Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: STK33 promotes TNBC cell proliferation by increasing the protein stability of CCAR1. A) The proteins immunoprecipitated by anti‐Flag antibody were analyzed by mass spectrometry, and the number of peptides for each protein identified was listed. B) 293T cells were transfected with Flag‐STK33 and HA‐CCAR1 plasmids, and then subjected to immunoprecipitation with anti‐Flag or anti‐HA antibodies. The lysates and immunoprecipitates were then blotted. C) MDA‐MB‐231 cells were transfected with an HA‐CCAR1 plasmid, and immunofluorescence staining was performed to detect the localization of HA (red) and STK33 (green) within the cells. D) The expression of STK33 and CCAR1 in MDA‐MB‐231 xenograft were measured by western blot. E) MDA‐MB‐231 and HCC1806 cells were transfected with STK33 siRNA or non‐targeting siRNA, and the expression of STK33 and CCAR1 was measured by western blot. F) 293T cells were transfected with Flag‐STK33 and HA‐CCAR1, and then subjected to immunoprecipitation with anti‐HA antibody, and the phosphorylation of CCAR1 was measured by western blot using p‐Ser/Thr antibody. G) MDA‐MB‐231 and HCC1806 cells were transfected with STK33 siRNA, followed by treatment with 10 µ m MG132 for 4h before harvest. The expression of STK33 and CCAR1 was measured by western blot. H) MDA‐MB‐231 cells were transfected with STK33 siRNA or non‐targeting siRNA, and followed treated with 10µg mL −1 of cycloheximide (CHX) and harvested at the indicated time points. The protein levels of CCAR1 and STK33 were detected by western blot. I) 293T cells were transfected with HA‐CCAR1, MYC‐Ub, or Flag‐STK33 plasmids, followed by treatment with MG132 (10 µ m ) for 10 h before harvest. Then the cell lysates were subjected to immunoprecipitation with anti‐HA antibody and blotted with anti‐MYC antibody. J) MDA‐MB‐231 cells were transfected with STK33 siRNA, and then transfected with HA‐CCAR1, the expression of STK33 and HA was measured by western blot. K) MDA‐MB‐231 cells were transfected with STK33 siRNA, and then transfected with HA‐CCAR1, and cell proliferation was measured by the colony formation assay. The data are presented as mean ± SD of three independent experiments. One‐way ANOVA was used to determine statistical significance, *** P < 0.001, **** P < 0.0001.

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Immunoprecipitation, Mass Spectrometry, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Expressing, Western Blot, Phospho-proteomics, Colony Assay

Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: High expression of the STK33‐CCAR1 axis is correlated with poor survival in patients with TNBC. A) Representative IHC staining for STK33 and CCAR1 in TNBC. Cases 1 and 2 are representative of a patient with STK33‐high TNBC. Cases 3 and 4 are representative of a patient with STK33‐low TNBC. B, C) Pearson's correlation analyses of STK33 and CCAR1. D) IHC analyses of STK33 and CCAR1 levels in breast cancer tissues from the Human Protein Atlas database (https://www.proteinatlas.org/). E) Pearson's correlation analyses of STK33 and CCAR1 mRNA levels in liver hepatocellular carcinoma from the TIMER web server (https://cistrome.shinyapps.io/timer/). F) Pearson's correlation analyses of STK33 and CCAR1 mRNA levels in diffuse large B‐cell lymphoma from the TIMER web server (https://cistrome.shinyapps.io/timer/). G) The correlation between STK33 expression and the histology grade in TNBC patients, the data are presented as mean ± SD, one‐way ANOVA was used to determine statistical significance, P < 0.05 was considered to be statistically significant. H) The correlation between CCAR1 expression and the histology grade in TNBC patients, the data are presented as mean ± SD, one‐way ANOVA was used to determine statistical significance, P < 0.05 was considered to be statistically significant. I) Kaplan–Meier plots of the overall survival based on CCAR1 expression in TNBC patients. J) Kaplan–Meier curves of overall survival based on STK33 and CCAR1 expression in TNBC patients.

