol.2025.15348

Figure Lengend Snippet: Independent prognostic value and immunotherapy relevance of the STING-related risk signature in PAAD. (A) Univariate Cox regression analysis of clinical parameters and risk score. (B) Multivariate Cox regression confirming the risk score as an independent prognostic factor after adjusting for covariates in (A). (C) Correlation analysis of risk score and activated dendritic cell abundance. ‘Dendritic cells activated’ represents proportion scores derived from the xCell algorithm (0–1 scale), quantifying relative abundance in the tumor microenvironment. Correlation was assessed using Spearman's rank correlation analysis (Spearman's ρ=0.28; P=0.015). (D) Higher risk scores in immunotherapy non-responders vs. responders (Mann-Whitney U test). Single-cell RNA sequencing ( GSE141017 ) visualization of STING1 signature gene expression: (E) t-SNE plot showing cell-type distribution. (F) Feature plots of signature gene expression across the PAAD tumor microenvironment. STING, stimulator of interferon genes; PAAD, pancreatic ductal adenocarcinoma; T, tumor; N, node; CD8T, T-cell surface glycoprotein CD8; ZDHHC1, zinc finger DHHC-type containing 1; IFIT2, interferon-induced protein with tetratricopeptide repeats 2; IFI16, interferon γ inducible protein 16; DTX4, deltex E3 ubiquitin ligase 4; MAVS, mitochondrial antiviral-signaling protein; PRKDC, protein kinase DNA-activated catalytic subunit; PAAD, pancreatic adenocarcinoma.

Article Snippet: For overexpression, full-length zinc finger DHHC-type containing 1 (ZDHHC1) complementary DNA (OriGene Technologies, Inc.) was cloned into the pCDH-CMV-MCS-EF1-Puro vector (cat. no. CD510B-1; System Biosciences, LLC).

Techniques: Derivative Assay, MANN-WHITNEY, RNA Sequencing, Gene Expression, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Stimulator of interferon genes signaling network-driven prognostic signature for pancreatic cancer: Hub gene discovery with multimodal validation

doi: 10.3892/ol.2025.15348

Figure Lengend Snippet: Functional roles of IFIT2 and ZDHHC1 in PAAD oncobiology. (A) Human Protein Atlas representative immunohistochemical staining of six prognostic signature genes in PAAD tissues (proteinatlas.org). Scale bar, 100 µm. (B) Western blot and semi-quantification confirming successful ZDHHC1 overexpression in Panc02 cells (C) Western blot and semi-quantification confirming IFIT2 knockout efficiency in Panc02 cells (****P<0.0001). (D) CCK-8 assay: ZDHHC1 overexpression enhances Panc02 proliferation (***P<0.001 and ****P<0.0001 vs OE-NC). (E) CCK-8 assay: IFIT2 knockout enhances Panc02 proliferation (**P<0.01 and ***P<0.001 vs. sh-NC). (F) TUNEL assay (scale bar, 50 µm) demonstrating reduced apoptosis in ZDHHC1-overexpressing Panc02 cells. (G) TUNEL assay (scale bar 50 µm) showing decreased apoptosis in IFIT2-knockout Panc02 cells (**P<0.01). IFIT2, interferon-induced protein with tetratricopeptide repeats 2; ZDHHC1, zinc finger DHHC-type containing 1; PAAD, pancreatic ductal adenocarcinoma; OE, overexpression; CCK-8, Cell Counting Kit-8; DTX4, deltex E3 ubiquitin ligase 4; IFI16, interferon γ inducible protein 16; MAVS, mitochondrial antiviral-signaling protein; PRKDC, protein kinase DNA-activated catalytic subunit; sh-NC, scrambled negative control shRNA; ns, not significant.

Article Snippet: For overexpression, full-length zinc finger DHHC-type containing 1 (ZDHHC1) complementary DNA (OriGene Technologies, Inc.) was cloned into the pCDH-CMV-MCS-EF1-Puro vector (cat. no. CD510B-1; System Biosciences, LLC).

Techniques: Functional Assay, Immunohistochemical staining, Staining, Western Blot, Over Expression, Knock-Out, CCK-8 Assay, TUNEL Assay, Cell Counting, Ubiquitin Proteomics, Negative Control, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Biochemical reconstitution of temozolomide-induced mutational processes

