Journal: Discover Oncology

Article Title: Lipid metabolism-related genes correlate with immune microenvironment and regulate the efficacy of immunotherapy via ferroptosis in melanoma

doi: 10.1007/s12672-025-04163-x

Figure Lengend Snippet: Overexpression ACSL4 increases the therapeutic efficacy of anti-PD-1 antibody in vivo. (A) A schematic view of the treatment plan, (B) Images of isolated tumors from xenograft mice that received indicated treatment. Tumor volumes and weights in each group were calculated and displayed on the right. (C) Immunofluorescence staining of ferroptosis marker PTGS2 in isolated xenograft tumors with indicated treatment. Scale bar = 100 μm. Data represent the mean ± SEM of triplicates. P value was calculated by two-tailed Student’s t-test. ** P < 0.01, *** P < 0.001. n = 6

Article Snippet: The primary antibodies and dilutions for western blotting (WB) and immunofluorescence (IF) staining analysis are listed below: ACSL4 (22401-1-AP, Proteintech, Wuhan, China; 1:100 for IHC, 1:100 for IF and 1:5 000 for WB), ALOX5 (10021-1-Ig, Proteintech; 1:400 for IHC, and 1:500 for WB), PTGS2 (66351-1-Ig, Proteintech; 1:400 for IF), CD8α(ab22378,, Abcam; 1:300 for IF), ACTIN (66009-1-Ig, Proteintech; 1:10 000 for WB).

Techniques: Over Expression, Drug discovery, In Vivo, Isolation, Immunofluorescence, Staining, Marker, Two Tailed Test

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: IAV induces NLRP3-caspase-1 mediated pyroptosis in macrophages in a time-dependent manner. J774A.1 cells uninfected and infected with the PR8 virus for 6 h, 12 h or 24 h. ( A ) The LDH levels in the culture supernatants from dead cells were measured using ELISA. ( B , C ) The expression levels of NLRP3 were detected by Western blot using β-actin as an internal control. ( D , E ) Flow cytometric analysis of the rates of pyroptosis mediated by caspase-1 using the FAM-FLICA Caspase-1 Kit. ( F ) The IL-1β levels in the culture supernatants were measured using ELISA. ( G , H ) The expression levels of GSDMD-N were detected by Western blot using β-actin as an internal control. ( I ) Representative images of immunofluorescence staining for GSDMD-N were observed by confocal laser scanning microscope. The nuclei are shown in blue using DAPI staining; while GSDMD-N is shown in red and detected by antibody. The white arrows indicate the fluorescent hub formed by GSDMD-N aggregation. ( J ) Bar diagram of the quantitative summary for GSDMD-N immunofluorescence. Around sixty cells in each group from three graphs were counted. * p < 0.05, ** p < 0.01 vs. the mock group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Infection, Virus, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Immunofluorescence, Staining, Laser-Scanning Microscopy

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: BBR inhibited GSDMD-mediated pyroptosis and cytokine release in macrophages infected with IAV. ( A ) The IL-1β and ( B ) TNF-α release levels in the culture supernatants from J774A.1 cells uninfected, infected with PR8 for 24 h, and infected with PR8 combined with BBR treatment at concentrations of 4.2, 8.4, and 16.8 μM were measured using ELISA. ( C ) Immunofluorescence staining for GSDMD-N was observed by confocal laser scanning microscope. J774A.1 cells were treated with the PR8 virus while BBR was at a concentration of 16.8 μM for 24 h. The nuclei are shown in blue using DAPI staining; GSDMD-N is shown in red and detected by antibody. The white arrows indicate the fluorescent hub formed by GSDMD-N aggregation. ( D ) Bar diagram of the quantitative summary for GSDMD-N immunofluorescence. Around sixty cells in each group from three graphs were counted. ** p < 0.01 vs. the mock group. # p < 0.05, ## p < 0.01 vs. the PR8 group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Laser-Scanning Microscopy, Virus, Concentration Assay