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Expressing, Immunohistochemistry

Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: Bufalin exerts anti‐cancer activity in animal TNBC model and in patient‐derived TNBC organoids. 4‐week‐old female nude mice were inoculated with MDA‐MB‐231 cells. The tumor‐bearing mice were subsequently given the indicated treatment. A) Subcutaneous tumors were excised and photographed at the end of the experiment. B) Tumor sizes were measured on the specified days, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance; P < 0.05 was considered to be statistically significant. C) Tumor weights were measured at the end of the experiments, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance; P < 0.05 was considered to be statistically significant. D) Representative immunohistostaining images for detecting Ki67 expression in the tumor specimens. E) Representative immunohistostaining images for detecting STK33 expression in the tumor specimens. F) Western blot analysis of the STK33 and CCAR1 protein expression in xenograft tumors following the indicated treatment. G) Mice liver functions were measured at the end of the experiments, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance, ns, P > 0.05. ALT, alanine aminotransferase; AST, aspartate aminotransferase. H) Mice kidney functions were measured at the end of the experiments, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance, ns, P > 0.05. BUN, blood urea nitrogen. I) Representative images of TNBC patient‐derived organoids (Scale bar 50µm). J–L) The proliferation curve of TNBC PDOs treated with Bufalin, the data are presented as mean ± SD of three independent experiments. One‐way ANOVA was used to determine statistical significance; P < 0.05 was considered to be statistically significant. M) Spearman correlation analysis between STK33 expression and the IC 50 of TNBC PDOs.

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Activity Assay, Derivative Assay, Expressing, Western Blot

Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: Regulatory signaling pathway of Bufalin in TNBC. In this study, we identified STK33 as a putative target of Bufalin. Bufalin disrupts the interaction between STK33 and HSP90, thereby promoting the ubiquitination and proteasomal degradation of STK33. Furthermore, STK33 is highly expressed in TNBC and enhances TNBC cell proliferation by phosphorylating and stabilizing CCAR1. Targeted degradation of STK33 by Bufalin significantly inhibits TNBC growth, highlighting its potential as a promising therapeutic candidate for TNBC treatment. Created in BioRender. Jiang, S. (2025) https://BioRender.com/00nde1g .

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Ubiquitin Proteomics

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: The mRNA expression of PIP4K2C in pan-cancer. (A) The mRNA expression of PIP4K2C in 33 tumors in TCGA GTEx samples (ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001). (B) PIP4K2C expression in the breast cancer tissues and unpaired normal samples. (C) The expression level of PIP4K2C in the breast cancer tissues and the paired normal samples. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical and endocervical cancers; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; STES, stomach and esophageal carcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Expressing

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: mRNA expression and protein levels of PIP4K2C in breast cancer cell lines and tissues. (A) The expression level of PIP4K2C in the normal mammary gland cell line MCF-10A and breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF7, ZR751 and BT20) was determined using qPCR. * P < 0.05, *** P < 0.01 vs MCF-10A. (B) The protein levels of PIP4K2C in cell lines were measured by western blot. (C) The expression level of PIP4K2C in the breast cancer tissues and the paired normal samples. (D) The protein levels of PIP4K2C in the breast cancer tissues and the paired normal samples. (E) The immunofluorescence staining of the breast cancer tissues and the paired normal samples. (F) The IHC images of PIP4K2C in normal and tumor tissues.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: PIP4K2C was knocked down by siRNA. (A-B) The transfection efficiency of MDA-MB-468 was detected at mRNA expression and protein levels, respectively. (C-D) The transfection efficiency of MCF7 was detected at mRNA expression and protein levels (48 h), respectively.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Transfection, Expressing

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: PIP4K2C was overexpressed in MCF 10A by transfection. (A) The transfection efficiency was detected at mRNA expression. (B) overexpression of PIP4K2C resulted in increased proliferation.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Transfection, Expressing, Over Expression

