Fig 2: Knockout of IRP1 activates the noncanonical NF-κB pathway through NFKB2(A) Clustering heatmap of mRNA from the NC and KO cells on the basis of differential expression analysis. Control group: n = 3; IRP1 KO group: n = 3.(B) Volcano plot displays differential genes expression in U87KO cells. Up (red), down (blue).(C) KEGG pathway analysis of upregulated genes. Tops of enriched pathway are shown.(D) Western blot to detect major protein levels in the canonical NF-κB pathway in U87NC, U87KO, U251NC and U251KO cells.(E) Western blot to detect major protein levels in the noncanonical NF-κB pathway in U87NC, U87KO, U251NC and U251KO cells.(F) qRT-PCR to detect the NFKB1 mRNA levels in U87NC, U87KO, U251NC and U251KO cells.(G) qRT-PCR to detect the NFKB2 mRNA levels in U87NC, U87KO, U251NC and U251KO cells.(H) IF confocal images of IRP1 (green), NFKB2 (red), and nuclei (blue). Scale bar, 20 μm.

Fig 3: Nrp1 upregulates the Nfkb1 and Smad3 pathway, suppressing Etv6 and Acox3 expression, leading to reduced levels of OXPHOS and TCA in TECs.A Transcription factor activity analysis heatmap of DT cells. B Western blotting analysis showing the changes in Nfkb1, Smad3, α-SMA and Pdgfrb after IR and Nrp1 knockout (n = 3 per group). C Dotplot of Tnfa, Nfkb1 and Smad3 in DT cells. D T-SNE plots showing the expression region of Nrp1 and transcription factor activity region of Nfkb1 and Etv6 in DT cells. E The amplification levels of Etv6 after binding with the anti-Nfkb1 monoclonal antibody using qPCR (n = 3 per group). F Dotplot of crotonyl-CoA-producing enzymes in kidney. G A schematic diagram illustrating the enzymes involved in the production of crotonyl-CoA. H Western blotting analysis showing the changes in crotonylation lysine modification after I-R and Nrp1 knockout. I Heatmap displaying the Kyoto Encyclopedia of Genes and Genomes (KEEG) pathways enriched in proteins with changing crotonylation modification sites before and after IR treatment, as well as before and after Nrp1 knockout. J Downregulated KEGG pathways in Nrp1 + DT cells compared to Nrp1-DT cells in scRNA-seq data. The statistical analyzes were two-sided and adjustments were made in P value. K Dotplot of genes related to mitochondrial functional status in DT cells. L Immunofluorescent staining of MitoTracker in primary renal tubular epithelial cells (pTECs) treated with TNF-α and Tmx. M Energy map showing increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in pTECs after Nrp1 knockout (n = 5 per group). Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by one-way ANOVA. Scale bar, 50 μm. Data represent mean ± SEM. Source data are provided as a Source Data file.

Fig 4: Depletion of CD4+ and CD8+ T cells accelerates tumor growth in p50(f/f);Lys‐Cre mice. (A) Peripheral blood cells from a WT mouse and from a p50(f/f);Lys‐Cre mouse treated with anti‐CD4 and anti‐CD8 antibodies twice weekly from day 21 to day 55 after 9464D tumor cell inoculation were subjected to flow cytometry for CD3, CD4, and CD8. The percent CD3+ cells among nucleated blood cells and the percent CD4+ or CD8+ cells among CD3+ cells are shown for each sample. (B) 9464D cells were inoculated into the flanks of p50(f/f);Lys‐Cre mice, which then received either no treatment (data from Fig. 1A) or anti‐CD4 and anti‐CD8 antibody (100 µg each) twice weekly starting on day 21. Tumor volumes (mean values and SEs) are shown at individual time points. The P‐value shown compares the entire growth curves using an exponential model. In addition, P was < 0.05 comparing volumes on days 49 to 56.

Fig 5: Loss of NFKB1 exacerbates PD phenotypes in MPTP-treated mice. (A-B). Animals (6 mice per group) were sacrificed on day 7 post MPTP injection and sections of SN were immunostained with antibody against tyrosine hydroxylase (TH). Representative illustrations were presented (A) and stereological cell counts were conducted (B) (Right, n = 6/group). (C-D) Characteristic staining of microglia was displayed (C) using antibody targeting IbaI and the Iba1-positive cells were enumerated (D) in SN sections from mice mentioned before. Double-blinded procedure was adopted and data is presented as mean ± S.E.M. (E-G) Behavioral examination results comparing MPTP-administrated mice with or without NFKB1 knockout. Swim test (E) and traction test (F) was performed on the 6th day subsequent to the final injection while the rotarod test (G) was conducted for 6 consecutive days after the last administration. There were 12 mice in each group. Data was presented as mean ± standard error and differences were analyzed by one-way analysis of variance followed by least significant difference post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001