Journal: Pharmaceutical Biology

Article Title: Indirubin regulates M1/M2 polarization and inhibits ferroptosis in dextran sulfate sodium induced colitis and in cultured THP-1 cells

doi: 10.1080/13880209.2025.2568215

Figure Lengend Snippet: IDR inhibited ferroptosis in THP-1 mocytes and in THP-1 derived macrophages with M1- or M2- polarized status. (a–d) IDR inhibited RSL3 induced cell loss in THP-1 derived macrophages, including M0 (a), M1 (b), and M2 (c) polarized macrophages, as well as in THP-1 monocytes (d). The viable cell counts were determined with CCK-8 assay. Data were presented as Mean ± S.D. (n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the RSL3 group. (e) The representative plots of IDR inhibiting RSL3 induced cell death determined by PI staining with flow-cytometry analysis. The proportion of PI-positive cells (f) and the preserved cell counts normalized to count beads (g) were also shown in the histogram plots. Data were presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01. (h) Reactive oxygen species (ROS) levels were determined with DHCF-DA. Data were presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01. (i) Malondialdehyde (MDA) levels were determined. Data were presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01. (j) Ferrous iron (Fe 2+ ) levels were quantified using a fluorometric assay. Data were normalized to cell counts and presented as Mean ± S.D. (n = 3). # p < 0.05 compared to the NC group. *** p < 0.05 compared to the DSS group. (k) The effects of IDR treatments on the mRNA expression levels of ferroptosis-associated genes, including Gpx4, Acsl4, Nrf2, and Slc7a11. Data were normalized to the level of reference gene Gapdh and presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 compared to the RSL3 group. (l) The representative images of Western blotting analysis of ferroptosis-associated proteins, including 4-HNE, GPX4, NRF2, FTL, and GAPDH. (m) Column plots of 4-HNE, GPX4, NRF2 and FTL abundance after normalizing to GAPDH. Mean ± S.D. (n = 3). * p < 0.05 compared to the RSL3 group.

Article Snippet: A Cell Ferrous Iron (Fe2+) Assay Kit (Fluorometric) (HY-K0322, MedChemExpress LLC, China) was applied to quantify iron concentration in THP-1 cells according to the manufacturer’s manual.

Techniques: Derivative Assay, CCK-8 Assay, Staining, Flow Cytometry, Expressing, Western Blot

Journal: Pharmaceutical Biology

Article Title: Indirubin regulates M1/M2 polarization and inhibits ferroptosis in dextran sulfate sodium induced colitis and in cultured THP-1 cells

doi: 10.1080/13880209.2025.2568215

Figure Lengend Snippet: IDR augmented M2-polarization and inhibited ferroptosis in peritoneal macrophages. (a) The effects of IDR treatments on the surface staining of Arg-1 in M2-polarized mouse peritoneal macrophages were determined by flow-cytometry analysis. (b) Histogram plot of the proportion and MFI (mean fluorescence intensity) of Arg-1 positive cells. Data were presented a Mean ± S.D. (n = 3). ## p < 0.01 compared to the NC group. * p < 0.05 and ** p < 0.01 compared to the M2 group. (c) The viable cell counts were determined with CCK-8 assay. Data were presented as Mean ± S.D. (n = 5). ## p < 0.01 compared to the NC group. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the M2 group. (d) The representative images of cellular ferrous iron fluorescence. (e) Histogram plot of the MFI of ferrous iron. Data were presented as Mean ± S.D. (n = 5). ### p < 0.01 compared to the NC group. ** p < 0.01 and *** p < 0.001 compared to the RSL3 group.

Article Snippet: A Cell Ferrous Iron (Fe2+) Assay Kit (Fluorometric) (HY-K0322, MedChemExpress LLC, China) was applied to quantify iron concentration in THP-1 cells according to the manufacturer’s manual.

Techniques: Staining, Flow Cytometry, Fluorescence, CCK-8 Assay

Journal: Viruses

Article Title: Infectious Spleen and Kidney Necrosis Virus Triggers Ferroptosis in CPB Cells to Enhance Virus Replication.

doi: 10.3390/v17050713

Figure Lengend Snippet: Figure 3. ISKNV infection induces ferroptosis in CPB cells. (A) Transmission electron microscopy of CPB cells treated with DMSO (72 h), erastin (10 µmol/L, 72 h), and ISKNV (100 MOI, 24 h, 48 h, 72 h). Scale bars = 1 µm. (B) Analysis of Fe2+ levels in CPB cells after treatment with ISKNV (100 MOI) using the fluorescent probe FerroOrange by laser scanning confocal microscopy. Scale bars = 20 µm. (C) Quantitative analysis of the mean fluorescence intensity of (B) using Image J. (D) Analysis of intracellular ROS levels using DCFH-DA staining, and laser scanning confocal microscopy of CPB cells treated with ISKNV (100 MOI) for 24–72 h. Scale bars = 10 µm. (E) Quantitative analysis of the mean fluorescence intensity of (D) using Image J. (F–H) Detection of Fe2+, ROS, and MDA levels in cell lysates treated with ISKNV (100 MOI) for 24–72 h by microplate reader. * p < 0.05, ** p < 0.01, and *** p < 0.001, with p > 0.05 considered not significant (ns).

Article Snippet: The Fe2+ content of the cells was detected by laser scanning confocal microscopy and a microplate reader using the fluorescent probe FerroOrange (Elabscience, E-BC-F101, Wuhan, China).

Techniques: Infection, Transmission Assay, Electron Microscopy, Confocal Microscopy, Fluorescence, Staining

Journal: JCI Insight

Article Title: Epithelial HO-1 regulates iron availability and promotes colonic tumorigenesis in a context-dependent manner

doi: 10.1172/jci.insight.181032

Figure Lengend Snippet: ( A ) Representative photomicrographs of immunofluorescence staining for 8-hydroxy-2′-deoxyguanosine (8-OHdG) and Ki-67, using paraffin-embedded mouse colonic individual tumor sections from 4 control and 4 Hmox1 ΔIEC mice undergoing AOM-DSS (original magnification, ×10). ( B ) Quantification of 8-OHdG staining intensity in tumor tissue. ( C ) 8-OHdG staining intensity in adjacent nontumor colonic tissue from control and KO mice. ( D ) Percentage of Ki-67 + cells based on immunofluorescence staining. ( E ) Ki-67 H-score analysis from tumor sections. ( F ) Representative photomicrographs of immunofluorescence staining for 4-HNE and EpCAM using colon tumor sections (original magnification, ×10). ( G ) Quantification of 4-HNE from tumor sections taken from control and KO mouse tumors. ( H ) Whole tumor iron concentration measured by colorimetric assay. ( I ) 4-HNE staining intensity normalized to mean tumor iron levels per genotype. ( J ) Tumoroids derived from pooled tumors from 2 Hmox1 fl/fl mice (control A and B ) and 1 Hmox1 ΔIEC (KO) mice were exposed to hemin (100 μM) for 24 hours. Lipid peroxidation measured by flow cytometry for 4-HNE, with median fluorescence intensity (MFI) values normalized to the mean of each vehicle-treated group. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and *** *P < 0.0001 by unpaired, Student’s t tests ( B - E, G - I ) and ANOVA ( J ) with correction for multiple comparisons.

Article Snippet: The Iron Assay Kit (Colorimetric) from Novus Biologicals was used to measure total iron content.

Techniques: Immunofluorescence, Staining, Control, Concentration Assay, Colorimetric Assay, Derivative Assay, Flow Cytometry, Fluorescence