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: Inhibition of PIP4K2C suppressed the proliferation, migration and invasion of MDA-MB-468 and MCF7 cells. (A) Knockdown of PIP4K2C by siRNA resulted in reduced proliferation. (B) The reduced cell migration rate was evaluated by wound healing assay. The percentage of wound closure at 24 and 48 h was calculated using ImageJ based on the change in scratch area from time 0 h. (C-D) The cell migration and invasion ability were detected by transwell assay.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Inhibition, Migration, Knockdown, Wound Healing Assay, Transwell Assay

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: Down-regulation of PIP4K2C enhanced the protein levels of LC3II/LC3I.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques:

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 5. Tight junction protein expression in subcutaneous adipose tissue. (A) Immunofluorescence staining of tight junction proteins (red) claudin-5, occludin, and JAM-1, nuclear staining with DAPI (blue), endothelial marker CD31 (green) in subcutaneous tissue of kidney transplant recipients and donors. Bar = 100 µm. Expression of tight junction proteins (B) claudin-5, (C) occludin and (D) JAM-1 in subcutaneous tissue of kidney transplant recipients and donors. Results expressed in medians and interquartile range. Statistical significance *p < 0.05, **p < 0.01.

Article Snippet: Primary antibodies used were claudin-5 (1:200, CSB-PA005507LA01HU, Cusabio Technology, USA), occludin (1:200, CSB-PA016263LA01HU, Cusabio Technology), and JAM-1 (1:400, CSB-PA897579LA01HU, Cusabio Technology), and incubated overnight at 4 °C in 3% Triton and 5% goat serum in 1X PBS.

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 6. TMAO incubation and tight junction protein expression in subcutaneous adipose tissue. (A) Immunofluorescence staining of tight junction proteins (red) claudin-5, occludin, and JAM-1, nuclear staining with DAPI (blue), endothelial marker CD31 (green) in subcutaneous tissue incubated with (A) TMAO and (B) control media. Bar = 100 µm. Expression of tight junction proteins (B) claudin-5, (C) occludin and (D) JAM-1 in subcutaneous tissue after incubation with TMAO and control media. Results expressed in medians and interquartile range. Statistical significance *p < 0.05, **p < 0.01.

Article Snippet: Primary antibodies used were claudin-5 (1:200, CSB-PA005507LA01HU, Cusabio Technology, USA), occludin (1:200, CSB-PA016263LA01HU, Cusabio Technology), and JAM-1 (1:400, CSB-PA897579LA01HU, Cusabio Technology), and incubated overnight at 4 °C in 3% Triton and 5% goat serum in 1X PBS.

Techniques: Incubation, Expressing, Immunofluorescence, Staining, Marker, Control

Journal: Journal of translational autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells.

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Fig. 3. Immunofluorescent antibody staining in naïve and treated HUVECs. AK. The mixture of APS total IgG and β2GPI induces a pronounced increase of the protein levels of the proinflammatory cytokines IL-6, IL-8 as well the transcription factor NF-κB1 and cell adhesion molecules Tissue Factor, ICAM-1, VCAM-1, Eselectin, P- selectin and TGFR1. There was no significant difference for the TGFR1 molecule (3 J). Visual analysis revealed that all the inflammatory mediators and adhesion molecules presented statistically significant difference between the untreated and treated endothelial cells (3 K).

Article Snippet: Coverslips were incubated overnight at 4 ◦C with primary antibodies against IL-6 (5 μg/ ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSBPA023451LA01HU, Cusabio).

Techniques: Staining

Journal: Journal of translational autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells.

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Fig. 5. Immunofluorescent antibody staining in placenta biopsies from APS patients and healthy women. A-K Placenta biopsies derived from APS patients as well as Healthy Donors show increased signal intensity for IL-6, IL-8, NF-κB1, ICAM1, VCAM-1, E-selectin, P-selectin, TGF-β2, and TGFR1 (5A-5D, 5F-5J). Slight difference in fluorescence intensity between HD and APS patient was observed for Tissue Factor (5E). Increased signal intensity was observed as well for the TNF-α molecule in the APS placenta biopsies (5 K).

Article Snippet: Coverslips were incubated overnight at 4 ◦C with primary antibodies against IL-6 (5 μg/ ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSBPA023451LA01HU, Cusabio).

Techniques: Staining, Derivative Assay, Fluorescence