Journal: Current Therapeutic Research, Clinical and Experimental

Article Title: The Berberine Derivative BBR684 Inhibits VDAC Oligomerization to Suppress Ferroptosis in Acute Kidney Injury

doi: 10.1016/j.curtheres.2026.100825

Figure Lengend Snippet: BBR684 attenuates Erastin-induced ferroptosis in HK-2 cells by suppressing lipid peroxidation and activating the antioxidant response. (A) Flow cytometric analysis of lipid peroxidation using the C11-BODIPY probe in HK-2 cells treated as indicated for 12 hours. Data are presented as mean fluorescence intensity (MFI). (B) Intracellular Fe²⁺ levels measured by a ferrous iron colorimetric assay. (C, D) Levels of malondialdehyde (MDA, C) and reduced glutathione (GSH, D) in cell lysates. (E, F) Representative immunofluorescence images (E) and quantification (F) of 4-hydroxynonenal (4-HNE) adducts (red) in HK-2 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. (G) Western blot analysis of key ferroptosis-related proteins: glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and nuclear factor erythroid 2–related factor 2 (NRF2). β-actin served as the loading control. Data are from 3 independent biological replicates (n = 3) and presented as mean ± SEM. Significance was determined by one-way ANOVA followed by Tukey's HSD post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001; n.s., not significant.

Article Snippet: Antibodies against GPX4 (Catalog #3F5G5), HO-1 (Catalog #10701-1-AP), NRF2 (Catalog #16396-1-AP), VDAC (Catalog #10866-1-AP), Cleaved Caspase-3 (Catalog #68773-1-Ig), and CD45 (Catalog #98035-1-RR) were purchased from Proteintech.

Techniques: Fluorescence, Colorimetric Assay, Immunofluorescence, Western Blot, Control

Journal: Current Therapeutic Research, Clinical and Experimental

Article Title: The Berberine Derivative BBR684 Inhibits VDAC Oligomerization to Suppress Ferroptosis in Acute Kidney Injury

doi: 10.1016/j.curtheres.2026.100825

Figure Lengend Snippet: BBR684 attenuates Erastin-induced ferroptosis in HK-2 cells by suppressing lipid peroxidation and activating the antioxidant response. (A) Flow cytometric analysis of lipid peroxidation using the C11-BODIPY probe in HK-2 cells treated as indicated for 12 hours. Data are presented as mean fluorescence intensity (MFI). (B) Intracellular Fe²⁺ levels measured by a ferrous iron colorimetric assay. (C, D) Levels of malondialdehyde (MDA, C) and reduced glutathione (GSH, D) in cell lysates. (E, F) Representative immunofluorescence images (E) and quantification (F) of 4-hydroxynonenal (4-HNE) adducts (red) in HK-2 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. (G) Western blot analysis of key ferroptosis-related proteins: glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and nuclear factor erythroid 2–related factor 2 (NRF2). β-actin served as the loading control. Data are from 3 independent biological replicates (n = 3) and presented as mean ± SEM. Significance was determined by one-way ANOVA followed by Tukey's HSD post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001; n.s., not significant.

Article Snippet: Antibodies against GPX4 (Catalog #3F5G5), HO-1 (Catalog #10701-1-AP), NRF2 (Catalog #16396-1-AP), VDAC (Catalog #10866-1-AP), Cleaved Caspase-3 (Catalog #68773-1-Ig), and CD45 (Catalog #98035-1-RR) were purchased from Proteintech.

