Journal: Food & Nutrition Research

Article Title: Indole-3-carbinol ameliorates ER stress-mediated hyperleptinemia in western diet-fed apoE -/- mice

doi: 10.29219/fnr.v70.12263

Figure Lengend Snippet: Effects of I3C on endoplasmic reticulum stress in the liver of ApoE -/- mice fed a WD. Representative protein levels of p-eIF2α, eIF2α, XBP1, CHOP, and GRP78 as measured by western blotting. Results are presented as the mean ± standard error ( n = 4–5 per group). * P < 0.05, ** P < 0.01 versus WD. p-eIF2α, phospho-eukaryotic initiation factor 2 subunit alpha; XBP1, X-box binding protein 1; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein.

Article Snippet: The membranes were incubated with primary antibodies against p-eIF2α (#3398), eIF2α (#2103), XBP1 (#27901), CHOP (#2895) (1:1,000; Cell Signaling Technology; Danvers, MA, USA), GRP78 (ab21685, Abcam Inc., Waltham, MA, USA) and β-actin (A5441, 1:10,000; Sigma-Aldrich).

Techniques: Western Blot, Binding Assay

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: Expression levels of XBP1 and XBP1s are positively correlated with resistance to CDK4/6 inhibitors and endocrine therapy in patients with HR+/HER2− metastatic breast cancer. A) Schematic for the study design and analytical workflow. PFS, progression‐free survival; RNA‐seq, RNA sequencing; DEG, differentially expressed gene; BRCA, breast invasive carcinoma. B) Venn diagram showing the shared numbers and overlaps of upregulated genes identified in HR+/HER2− breast cancer tumors from the TCGA and METABRIC datasets, and the treatment‐resistant cohort of patients with HR+/HER2− metastatic breast cancer. ET, endocrine therapy. C) Kaplan–Meier curves of progression‐free survival (PFS) in the validation cohort of patients with high and low levels of XBP1 and TMEM26 expressions based on qRT‐PCR assay. P ‐values are indicated and were calculated using the log‐rank test. D) XBP1 and XBP1s expression levels in primary breast tumors of different molecular subtypes (HR+/HER2− subtype, n = 152; HER2+ subtype, n = 84; Triple‐negative subtype, n = 38). * p < 0.05, *** p < 0.001, **** p < 0.0001. E) Kaplan–Meier curves of PFS in the validation cohort of patients with high and low levels of XBP1s expression. P ‐values are indicated and were calculated using the log‐rank test. F) Immunohistochemistry (IHC) staining for XBP1s and XBP1 in representative tumor samples from patients who were sensitive (PFS > 6 months) or resistant (PFS ≤ 6 months) to combination therapy of CDK4/6 inhibitors and endocrine therapy. Scale bar, 50 µm. G) The immunoreactive score of IHC staining for XBP1s and XBP1 in tumors from the validation cohort of patients. ** p < 0.01. For statistical analysis, the log‐rank test was employed in (C) and (E); one‐way ANOVA with Tukey's post hoc test was utilized for (D); the Mann–Whitney U test was employed in (G). Data are represented as the mean ± standard deviation.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: Expressing, RNA Sequencing, Biomarker Discovery, Quantitative RT-PCR, Immunohistochemistry, MANN-WHITNEY, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: XBP1s attenuated the therapeutic efficacy of CDK4/6 inhibitors and endocrine therapy in vivo. A) Schematic diagram illustrating the workflow of the in vivo study. B–H) Mice with cell‐derived xenografts of MCF7/pCDH or MCF7/XBP1s were randomly divided into four treatment groups: vehicle, palbociclib, fulvestrant, or their combination. B) Representative images of IHC staining for XBP1s in MCF7/pCDH and MCF7/XBP1s cell‐derived xenografts from different treatment groups. Scale bar, 50 µm. C) Tumor growth curve plots of MCF7/pCDH tumor in each treatment group after treatment administration. n = 6 per treatment group. Each data point represents the mean tumor volume ± standard deviation. D) Waterfall plot showing the change in tumor volume of individual MCF7/pCDH