Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Citrulline Inhibits Clostridioides difficile Infection With Anti-inflammatory Effects

doi: 10.1016/j.jcmgh.2025.101474

Figure Lengend Snippet:

Article Snippet: One-way ANOVA was used, but no statistically significant difference was found. ( E–F ) Macrophages were transiently transfected with control siRNA (sc-37007) or HSP27 siRNA (sc-29350) from Santa Cruz Biotechnology overnight. ( E ) Total HSP27 ELISA.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Citrulline Inhibits Clostridioides difficile Infection With Anti-inflammatory Effects

doi: 10.1016/j.jcmgh.2025.101474

Figure Lengend Snippet: Citrulline promoted IL-10 expression with HSP27 phosphorylation in toxin B-treated macrophages. ( A–B ) Phospho-kinase array. Primary human macrophages were treated with or without citrulline for 30 minutes, followed by either PBS or toxin B for 2 hours. The cells were lysed, and the lysates were assayed with the protein Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R&D Systems). The images were captured by the Bio-Rad ChemiDoc Imaging system and analyzed by Bio-Rad Image Lab software. The cell lysates were collected for the protein arrays. Citrulline increased phosphorylated HSP27 levels in toxin B-treated macrophages. Results were pooled from 4 experiments (mean ± SD). One-way ANOVA was used. ( C ) Phosphorylated HSP27 levels. The fresh human colonic explants were pretreated with 10 μM citrulline for 30 minutes, followed by incubation with 0.1 mg/mL toxin B for 2 hours. The phosphorylated HSP27 levels were determined by ELISA. Results were pooled from 4 tissue donors (mean ± SD). One-way ANOVA was used. ( D ) Total HSP27 levels. The fresh human colonic explants were pretreated with 10 μM citrulline for 30 minutes, followed by 6 hours of incubation with 0.1 μg/mL toxin A or toxin B. Total HSP27 levels in conditioned media were measured by ELISA. Results were pooled from 4 tissue donors (mean ± SD). One-way ANOVA was used, but no statistically significant difference was found. ( E–F ) Macrophages were transiently transfected with control siRNA (sc-37007) or HSP27 siRNA (sc-29350) from Santa Cruz Biotechnology overnight. ( E ) Total HSP27 ELISA. After overnight transfection, the macrophages were incubated with serum-free RPMI1640 media for 24 hours. Secreted total HSP27 levels of transfected macrophages were measured by ELISA. Results were pooled from 4 experiments (mean ± SD). One-way ANOVA was used. ( F ) IL-10 ELISA. Macrophages (with or without siRNA transfection) were pretreated with citrulline or recombinant human HSP27 (1580-HS-050, R&D Systems) for 30 minutes and incubated with toxin B for 6 hours. IL-10 levels in conditioned media were detected by ELISA. Results were pooled from four experiments (mean ± SD). One-way ANOVA was used.

Article Snippet: One-way ANOVA was used, but no statistically significant difference was found. ( E–F ) Macrophages were transiently transfected with control siRNA (sc-37007) or HSP27 siRNA (sc-29350) from Santa Cruz Biotechnology overnight. ( E ) Total HSP27 ELISA.

Techniques: Expressing, Imaging, Software, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Control, Recombinant

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Citrulline Inhibits Clostridioides difficile Infection With Anti-inflammatory Effects

doi: 10.1016/j.jcmgh.2025.101474

Figure Lengend Snippet: Information on Fresh Human Colonic Tissues, Primary Macrophages, PBMCs, and Reagents

Article Snippet: One-way ANOVA was used, but no statistically significant difference was found. ( E–F ) Macrophages were transiently transfected with control siRNA (sc-37007) or HSP27 siRNA (sc-29350) from Santa Cruz Biotechnology overnight. ( E ) Total HSP27 ELISA.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

doi: 10.1186/bcr3042

Figure Lengend Snippet: Knockdown of Hsp27 decreased BCSCs of breast cancer cells . ( A ) AS-B145 cells were transfected with Hsp27 siRNA (si-Hsp27) or negative control siRNA (ctrl-siRNA) for 48 h and detected a ALDH+ population by ALDEFLUOR assay. The percentage of ALDH+ cells were calculated from samples without DEAB treatment (w/o DEAB) after gating with a cutoff line which was set according to DEAB treated (w/DEAB) cells. The quantitative results were presented as relative percentage of ctrl siRNA transfected cells. *, P < 0.05. ( B ) Primary (1 st ) mammosphere formation capacity of AS-B145 or AS-B244 cells was also analyzed after knockdown of Hsp27 for 48 h. For secondary (2 nd ) mammosphere cultivation, 1 st spheres were collected, digested with accutase and subjected to mammosphere cultivation at 48 h post-transfected with si-Hsp27 or negative control siRNA (ctrl-siRNA). Data were collected at Day 7 post starting the cultivation and presented as relative percentage of ctrl siRNA. Bar, 100 μm. #, P < .01; *, P < 0.05. ( C ) The tumorigenicity of AS-B145 sphere cells after knockdown of Hsp27 (si-Hsp27) or negative control siRNA (ctrl-siRNA) was determined by xenograftment assay in NOD/SCID mice. The indicated numbers of cells were injected into mammary fat pads for 44 days and the tumor formation was monitored weekly. The cancer stem cell (CSC) frequency was calculated with ELDA software . *, P < 0.05. ALDH, aldehyde dehydrogenase; BCSCs, breast cancer stem cells.