Techniques: Fluorescence, Colorimetric Assay, Immunofluorescence, Western Blot, Control

Journal: Xenotransplantation

Article Title: CMAH‐Targeted Knock‐In of Inducible Heme Oxygenase‐1 and Constitutive CD47 in GGTA1‐Knockout Pigs for Xenotransplantation

doi: 10.1111/xen.70124

Figure Lengend Snippet: Stimulus‐responsive expression of HO1 and CD47 in transduced HEK‐293T cells under xenogeneic and inflammatory stimuli. (A) Schematic diagram of the dual‐promoter expression construct: HO1 cDNA under the human HO1 promoter and CD47 cDNA under the pig EF1α promoter. (B) Western blot analysis of HO1 and CD47 protein expression in HEK‐293T cells transduced with the construct in (A) and treated with control medium, human serum, or PMA. (C) qPCR showing time‐dependent mRNA expression levels of HO1 and CD47 in HEK‐293T cells transduced with the construct in (A) and subjected to the same treatments. HO1 exhibited strong inducibility, while CD47 , already expressed at high baseline levels, was further upregulated following stimulation. Data are presented as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001). HO1, heme oxygenase‐1; PMA, phorbol 12‐myristate 13‐acetate.

Article Snippet: Thereafter, the sections were incubated overnight at 4°C with a primary antibody against HO‐1 (sc‐136960; Santa Cruz Biotechnology) at a 1:200 dilution.

Techniques: Expressing, Construct, Western Blot, Transduction, Control

Journal: Xenotransplantation

Article Title: CMAH‐Targeted Knock‐In of Inducible Heme Oxygenase‐1 and Constitutive CD47 in GGTA1‐Knockout Pigs for Xenotransplantation

doi: 10.1111/xen.70124

Figure Lengend Snippet: CRISPR/Cas9‐mediated knock‐in of HO1/CD47 expression cassette into pig CMAH locus and functional evaluation of knock‐in cell clones. (A) Knock‐in strategy and PCR‐based genotyping scheme for integrating the HO1/CD47 expression cassette into exon 4 of the pig CMAH locus using CRISPR/Cas9‐mediated homology‐directed repair. The diagram illustrates the structure of the inserted cassette and the location of PCR primer pairs (HO1‐F/CD47‐R and CMAH‐F/HO1‐R) used for genotyping. (B) PCR‐based genotyping analysis of donor cell clones confirmed the targeted integration of the HO1/CD47 expression cassette into the CMAH locus. The presence of the cassette was verified by amplification of its internal region (HO1‐F/CD47‐R), and site‐specific integration at the 5′ junction with the CMAH gene was confirmed using the CMAH‐F/HO1‐R primer pair. Multiple clones showed positive bands for both primer sets. The expected amplicon sizes were 1763 bp (HO1‐F/CD47‐R) and 949 bp (CMAH‐F/HO1‐R). (C) Analysis of cytotoxicity in GTKO/CMAH‐KO/HO1/CD47 knock‐in fibroblast clones following exposure to 10% human serum. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001. CMAHKO, CMAH knockout; GTKO , GGTA1 ‐knockout; HO1, heme oxygenase‐1; SD, standard deviation.

Article Snippet: Thereafter, the sections were incubated overnight at 4°C with a primary antibody against HO‐1 (sc‐136960; Santa Cruz Biotechnology) at a 1:200 dilution.

Techniques: CRISPR, Knock-In, Expressing, Functional Assay, Clone Assay, Amplification, Knock-Out, Standard Deviation

Journal: Xenotransplantation

Article Title: CMAH‐Targeted Knock‐In of Inducible Heme Oxygenase‐1 and Constitutive CD47 in GGTA1‐Knockout Pigs for Xenotransplantation

doi: 10.1111/xen.70124

Figure Lengend Snippet: Generation and genotyping of a GTKO/CMAH‐KI (HO1/CD47)/+ cloned piglet. (A) Image of the cloned piglet produced using knock‐in clone #229. (B) PCR analysis confirming HO1/CD47 knock‐in and GGTA 1‐knockout. Primer information is detailed in Section and Figure . Lanes 1 and 4 show GGTA1 ‐knockout. Lanes 2 and 5 indicate the presence of the HO1/CD47 cassette. Lanes 3 and 6 show the 5′ junction between the CMAH locus and the HO1/CD47 cassette. HO1, heme oxygenase‐1.