cell‐derived xenografts at the end of the experiment compared with the baseline tumor volume on day 0 of the treatment. n = 6 per treatment group. E) Percentage of tumors exhibiting different drug responses (PD, SD, R) in MCF7/pCDH cell‐derived xenografts. PD, progression disease; SD, stable disease; R, regression. F) Tumor growth curve plots of MCF7/XBP1s tumor in each treatment group after treatment administration. n = 6 per treatment group. Each data point represents the mean tumor volume ± standard deviation. G) Waterfall plot showing the change in tumor volume of individual MCF7/ XBP1s cell‐derived xenografts at the end of the experiment compared with the baseline tumor volume on day 0 of the treatment. n = 6 per treatment group. H) Percentage of tumors exhibiting different drug responses (PD, SD, R) in MCF7/XBP1s cell‐derived xenografts.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: Drug discovery, In Vivo, Derivative Assay, Immunohistochemistry, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: Overexpression of XBP1s confers resistance to fulvestrant and palbociclib and impairs the synergistic effect of palbociclib combined with fulvestrant in vitro. A) The dose‐response curves and half‐maximal inhibitory concentration (IC 50 ) values of palbociclib in MCF7 and T‐47D cells transfected with the control or XBP1s‐overexpressing plasmid after being treated for 96 h. B) Representative images and quantification of colony formation assay in MCF7 and T‐47D cells treated with vehicle or palbociclib (3.5 × 10 −6 m for MCF7 cells, 1 × 10 −6 m for T‐47D cells). ns, not significant; *** p < 0.001. C) Western blotting of the indicated proteins in MCF7 cells with or without XBP1s overexpression after being treated with the indicated concentration of palbociclib. D) Cell cycle distribution of MCF7 cells with or without XBP1s overexpression after being treated with palbociclib (3.5 × 10 −6 m ). E) The dose‐response curves and IC 50 values of fulvestrant in MCF7 and T‐47D cells transfected with the control or XBP1s ‐overexpressing plasmid after being treated for 96 h. F) Representative images and quantification of colony formation assay after treatment with vehicle or fulvestrant in MCF7 (15 × 10 −9 m ) cells with or without XBP1s overexpression. * p < 0.05, *** p < 0.001. G) Representative images and quantification of colony formation assay after treatment with vehicle or fulvestrant in T‐47D cells (120 × 10 −9 m ) with or without XBP1s overexpression. ns, not significant; ** p < 0.01. H) The dose‐response curves and IC 50 values of palbociclib in patient‐derived organoids (PDOs) after being treated for 96 h. ** p < 0.01. I) The dose–response curves and IC 50 values of palbociclib in PDOs after being treated for 96 h. ** p < 0.01. J,K) Relative spheroid area in PDO‐8912 transfected with the control or XBP1s ‐overexpressing plasmid following treatment with palbociclib and fulvestrant for the indicated days. The culture medium was replaced every 3 d. * p < 0.05, ** p < 0.01, *** p < 0.001. For statistical analysis, two‐tailed unpaired Student's t ‐test was utilized for (B), (F), (G), (H), (I), (J), and (K). Data are presented as the mean ± standard deviation.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: Over Expression, In Vitro, Concentration Assay, Transfection, Control, Plasmid Preparation, Colony Assay, Western Blot, Derivative Assay, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: Epigenetic and pharmacological inhibition of XBP1s enhances the therapeutic sensitivity of fulvestrant and palbociclib in HR+/HER2− breast cancer cells. A) The dose–response curves and IC 50 values of palbociclib in MCF7 cells with or without XBP1 knockdown after being treated for 96 h. B) The dose–response curves and IC 50 values of fulvestrant in MCF7 cells with or without XBP1 knockdown after being treated for 96 h. C) Schematic illustrating the