doi: 10.1016/j.jbc.2025.110676

Figure Lengend Snippet: Influence of TLS polymerases on the TMZ-induced mutation spectra in the presence and absence of hMGMT. A, illustration of the experimental system. dsDNA substrates were incubated with 400 μM TMZ three times. When indicated, damaged templates were treated with hMGMT. The DNA was heated and reannealed with NGS primer and 10x excess competitor to sequester the top strand. Then the primer extension was started by adding yPol δ. After 30 min of incubation with yPol δ, the second polymerase (either yPol ζ, hPol κ, or hPol η) was added, and the reaction was continued for another 30 min. B – D, mutation spectra produced on the TMZ-damaged DNA in the presence of the indicated second polymerase without hMGMT treatment. E–G, influences of the second polymerase on the C>T mutations were expressed as a ratio of the mutation frequencies at individual sites. CpC>T and CpT>T mutations (SBS11), other mutations (Others), and all mutations (All) are plotted as separate groups. H – J, the same experiments as in B – D were carried out using the templates that were treated with hMGMT. K, influences of hMGMT on the C>T and C>A mutations that were produced in the presence of indicated second polymerases. For hPol η reactions, only C>T mutations were analyzed because this polymerase did not produce considerable C>A mutations. Mutation frequencies mapped on the templates are shown in . hMGMT, human methylguanine methyltransferase; hPol κ, human Pol κ; hPol η, human Pol η; NGS, next-generation sequencing; SBS11, substitution signature 11; TLS, translesion synthesis; TMZ, temozolomide; yPol δ, yeast Pol δ.

Article Snippet: Human complementary DNA of MGMT (hMGMT) was obtained from SinoBiological , amplified by PCR, and cloned into pET21a to express MGMT with a C-terminal His6-tag.

Techniques: Mutagenesis, Incubation, Produced, Next-Generation Sequencing, Translesion Synthesis

Journal: Journal of Korean Medical Science

Article Title: Effect of SLC5A8 Missense Variants on Its Tumor-Suppressive Function

doi: 10.3346/jkms.2025.40.e146

Figure Lengend Snippet: EV = empty vector, WT = wild-type, SLC5A8 = solute carrier family-5 member-8. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT.

Article Snippet: Wild-type SLC5A8 complementary DNA (Origene Technologies, Inc., Rockville, MD, USA) was subcloned into the p3XFLAG-CMV-10 vector.

Techniques: Plasmid Preparation

Journal: Journal of Korean Medical Science

Article Title: Effect of SLC5A8 Missense Variants on Its Tumor-Suppressive Function

doi: 10.3346/jkms.2025.40.e146

Figure Lengend Snippet: EV = empty vector, WT = wild-type, SLC5A8 = solute carrier family-5 member-8. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. native SLC5A8 expression without MG132 or bafilomycin A 1 treatment.

Article Snippet: Wild-type SLC5A8 complementary DNA (Origene Technologies, Inc., Rockville, MD, USA) was subcloned into the p3XFLAG-CMV-10 vector.

Techniques: Plasmid Preparation, Expressing

Journal: Journal of Korean Medical Science

Article Title: Effect of SLC5A8 Missense Variants on Its Tumor-Suppressive Function

doi: 10.3346/jkms.2025.40.e146

Figure Lengend Snippet: EV = empty vector, WT = wild-type, SLC5A8 = solute carrier family-5 member-8.

Article Snippet: Wild-type SLC5A8 complementary DNA (Origene Technologies, Inc., Rockville, MD, USA) was subcloned into the p3XFLAG-CMV-10 vector.

Techniques: Plasmid Preparation

Journal: Journal of Korean Medical Science

Article Title: Effect of SLC5A8 Missense Variants on Its Tumor-Suppressive Function

doi: 10.3346/jkms.2025.40.e146

Figure Lengend Snippet: EV = empty vector, WT = wild-type, SLC5A8 = solute carrier family-5 member-8. ### P < 0.001 vs. EV; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT.

Article Snippet: Wild-type SLC5A8 complementary DNA (Origene Technologies, Inc., Rockville, MD, USA) was subcloned into the p3XFLAG-CMV-10 vector.

Techniques: Plasmid Preparation

Journal: Journal of Korean Medical Science

Article Title: Effect of SLC5A8 Missense Variants on Its Tumor-Suppressive Function

doi: 10.3346/jkms.2025.40.e146

Figure Lengend Snippet: EV = empty vector, WT = wild-type, SLC5A8 = solute carrier family-5 member-8. ### P < 0.001 vs. EV, ** P < 0.01, *** P < 0.001 vs. WT.

Article Snippet: Wild-type SLC5A8 complementary DNA (Origene Technologies, Inc., Rockville, MD, USA) was subcloned into the p3XFLAG-CMV-10 vector.

Techniques: Plasmid Preparation

Journal: Journal of Korean Medical Science

Article Title: Effect of SLC5A8 Missense Variants on Its Tumor-Suppressive Function

doi: 10.3346/jkms.2025.40.e146

Figure Lengend Snippet: EV = empty vector, WT = wild-type, SLC5A8 = solute carrier family-5 member-8. ## P < 0.01, ### P < 0.001 vs. EV, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT.

Article Snippet: Wild-type SLC5A8 complementary DNA (Origene Technologies, Inc., Rockville, MD, USA) was subcloned into the p3XFLAG-CMV-10 vector.

Techniques: Plasmid Preparation