Article Snippet: Thereafter, the sections were incubated overnight at 4°C with a primary antibody against HO‐1 (sc‐136960; Santa Cruz Biotechnology) at a 1:200 dilution.

Techniques: Clone Assay, Produced, Knock-In, Knock-Out

Journal: Xenotransplantation

Article Title: CMAH‐Targeted Knock‐In of Inducible Heme Oxygenase‐1 and Constitutive CD47 in GGTA1‐Knockout Pigs for Xenotransplantation

doi: 10.1111/xen.70124

Figure Lengend Snippet: Western blot analysis of HO1 and CD47 expression in tissues of GTKO/CMAH‐KI (HO1/CD47)/+ cloned piglet. Protein lysates were prepared from six tissues (heart, liver, lung, spleen, kidney, and aorta) of GTKO/ CMAH/HO1/CD47/+ cloned pigs and analyzed using Western blotting. HO1 expression (top panel) was predominantly detected in the liver, lung, and spleen, whereas CD47 (middle panel) was strongly expressed across all tissues. Vinculin was used as the loading control (bottom panel). HO1, heme oxygenase‐1.

Article Snippet: Thereafter, the sections were incubated overnight at 4°C with a primary antibody against HO‐1 (sc‐136960; Santa Cruz Biotechnology) at a 1:200 dilution.

Techniques: Western Blot, Expressing, Clone Assay, Control

Journal: Xenotransplantation

Article Title: CMAH‐Targeted Knock‐In of Inducible Heme Oxygenase‐1 and Constitutive CD47 in GGTA1‐Knockout Pigs for Xenotransplantation

doi: 10.1111/xen.70124

Figure Lengend Snippet: Flow cytometric analysis of human CD47 expression in pEFs and PBMCs from GTKO and GTKO/CMAH‐KI (HO1/CD47)/+ pigs. (A) Knock‐in pigs showed a substantial increase in CD47 + pEFs compared with those in GTKO pigs. (B) Both monocytes and lymphocytes from knock‐in pigs exhibited a marked increase in CD47 + cells compared with those in GTKO pigs. CD47 was stained with APC‐conjugated anti‐human CD47 antibody. Monocytes and lymphocytes were gated based on FSC/SSC profiles. HO1, heme oxygenase‐1; PBMCs, peripheral blood mononuclear cells; PEFs, pig ear fibroblasts.

Article Snippet: Thereafter, the sections were incubated overnight at 4°C with a primary antibody against HO‐1 (sc‐136960; Santa Cruz Biotechnology) at a 1:200 dilution.

Techniques: Expressing, Knock-In, Staining

Journal: Xenotransplantation

Article Title: CMAH‐Targeted Knock‐In of Inducible Heme Oxygenase‐1 and Constitutive CD47 in GGTA1‐Knockout Pigs for Xenotransplantation

doi: 10.1111/xen.70124

Figure Lengend Snippet: Immunohistochemical detection of HO1 protein in tissues from GTKO control and GTKO/CMAH‐KI (HO1/CD47)/+ pigs. Tissue sections from the heart, liver, lungs, and spleen were analyzed using immunohistochemistry to assess HO1 protein expression. The upper row shows sections from knock‐in pigs, while the lower row shows the corresponding tissues from GTKO control pigs. Positive HO1 staining (brown) was observed in the liver and lungs of transgenic pigs, consistent with inflammation‐inducible promoter activity, whereas no signal was detected in the heart and spleen or any tissue from GTKO controls. Tissues were obtained from the neonatal knock‐in piglet (#2) that died at 3 days of age; therefore, the data represent a single biological specimen. All sections were counterstained with hematoxylin. Scale bar, 50 µm; magnification, 40×. HO1, heme oxygenase‐1.

Article Snippet: Thereafter, the sections were incubated overnight at 4°C with a primary antibody against HO‐1 (sc‐136960; Santa Cruz Biotechnology) at a 1:200 dilution.

Techniques: Immunohistochemical staining, Control, Immunohistochemistry, Expressing, Knock-In, Staining, Transgenic Assay, Activity Assay