mechanism through which 4µ8C and MKC8866 hinder the synthesis of XBP1s. D) The mRNA levels of XBP1s downstream target genes were assessed in MCF7 cells by the qRT‐PCR assay following 6 h of treatment with the vehicle control (dark red bars), 0.5 × 10 −6 m of thapsigargin (Tg) alone (orange bars), or 0.5 × 10 −6 m of Tg combined with increasing concentrations of 4µ8C (light yellow bars), from 1.25 to 40 × 10 −6 m (twofold). E) The mRNA levels of XBP1s downstream target genes were assessed in MCF7 cells by the qRT‐PCR assay following 24 h of treatment with the vehicle control (dark red bars), 0.5 × 10 −6 m of thapsigargin (Tg) alone (orange bars), or 0.5 × 10 −6 m of Tg combined with increasing concentrations of MKC8866 (light yellow bars), from 0.125 to 4 × 10 −6 m (twofold). F,G) Cell proliferation analysis and cell cycle distribution for MCF7 cells treated with or without 4µ8C and MKC8866. * p < 0.05, ** p < 0.01. H) The dose–response curves and IC 50 values of palbociclib in MCF7 cells transfected with or without 4µ8C (left) and MKC8866 (right) after being treated for 96 h. I) The dose–response curves and IC 50 values of fulvestrant in MCF7 cells transfected with or without 4µ8C (left) and MKC8866 (right) after being treated for 96 h. J) Dose–response matrix (relative cell viability) of 4µ8C combined with palbociclib (left) or fulvestrant (right) in MCF7 cells based on the CCK8 assay. K) Combination index (CI) values for the antiproliferation effects of 4µ8C combined with palbociclib or fulvestrant in MCF7 cells. CI values were calculated based on the dose–response matrix in (J). For statistical analysis, two‐way ANOVA with Bonferroni's method correction was applied in (F). Data are presented as the mean ± standard deviation.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: Inhibition, Knockdown, Quantitative RT-PCR, Control, Transfection, CCK-8 Assay, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: XBP1s overexpression promotes cell proliferation and cell cycle progression by regulating the G1/S transition in HR+/HER2− breast cancer cells. A) Enrichment plots for hallmarks of the E2F target, estrogen response, and the MYC target generated using gene set enrichment analysis (GSEA) based on the results of RNA sequencing of MCF7/pCDH and MCF7/XBP1s cells. FDR, false discovery rate. NES, normalized enrichment score. B) Representative images and quantification of EdU‐positive MCF7 and T‐47D cells with or without XBP1s overexpression. Scale bar, 50 µm. ** p < 0.01, *** p < 0.001. C) Images of tumors from the MCF7/pCDH group and MCF7/XBP1s group at the end of the experiments. D) IHC staining for XBP1s in tumor slides from the MCF7/pCDH and MCF7/XBP1s groups. Scale bar, 50 µm. E) Tumor growth curves of cell‐derived xenografts of MCF7/pCDH and MCF7/XBP1s cells. Tumor volumes were recorded every 4 d starting from day 12 after the injection of cells. n = 6 per group. * p < 0.05, ** p < 0.01. F–I) MCF7/pCDH and MCF7/XBP1s cells were synchronized to the G1/S phase by double thymidine blockage, then released to a fresh medium and collected for the indicated time. F) Cell cycle distribution was measured using flow cytometry. G) Representative time‐lapse images of MCF7 cells, with or without XBP1s overexpression, were presented at regular 3‐h intervals after synchronization. Scale bar, 25 µm. H) Quantification of the cell cycle duration based on time‐lapse live‐cell imaging. ** p < 0.01. I) The levels of proteins related to cell cycle determination were detected by western blotting in MCF7 cells after synchronization and subsequent release for the indicated duration. J) Western blotting for the indicated proteins related to G1 to S phase transition in MCF7 and T‐47D cells with or without XBP1s overexpression. For statistical analysis, two‐tailed unpaired Student's t ‐test was used for (B) and (H); two‐way ANOVA with Bonferroni's method correction was applied in (E). Data are presented as the mean ± standard deviation.