Fig 1: IGF2BP1 induces pyroptosis via targeting MIF. (A & B) MIF interacted with NLRP3. The co-localization of MIF protein and NLRP3 protein in HK2 cells was measured by immunofluorescence (A). Immunofluorescent staining of NLRP3 (red), MIF (green), and nuclear staining with DAPI (blue) are shown. Scale bar, 50 µm. The combination of MIF protein and NLRP3 protein in HK2 cells with or without LPS stimulation was measured by co-IP (B). (C-E) MIF protein levels in the CLP and Sham mice, as measured by IHC (C; Scale bar, 100 µm), WB(D), and ELISA (E). (F & G) MIF protein levels in HK2 cells treated with the indicated concentration of LPS for 12 h, as measured by immunofluorescence (F; Scale bar, 50 µm) and WB (G). Immunofluorescent staining of MIF (green) and nuclear staining with DAPI (blue) are shown. (H) MIF protein levels in sh-MIF/sh-NC-expressing HK2 cells treated with the indicated concentration of LPS for 12 h, as measured by WB. (I-K) Pyroptosis levels in sh-MIF/sh-NC-expressing HK2 cells following the induction with or without the indicated concentration of LPS for 12 h. Pyroptosis-related proteins (I), LDH release (J), secretory IL-1ß, and IL-18 levels (K) were measured. (L-N) Serum MIF (L), creatinine (M), and IL-1ß (N) levels in CLP mice treated with or without ISO-1, as measured by ELISA. (O-Q) Pyroptosis level in sh-IGF2BP1-expressing HK2 cells with MIF over-expression following the induction with the indicated concentration of LPS for 12 h. Pyroptosis-related proteins (O), secretory IL-1ß level (P), and secretory IL-18 level (Q) were measured. Statistical analysis was performed using a t-test (E, K-N, P-Q). *P < 0.05, **P < 0.01 and ns, non-significance.
Fig 2: OB targets RANKL-induced signaling by binding to MIF. (A) In Silico docking analysis of OB in the active pocket of MIF (PDB code 3DJH). The potential interactive residues were displayed in 3D and 2D. (B) Surface plasmon resonance (SPR) assays showing the binding of OB to MIF protein. (C) Representative blotting images showing OB’s effect on MIF and its related osteoclast markers following the treatment of RANKL in BMMs. BMMs: bone marrow macrophages; MIF, macrophage migration inhibitory factor; OB: obacunone; RANKL: receptor activator of nuclear factor-?B ligand.
Fig 3: Inhibiting IGF2BP1 is a potential strategy to protect against renal septic injury in vivo. (A) Representative images of H&E and PAS staining of kidney tissues from CLP mice with or without the knockdown of IGF2BP1. Scale bar, 50 µm. (B) Expression of IGF2BP1 in the CLP kidney injected with Lv-IGF2BP1 or Lv-NC, as measured by WB. (C) Renal function serum creatinine (Scr) level in mice receiving CLP surgery after injection of Lv-IGF2BP1 or Lv-NC. (D-G) The expression level of E2F1 mRNA (D), MIF mRNA (E), IL-6 mRNA (F), and TNF-a mRNA (G) in kidney tissues from mice injected with Lv-IGF2BP1 or Lv-NC, as measured by qPCR. (H) Serum IL-1ß levels in mice injected with Lv-IGF2BP1 or Lv-NC, as measured by ELISA. Group comparisons were performed by t-test (C-H). N = 6/group, *P<0.05, **P < 0.01.
Fig 4: MIF is transcriptionally promoted by IGF2BP1. (A) MIF mRNA levels in sh-IGF2BP1/sh-NC-expressing HK2 cells following treatment with or without LPS for 12 h, as measured by qPCR (A). (B) MIF protein levels in sh-IGF2BP1/sh-NC-expressing HK2 cells following treatment with or without LPS for 12 h, as measured by WB. The quantification of the relative MIF protein levels was shown in the right panel. (C) Actinomycin D (Act D) chase experiments for MIF mRNA expression in sh-IGF2BP1/sh-NC-expressing HK2 cells following treatment with or without the indicated concentration of LPS for 12 h. (D) MIF promoter activities in HK2 cells following the treatment with or without LPS for 12 h. (E) MIF promoter activities in HK2 cells in the absence or presence of a-Amanitin, as measured by luciferase assays. (F & G) IGF2BP1 did not affect the protein stability of MIF. HK2 cells were treated with CHX (100 µM) during administration with or without LPS for 12 h. The ratio between MIF and GAPDH was graphed in the middle panel, and the relative MIF level with or without CHX was also calculated in HK2 cells treated with or without LPS (right panel). (H) MIF mRNA levels in AKI patients (n=38) and control samples (n=12), as measured in qPCR. (I) IGF2BP1 mRNA levels in AKI patients (n=38) and control samples (n=12), as measured in qPCR. (J) Correlation between IGF2BP1 mRNA and MIF mRNA in AKI patients (n=38) and control samples (n=12), as analyzed by Pearson analysis. Statistical analysis was performed using one-way ANOVA (A, D, E), two-way ANOVA (B, C), t-test (G), Wilcoxon rank sum test (H, I), and Pearson analysis (J). **P < 0.01, ***P < 0.001, and ns, non-significance.
Fig 5: IGF2BP1 promotes MIF transcription via E2F1. (A) The specific binding site bases of transcription factor E2F1, downloaded from the JASPAR website (https://jaspar.genereg.net/). (B) The conserved E2F1 binding motif of the MIF promoter between humans and mice. (C) MIF promoter activities with or without intact E2F1 motif with E2F1 overexpressed or knocked down in HK2 cells, as measured by luciferase assay. (D & E) MIF mRNA and protein levels with E2F1 overexpressed or knocked down in HK2 cells, as measured by qPCR (D) and WB (E) in HK2 cells respectively. (F) The interaction between probes compassing the E2F1 motif and nuclear exacts (NE) from HK2 cells, as measured by EMSA. The proportion of wild-type (Wt) probes and mutant (mut) probes in each assay was different. (G) The interaction between probes compassing the E2F1 motif and nuclear exacts (NE) from HK2 cells, as measured by EMSA. The type or dose of antibodies was different for each assay. (H) ChIP-seq of E2F1 related to MIF in HeLa cells, as shown on the Cistrome Data Browser website (http://cistrome.org/db). (I & J) ChIP assays using anti-E2F1 antibody and control IgG antibody in HK2 cells with or without the indicated concentration of LPS for 12 h (I). QPCR was used to quantify immunoprecipitated chromatin fragments using primers specific to E2F1 binding sites (J). (K & L) ChIP assays using anti-E2F1 antibody and IgG antibody in HK2 cells with E2F1 knocked down (K). qPCR was used to quantify immunoprecipitated chromatin fragments (L). F1/R1 region compassed the E2F1 motif in the MIF promoter, while F2/R2 region represented an unrelated region in the MIF promoter. (M-O) IGF2BP1 promotes MIF promoter activities and expression via E2F1 in HK2 cells. MIF promoter activities, mRNA levels, and protein levels in sh-E2F1/sh-NC-expressing HK cells with or without inducible IGF2BP1 expression, as measured by luciferase assay (M), qPCR (N), and WB (O) respectively. Statistical analysis was performed using one-way ANOVA (C, D) and t-test (J, L-N). **P < 0.01, ***P < 0.001, and ns, non-significance.
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