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: BBR attenuates NLRP3 inflammasome activation to reduce macrophage pyroptosis triggered by IAV. J774A.1 cells were infected with the PR8 virus for 24 h in the presence of BBR (16.8 µM) treatment. As a positive control, cells were pretreated with the NLRP3 inhibitor MCC950 (50 μM) for 3 h and then infected with the PR8 virus for an additional 24 h. ( A ) The LDH levels in the culture supernatants from the dead cells were measured using ELISA. ( B , C ) The expression levels of NLRP3 were detected by Western blot using β-actin as an internal control. ( D ) Immunofluorescence staining for the NLRP3 inflammasome was observed by confocal laser scanning microscope. NLRP3 and ASC are shown in green and blue, detected by antibody, respectively, while the mitochondria are shown in red using Mito Tracker staining. The white arrows represent regions with ASC expression. ( E ) Bar diagram of the quantitative summary for the NLRP3 immunofluorescence. ( F ) Bar diagram of the quantitative summary for ASC immunofluorescence. Around sixty cells in each group from three graphs were counted. ( G , H ) Flow cytometric analysis of the rates of pyroptosis mediated by caspase-1 using the FAM-FLICA Caspase-1 Kit. ( I ) The IL-1β levels in the culture supernatants were measured using ELISA. ( J , K ) The expression levels of GSDMD-N were detected by Western blot using β-actin as an internal control. * p < 0.05, ** p < 0.01 vs. the mock group. # p < 0.05, ## p < 0.01 vs. the PR8 group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Activation Assay, Infection, Virus, Positive Control, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Immunofluorescence, Staining, Laser-Scanning Microscopy

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: BBR suppresses mitochondrial ROS release in macrophages infected with IAV. ( A ) Immunofluorescence staining for MAVS was observed by confocal laser scanning microscope. MAVS and NLRP3 are shown in green and blue, detected by antibody, respectively, while the mitochondria are shown in red using Mito Tracker staining. The white arrows represent regions with higher NLRP3 expression. ( B ) Bar diagram of the quantitative summary for NLRP3 immunofluorescence. ( C ) Bar diagram of the quantitative summary for MAVS immunofluorescence. ( D ) Immunofluorescence staining for mtROS was observed by confocal laser scanning microscope. The ROS are shown in green marked by DCFH-DA, while the mitochondria are shown in red using Mito Tracker staining. The white arrows represent regions with higher NLRP3expression. ( E ) Bar diagram of the quantitative summary for ROS immunofluorescence. ( F , G ) Detection of the mtROS levels by flow cytometry with DCFH-DA. * p < 0.05, ** p < 0.01 vs. the mock group. ## p < 0.01 vs. the PR8 group. △△ p < 0.01 vs. the PR8 + NAC group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Infection, Immunofluorescence, Staining, Laser-Scanning Microscopy, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

doi: 10.1101/2025.03.29.646044

Figure Lengend Snippet: (a) (left) Schematic of modified Takasato protocol for iPSC-derived kidney organoids. (right) Brightfield images of DD10, DD12 and DD14 organoids. Scale bar: 500 microns. (b) Human kidney immunofluorescence stains for proximal, medial, and distal domain markers at the PTA, RV and SSB stages. Corresponding stage-matched kidney organoids at DD10, DD12 and DD14 are stained for the same markers. Yellow arrows denote polarized nephrons. Scale bar: 50 microns. (c) Bar graph showing percentage of total nephrons per organoid symmetrical vs asymmetrical WT1 expression on DD10, DD12 and DD14. Numbers above graph denote average nephroids counted per organoid (n = 2). (d) Schematic of kidney organoid differentiation trajectory from DD10-DD28. (e) Immunofluorescence stains of human kidneys at the RV, CSB, SSB and CLSN stages showing transcription factors marking the distal nephron domains. The SSB includes labeled positional relationships of precursors of the CNT (pCNT), DCT (pDT), loop of Henle (pLOH), proximal tubule (pPT) and podocyte (pPod). (f) Immunofluorescence stains of DD12 organoids showing distal transcription factor expression in polarized vs unpolarized nephrons. Scale bar: 50 microns. (g) Heatmaps of TPM value z-scores of nephron cell type markers at the SSB stage, expressed across 10 differentiation conditions in DD14 organoids (n = 2). Blue arrow denotes condition 8. (h-i) Immunofluorescence stains of DD14 control (condition 1) vs DE (condition 8) organoids showing proximal-distal marker expression. Scale bar: 50 microns. (j) Volcano plot of DESeq analysis comparing DD14 DMSO control and DE organoid total mRNA sequencing data. Dashed lines show 0.05 p-value cutoff (horizontal) and ±1.5-fold change cutoff (vertical).

