Journal: The American Journal of Pathology

Article Title: A Protein C Deficiency Exacerbates Inflammatory and Hypotensive Responses in Mice During Polymicrobial Sepsis in a Cecal Ligation and Puncture Model

doi:

Figure Lengend Snippet: Plasma coagulation factor assays of WT and PC+/− mice after CLP surgery. a: Plasma PC levels. The plasma PC levels were determined by ELISA before (WT, n = 4; PC+/−, n = 5), and 6 hours (WT, n = 5; PC+/−, n = 7) and 24 hours (WT, n = 5; PC+/−, n = 5) after CLP surgery in WT and PC+/− male mice. *, P < 0.001; #, P < 0.01. b: Plasma fibrinogen levels were determined by a coagulometric assay at 6 hours (WT, n = 4; PC+/−, n = 5) and 24 hours (WT, n = 15; PC+/−, n = 14) after, CLP surgery and in resting (WT, n = 11; PC+/−, n = 9) WT and PC+/− male mice. *, P = 0.016. c: Plasma PAI-1 levels were determined by ELISA before (WT, n = 3; PC+/−, n = 4), and 6 hours (WT, n = 3; PC+/−, n = 7) and 24 hours (WT, n = 5; PC+/−, n = 4) after, CLP surgery in WT and PC+/− male mice. d: Plasma FXII levels relative to the resting WT group, as measured by a coagulometric assay, before (WT, n = 4; PC+/−, n = 3) and 24 hours after (WT, n = 5; PC+/−, n = 3), CLP surgery. *, P = 0.05 (Kruskal-Wallis). e: Plasma FXI levels relative to the resting WT group as measured by a coagulometric assay, before (WT, n = 7; PC+/−, n = 5) and 24 hours after (WT, n = 5; PC+/−, n = 3), CLP surgery. No significant differences were observed.

Article Snippet: After four washes with PBS/Tween-20, 100 μl of 4 μg/ml rabbit anti-mouse PAI-1 polyclonal antibody (Molecular Innovations) in PBS was added to the wells and incubated for 2 hours.

Techniques: Coagulation, Enzyme-linked Immunosorbent Assay

Journal: The American Journal of Pathology

Article Title: A Protein C Deficiency Exacerbates Inflammatory and Hypotensive Responses in Mice During Polymicrobial Sepsis in a Cecal Ligation and Puncture Model

doi:

Figure Lengend Snippet: Gene expression ratio to RPL-19 of PAI-1 (a), MPO (b), IL-1β (c), and IL-6 (d) relative to the WT resting group (n = 3) in liver, kidney, and lung, as determined by RT-PCR in WT mice 24 hours after CLP (n = 3), PC+/− resting mice (n = 3), and PC+/− mice 24 hours after CLP (n = 4). a: In liver, PC+/− mice 24 hours after CLP were different from PC+/− resting and WT resting groups (P = 0.025, K-W), in kidney, differences between groups were not significant (K-W, multicomparison test). b: In liver, PC+/− resting mice were different from PC+/− mice 24 hours after CLP and WT resting groups (P = 0.055); in kidney, PC+/− mice 24 hours after CLP were different from all of the other groups (P = 0.02, K-W); in lung, PC+/− resting mice were different from PC+/− and WT mice 24 hours after CLP (P = 0.08, K-W). c: In liver, no significant differences between groups were found; in kidney: PC+/− group 24 hours after CLP was different from WT and PC+/− resting groups (P = 0.04, K-W); in lung: the PC+/− group after CLP was not different form the PC+/− resting group (T-K); P = not significant (K-W). d: In liver, PC+/− mice 24 hours after CLP were different from the WT resting group (P = 0.11, K-W); in kidney, PC+/− mice 24 hours after CLP were different from the WT and PC+/− resting groups (P = 0.032, K-W); in lung: no significant differences between the groups were found (K-W).

Article Snippet: After four washes with PBS/Tween-20, 100 μl of 4 μg/ml rabbit anti-mouse PAI-1 polyclonal antibody (Molecular Innovations) in PBS was added to the wells and incubated for 2 hours.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Materials Today Bio

Article Title: Porcine pericardial decellularized matrix bilayer patch containing adipose stem cell-derived exosomes for the treatment of diabetic wounds

doi: 10.1016/j.mtbio.2024.101398

Figure Lengend Snippet: Fig. 10. The exosome-loaded bilayer decellularized pericardial patches promote the healing of diabetic refractory wounds by enhancing the activation of M2 macrophages in the wound and activating the cAMP signaling pathway. (A) On day 7, dual immunofluorescence staining for F4/80 (M0 marker), iNOS (M1 marker), and CD206 (M2 marker) was performed to assess the numbers of activated M1 and M2 macrophages in the wound and surrounding skin tissue, scale bar = 50 μm. (B) On day 7, RT-qPCR was used to detect the expression levels of M1 (cd86, il-6) and M2 (cd163, arg-1) macrophage-related genes in the wound tissue of each group. (C) On day 7, Western Blotting experiments were conducted to measure the expression levels of key proteins in the cAMP signaling pathway (Atp1a2, Calm4, and Cngb1) in the wound tissue of each group, with quantitative analysis of protein density, n = 3 (D). *, p < 0.05, **, p < 0.01, and ***, p < 0.001, compared with the PBS group.

Article Snippet: The primary antibodies used were as follows: Ki67 (13180-T48, Sino Biological Inc, China), 1:500; VEGF (ab32152, Abcam, USA), 1:2000; Atp1a2 (ab166888, Abcam, USA), 1:2000; Calm4 (CSB-PA004454GA01HU, Cusabio), 1:1000; Gngb1 (PA5-101543, Invitrogen), 1:1000; GAPDH (#2118S, CST), 1:1000.

Techniques: Activation Assay, Immunofluorescence, Staining, Marker, Quantitative RT-PCR, 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: 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