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: Over Expression, Generated, RNA Sequencing, Immunohistochemistry, Derivative Assay, Injection, Flow Cytometry, Live Cell Imaging, Western Blot, Sublimation, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: SND1 is a direct target of XBP1s and is responsible for the effects of XBP1s on cell proliferation, G1/S transition, and resistance to therapy. A) The Venn diagram shows the intersection of hallmark gene sets identified by GSEA based on the RNA‐seq data. B) KEGG pathway enrichment analysis of ChIP‐seq data obtained from MCF7 cells. C) Scatterplot showing the correlation between the expression of XBP1s and SND1 in tumors obtained from patients with HR+/HER2− early breast cancer ( n = 152). The correlation was assessed using Pearson's correlation coefficient. D,E) MCF7 and T‐47D cells were transfected with the control or XBP1s ‐overexpressing plasmid. The mRNA expression of SND1 was detected using qRT‐PCR assay (D), and the protein level of SND1 was measured via western blotting assay (E). ** p < 0.01, ### p < 0.001. F) ChIP‐qPCR of predicted XBP1s binding sites on SND1 promoters in MCF7 and T‐47D cells. ** p < 0.01, ### p < 0.001. G) The upper panel shows the XBP1s binding motif; the lower panel shows a schematic diagram of dual‐luciferase reporter vectors containing wild‐type or mutant XBP1s binding sites on the SND1 promoter. H) The activity of wild‐type (pGL3–SND1–WT) and mutant (pGL3–SND1–MUT) SND1 promoter in MCF7 cells, as indicated, was measured by dual‐luciferase reporter assay. * p < 0.05, *** p < 0.001, **** p < 0.0001. I) The protein level of SND1 was detected using western blotting in MCF7 and T‐47D cells treated with the vehicle control, 0.5 × 10 −6 m of Tg alone, or 0.5 × 10 −6 m of Tg combined with increasing concentrations of 4µ8C. J–M) Cells were treated with shNC, SND1‐sh2, or SND1‐sh3 after being stably transfected with the control or XBP1s ‐overexpressing plasmid. The cell proliferation analysis (J), cell cycle distribution (K), dose–response curves and IC 50 values of palbociclib (L) and fulvestrant (M) in MCF7 and T‐47D cells. * p < 0.05, ** p < 0.01. For statistical analysis, two‐tailed unpaired Student's t ‐test was used for (D) and (F); one‐way ANOVA with Turkey's post hoc test was utilized for (H); two‐way ANOVA with Turkey's post hoc test was applied in (J). Data are presented as the mean ± standard deviation.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: RNA Sequencing, ChIP-sequencing, Expressing, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, ChIP-qPCR, Binding Assay, Luciferase, Mutagenesis, Activity Assay, Reporter Assay, Stable Transfection, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: SND1 facilitates E2F1‐mediated transcriptional activation of target genes in HR+/HER2− breast cancer cells. A) Whole‐cell extracts of MCF7 and T‐47D cells were subjected to co‐immunoprecipitation (co‐IP) using anti‐SND1 antibodies, and the interaction between E2F1 and SND1 was identified via western blotting. B) Whole‐cell extracts of MCF7 and T‐47D cells were subjected to co‐IP using anti‐E2F1 antibodies, and the interaction between E2F1 and SND1 was identified by western blotting. C) Immunofluorescence (IF) staining assays exhibited the subcellular localization of SND1 (yellow) and E2F1 (green) in MCF7 and T‐47D cell lines. Nuclei were stained with DAPI (blue). D) The mRNA levels of E2F1 downstream target genes in MCF7 and T‐47D cells after transfection with the control or SND1 ‐overexpressing plasmid were determined by qRT‐PCR assay. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. E) Scatterplot showing the correlation between the expression of XBP1s and E2F1 , CCNE1 , CCNA2 , and RRM2 in tumors obtained from patients with HR+/HER2− early breast cancer ( n = 152). The correlation was assessed using Pearson's correlation coefficient. F,G) After being treated with or without 0.1 × 10 −6 of palbociclib for 24 h, whole‐cell extracts of MCF7 and T‐47D cells were immunoprecipitated with anti‐SND1 antibodies or anti‐E2F1 antibodies, followed by western blotting was conducted to investigate the interaction between SND1 and E2F1. H) The qRT‐PCR assay was used to detect the mRNA levels of E2F1 downstream target genes in MCF7 and T‐47D cells treated with or without palbociclib following stable transfection with either the control or XBP1s ‐overexpressing plasmid. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. For statistical analysis, two‐tailed unpaired Student's t ‐test was used for (D); one‐way ANOVA with Turkey's post hoc test was utilized for (H). Data are presented as the mean ± standard deviation.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: Activation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Stable Transfection, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: 4µ8C sensitizes HR+/HER2− breast cancer to the combination therapy of fulvestrant and palbociclib in PDO models. A) Dose–response matrix (relative cell viability) and combination index (CI) for the effect of palbociclib (left) or fulvestrant (right) combined with 4µ8C in PDO‐0912. B) Dose–response matrix (relative cell viability) and CI for the effect of palbociclib (left) or fulvestrant (right) combined with 4µ8C in PDO‐3890. C) Acridine orange/propidium iodide (AO/PI) staining was performed on HR+/HER2− patient‐derived organoids PDO‐0912 and PDO‐3890 following treatment, as indicated. The left panel shows the representative images of live/dead organoids 96 h after the indicated treatment; the right panel shows the quantification results of live (green)/dead (red) analysis. PDO‐0912 was subjected to the vehicle, 10 × 10 −6 m 4µ8C, a combination of 5 × 10 −6 m palbociclib and 0.5 × 10 −6 m fulvestrant (Pal+Ful), or their combination with 10 × 10 −6 m 4µ8C (Pal+Ful+4µ8C); PDO‐3890 was subjected to the vehicle, 10 × 10 −6 m 4µ8C, a combination of 14 × 10 −6 m palbociclib and 1 × 10 −6 m fulvestrant (Pal+Ful), or their combination with 10 × 10 −6 m 4µ8C (Pal+Ful+4µ8C). Scale bar, 50 µm. D,E) Following transfection with the control or XBP1s ‐overexpressing plasmid, cell viability assay was performed to determine the organoid growth following treatment with the vehicle and Pal+Ful for the indicated days in PDO‐0912 and PDO‐3890. * p < 0.05, ** p < 0.01. F) Cell viability assay was performed to determine the organoid growth following treatment with the vehicle, 4µ8C, Pal+Ful, or Pal+Ful+4µ8C for the indicated days in PDO‐1553 and PDO‐4576. G–I) Mice with MCF7 cell‐derived xenografts were randomly divided into four treatment groups: vehicle, 4µ8C, Pal+Ful, or Pal+Ful+4µ8C. G) Representative images of IHC staining for XBP1s, SND1, and E2F1 in MCF7 cell‐derived xenografts from different treatment groups. Scale bar, 50 µm. H) Tumor growth curve plots of the MCF7 tumor in each treatment group after treatment administration. n = 6 per treatment group. I) Images of MCF7 cell‐derived xenografts at the end of the experiment. n = 6 per treatment group. For statistical analysis, two‐way ANOVA with Bonferroni's method correction was applied in (D) and (E). Data are presented as the mean ± standard deviation.

Article Snippet: Then, the slides were incubated with primary antibodies against XBP1u (25997‐1‐AP, Proteintech), XBP1s (27901, Cell Signaling Technology), ER (21244‐1‐AP, Proteintech), PR (25871‐1‐AP, Proteintech), HER2 (18299‐1‐AP, Proteintech), Ki67 (27309‐1‐AP, Proteintech), SND1 (ab65078, Abcam), Phospho‐Rb S780 (ab173289, Abcam), or E2F1 (ab288369, Abcam) in a humidified at 4 °C overnight.

Techniques: Staining, Derivative Assay, Transfection, Control, Plasmid Preparation, Viability Assay, Immunohistochemistry, Standard Deviation