Article Snippet: Antigen retrieval was performed as described in the “ Immunofluorescence Analysis ” section, and the sections were incubated in 0.1% TBS-T (0.1% Tween-20 in 1X TBS (Cell Signaling Technology)) + 1.5% SEA BLOCK blocking buffer for 1 hour at RT.

Techniques: Modification, Derivative Assay, Immunofluorescence, Staining, Expressing, Labeling, Control, Marker, Sequencing

Journal: bioRxiv

Article Title: Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

doi: 10.1101/2025.03.29.646044

Figure Lengend Snippet: (a-c) Immunofluorescence stains of DD10 organoids illustrating expression of select induced nephron progenitor markers. Scale bar: 50 microns. (d) Immunofluorescence stain of DD14 control organoids illustrating emergence of MAFB + podocyte precursors. (e) Immunofluorescence stain of DD28 control organoids showing MAFB + glomerular-like structures and surrounding vasculature-like structures and sporadic GATA3 + tubules (yellow arrows). Scale bar: 50 microns. (f) Immunofluorescence stain of DD12 control organoid tile scan showing TFAP2A emergence. Scale bar: 50 microns. (g) Schematic of SMI treatment screening to optimize nephron distalization. Top bar shows timeline for SMI treatment for each condition. (h) Immunofluorescence stains of DD14 organoids generated from the 10 condition-screen showing distal transcription factor. Red box denotes condition 8. Scale bars: 50 microns. (i) Bar graph of TPM values show expression levels of WT1, CDH1, HNF1B, POU3F3 and TFAP2A in DD14 organoids (n = 2) treated with different SMIs. Error bars show standard deviation, and top bars show statistical significance between each SMI treatment against control based on non-parametric t test (* - p<0.05, ** - p<0.01). (j-k) Heatmaps of TPM value z-scores of nephron cell type markers at the SSB stage, expressed across 10 differentiation conditions in (j) DD14 and (k) DD20 organoids (n = 2). (l) Bar graph of TPM values show expression levels of WT1, POU3F3 and TFAP2A in DD14 organoids (n = 2) from all 10 conditions. Data are represented as mean ± SD with statistical significance based on non-parametric t test between condition 8 and DMSO control (* - p<0.05, *** - p<0.001).

Article Snippet: Antigen retrieval was performed as described in the “ Immunofluorescence Analysis ” section, and the sections were incubated in 0.1% TBS-T (0.1% Tween-20 in 1X TBS (Cell Signaling Technology)) + 1.5% SEA BLOCK blocking buffer for 1 hour at RT.

Techniques: Immunofluorescence, Expressing, Staining, Control, Generated, Standard Deviation

Journal: bioRxiv

Article Title: Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

doi: 10.1101/2025.03.29.646044

Figure Lengend Snippet: (a) Annotated unsupervised clustering of PAX2 + nephron-like DD14 control and DE organoid cells represented in a UMAP. (Inset) Sample contribution of each condition. (b) Violin plot with annotations of select gene markers (top x axis) showing expression levels (bottom x axis) in the DD14 organoid dataset. The y axis shows cluster numbers and sample origin (gray square – control organoids, pink square – DE organoids). (c) Volcano plot of DESeq analysis comparing DD14 DMSO control and DE organoid nephron scRNA sequencing data. Dashed lines show 0.05 p-value cutoff (horizontal) and ±1.5-fold change cutoff (vertical). (d) Bar plot showing normalized percent contribution of cells expressing select nephron markers (log 2 FC > 0) separated by sample origin. Numbers in bars show raw cell counts. (e) Feature plots of select genes in the DD14 DE organoid dataset. (f) Immunofluorescence stain of week 16 human kidney from SSB-CLSN stage marking DCT/CNT precursor and loop of Henle/macula densa precursor segments. (g) Immunofluorescence stains of DD14 control and DE organoids showing expression of DCT/CNT precursor and loop of Henle/macula densa precursor markers. Yellow arrow – autofluorescence. Scale bar: 50 microns.