Article Snippet: The specific siRNA oligos of Hsp27 (sc-29350) or IκBα (sc-29360), or negative control siRNA oligos was purchased from Santa Cruz Biotechnologies, Inc.

Techniques: Transfection, Negative Control, Injection, Software

Journal: Breast Cancer Research : BCR

Article Title: Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

doi: 10.1186/bcr3042

Figure Lengend Snippet: Inhibition of Hsp27 suppressed EMT signature of BCSCs . ( A ) Cell migration ability of AS-B145, ALDH+ AS-B244, MDA-MB-231 or Sca-1+ 4T1 cells in present of quercetin (Q, 12.5, 25 or 50 μM) or 0.1% DMSO or was analyzed with wound-healing assay. *, P < 0.05; #, P < 0.01. ( B ) Cell migration ability of AS-B145, MDA-MB-231 or ALDH+ AS-B244 cells after transfection of Hsp27 siRNA (si-Hsp27) or negative control siRNA (ctrl siRNA) was analyzed with wound-healing assay. *, P < 0.05; #, P < 0.01. ( C ) EMT related proteins (E-cadherin, vimentin or snail) of AS-B145 or ALDH+ AS-B244 cells were analyzed by Western blot. Inserted values indicated relative expression of proteins in negative control siRNA (ctrl) or si-Hsp27 (KD) transfected cells. ALDH: aldehyde dehydrogenase; BCSCs: breast cancer stem cells; EMT, epithelial-mesenchymal transition.

Article Snippet: The specific siRNA oligos of Hsp27 (sc-29350) or IκBα (sc-29360), or negative control siRNA oligos was purchased from Santa Cruz Biotechnologies, Inc.

Techniques: Inhibition, Migration, Wound Healing Assay, Transfection, Negative Control, Western Blot, Expressing

Journal: Breast Cancer Research : BCR

Article Title: Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

doi: 10.1186/bcr3042

Figure Lengend Snippet: Knockdown of Hsp27 decreased the activity of NF-κB in BCSCs . ( A ) AS-B145 or ALDH+ AS-B244 cells were transfected with negative control siRNA (Ctrl) or Hsp27 siRNA (KD) for 24 h or 48 h and total proteins were harvested to detect the expression of IκBα, phosphor-IκBα (p-IκBα), Hsp27 and β-actin by Western blot. Inserted values indicated relative proteins expression in comparison with ctrl siRNA. ( B ) AS-B145 or ALDH+ AS-B244 cells were transfected with negative control siRNA (Ctrl) or Hsp27 siRNA (KD) for 48 h and nuclear (Nuc) and cytosolic (Cyto) proteins were isolated to detect NF-κB, histone H1, Hsp27 and β-actin by Western blot. Inserted values indicated relative proteins expression in comparison with ctrl siRNA. ( C ) AS-B145 or ALDH+ AS-B244 cells were co-transfected with NF-κB reporter vector, Renilla luciferase vector and negative control siRNA (ctrl-si)/or Hsp27 siRNA (si-Hsp27) for 48 h. Luciferase activity was determined by dual luciferase assay. Data were presented as relative luciferase activity of ctrl siRNA. *, P < 0.05. ( D ) AS-B145 or AS-B244 cells were treated with JSH-23 (20, 10 or 5 μM) or 0.1% DMSO for 48 h and ALDH+ population were determined by ALDEFLUOR assay. Data were presented as relative percentage of DMSO. *, P < 0.05; #, P < 0.01. ALDH, aldehyde dehydrogenase; BCSCs, breast cancer stem cells; NF-κB, nuclear factor kappa B

Article Snippet: The specific siRNA oligos of Hsp27 (sc-29350) or IκBα (sc-29360), or negative control siRNA oligos was purchased from Santa Cruz Biotechnologies, Inc.

Techniques: Activity Assay, Transfection, Negative Control, Expressing, Western Blot, Isolation, Plasmid Preparation, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

doi: 10.1186/bcr3042

Figure Lengend Snippet: Restoring NF-κB activity attenuates the suppressive effect of Hsp27 knockdown in ALDH+ breast cancer cells . AS-B145 or AS-B244 cells were transfected with negative control siRNA (Ctrl-siRNA, 100 nM), ctrl-siRNA (50 nM)+si-Hsp27 (50 nM) or si-Hsp27 (50 nM) +si-IκBα (50 nM) for 48 h. ( A ) The expression of Hsp27 and IκBα was determined by Western blot. Inserted values indicated relative expression in comparison with ctrl-siRNA. ( B ) The NF-κB activity was determined by co-transfected with NF-κB-Luc reporter vector (0.5 μg) and RLuc (0.05 μg) vector. The results were presented as relative luciferase activity to ctrl-siRNA. ( C, D ) The percentage of ALDH+ population among groups was determined by ALDEFLUOR assay. DEAB was used to set the cutoff line for ALDH+ cells. The quantitative results were presented as relative percentage of negative control siRNA (ctrl-siRNA) group and were shown in bar graph (D). *, P < 0.05; #, P < 0.01. ALDH, aldehyde dehydrogenase; NF-κB, nuclear factor kappa B.

Article Snippet: The specific siRNA oligos of Hsp27 (sc-29350) or IκBα (sc-29360), or negative control siRNA oligos was purchased from Santa Cruz Biotechnologies, Inc.

Techniques: Activity Assay, Transfection, Negative Control, Expressing, Western Blot, Plasmid Preparation, Luciferase