Article Snippet: Antigen retrieval was performed as described in the “ Immunofluorescence Analysis ” section, and the sections were incubated in 0.1% TBS-T (0.1% Tween-20 in 1X TBS (Cell Signaling Technology)) + 1.5% SEA BLOCK blocking buffer for 1 hour at RT.

Techniques: Control, Expressing, Sequencing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

doi: 10.1101/2025.03.29.646044

Figure Lengend Snippet: (a) Brightfield images and immunofluorescence stains on DD14 control and organoids treated with dosages indicated (left) showing detection of select distal nephron markers. (b-d) Immunofluorescence stains on DD14 control and DE protocol-treated organoids cultured from HNF4A-YFP iPSCs. Immunostains show detection of select podocyte and distal nephron markers. Scale bar: 50 microns. (e) Live imaging of YFP fluorescence in DD14 control and DE protocol-treated organoids cultured from HNF4A-YFP iPSCs.

Article Snippet: Antigen retrieval was performed as described in the “ Immunofluorescence Analysis ” section, and the sections were incubated in 0.1% TBS-T (0.1% Tween-20 in 1X TBS (Cell Signaling Technology)) + 1.5% SEA BLOCK blocking buffer for 1 hour at RT.

Techniques: Immunofluorescence, Control, Cell Culture, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

doi: 10.1101/2025.03.29.646044

Figure Lengend Snippet: A. schematic showing reference mapping of DD14 organoid scRNA sequencing dataset to reanalyzed week 14 and week 17 merged human nephron and ureteric scRNA sequencing data , . (Right) UMAP of human kidney (reference) overlaid with control and DE organoid mapping. B. Bar plot showing number of organoid cells mapping to each predicted cell type, split by condition. Numbers above bars denote prediction score for each cell type. (c) Immunofluorescence stain of DD14 control and DE organoids showing detection of IRX1. (d-e) Immunofluorescent stains of DD20 control and DE organoids showing detection of select loop of Henle/macula densa precursor and podocyte precursor markers. Abbreviations: NP – nephron progenitors, IP – induced progenitors, PTA – pretubular aggregate, pod - podocytes, PEC – parietal epithelial cells, dist prec – distal nephron precursors, PT prec – proximal tubule precursors, PT – proximal tubule, LOH – loop of Henle, DCT/CNT – distal convoluted tubule/connecting tubule.

Article Snippet: Antigen retrieval was performed as described in the “ Immunofluorescence Analysis ” section, and the sections were incubated in 0.1% TBS-T (0.1% Tween-20 in 1X TBS (Cell Signaling Technology)) + 1.5% SEA BLOCK blocking buffer for 1 hour at RT.

Techniques: Sequencing, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

doi: 10.1101/2025.03.29.646044

Figure Lengend Snippet: (a) Violin plots representing nephron cell type markers used to annotate cell types of single cell RNA sequencing performed on week 14, week 17 human kidney. (b) Feature plots of TFAP2A, HNF1B and POU3F3 detection in human kidney dataset. (c) Annotated mapping of mapped control (left) and DE (right) organoid clusters using the developing human kidney dataset as a reference. (d-e) Dot plot of distal-enriched transcription factor expression in (d) human and (e) organoid scRNA sequencing datasets. Purple box marks nephrogenic TFAP2A + clusters. (f) Feature plots showing co-expression of transcription factors IRX1 (red) and SIM2 (green) in distalizing cells of human kidney and organoid datasets. (g) Horizontal bar plot showing TPM values of select distal nephron markers in DD14 and DD20 DE organoids. Data are represented as mean ± SD with statistical significance based on non-parametric t test (* - p < 0.05, ** - p < 0.01). FC = fold change. (h) Immunofluorescence stain of DD28 control and DE organoids showing detection of loop of Henle/macula densa precursor markers. Scale bar: 50 microns.

Article Snippet: Antigen retrieval was performed as described in the “ Immunofluorescence Analysis ” section, and the sections were incubated in 0.1% TBS-T (0.1% Tween-20 in 1X TBS (Cell Signaling Technology)) + 1.5% SEA BLOCK blocking buffer for 1 hour at RT.

Techniques: RNA Sequencing, Control, Expressing, Sequencing, Immunofluorescence, Staining