Journal: International journal of biological sciences

Article Title: Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.

doi: 10.7150/ijbs.78348

Figure Lengend Snippet: Figure 2. 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-1β, and IL-18 levels (K) were measured. (L-N) Serum MIF (L), creatinine (M), and IL-1β (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-1β 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.

Article Snippet: Mouse MIF (#KE10027, Proteintech, Wuhan, China), Scr (#C011-2-1, Jiancheng, Nanjing, China), IL-1β (#KE10003, Proteintech, Wuhan, China) levels in mice were determined.

Techniques: Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Over Expression

Journal: International journal of biological sciences

Article Title: Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.

doi: 10.7150/ijbs.78348

Figure Lengend Snippet: Figure 5. IGF2BP1 m6A-dependently stabilizes E2F1 mRNA. (A & B) E2F1 protein expression in the CLP and Sham mice, as measured by IHC (A; Scale bar, 100 μm) and WB (B). (C) Act D chase experiments for E2F1 mRNA in HK2 cells with or without knockdown of IGF2BP1 following treatment with or without LPS for 12 h. (D) Act D chase experiments for E2F1 mRNA in HK2 cells with or without knockdown of METTL3 following treatment with or without LPS for 12 h. (E & F) The mRNA and protein levels of E2F1 and MIF were measured using qPCR (F) and WB (G) in HK2 cells with IGF2BP1 knocked down, treated with or without DAA for 12 h. (G) Cell viabilities of sh-METTL3/sh-NC-expressing HK2 cells with the treatment of LPS in the absence or presence of VX765, as measured by CCK-8 assay. (H-J) Pyroptosis level in sh-METTL3/sh-NC-expressing HK2 cells with or without the indicated concentration of LPS in the absence or presence of VX765. Pyroptosis-related proteins (H), LDH release (I), secretory IL-1β and IL-18 levels (J) were measured. Statistical analysis was performed using two-way ANOVA (C-E) and one-way ANOVA (G, I, J). *P < 0.05, **P < 0.01, ***P < 0.001, and ns, non-significance.

Article Snippet: Mouse MIF (#KE10027, Proteintech, Wuhan, China), Scr (#C011-2-1, Jiancheng, Nanjing, China), IL-1β (#KE10003, Proteintech, Wuhan, China) levels in mice were determined.

Techniques: Expressing, Knockdown, CCK-8 Assay, Concentration Assay

Journal: International journal of biological sciences

Article Title: Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.

doi: 10.7150/ijbs.78348

Figure Lengend Snippet: Figure 7. 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-α mRNA (G) in kidney tissues from mice injected with Lv-IGF2BP1 or Lv-NC, as measured by qPCR. (H) Serum IL-1β 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.

Article Snippet: Mouse MIF (#KE10027, Proteintech, Wuhan, China), Scr (#C011-2-1, Jiancheng, Nanjing, China), IL-1β (#KE10003, Proteintech, Wuhan, China) levels in mice were determined.

Techniques: In Vivo, Staining, Knockdown, Expressing, Injection, Enzyme-linked Immunosorbent Assay

Journal: Journal of proteome research

Article Title: Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

doi: 10.1021/acs.jproteome.4c00271

Figure Lengend Snippet: Figure 1. Native mass spectra of the Cu(I)/Zn(II)-MT3ox complexes obtained after incubation of Zn7MT3red with 4 CuCl2 mol eq at increasing concentration of NEM. The m/z region corresponds to 5+ ions (full spectra in Figure S5). The proteins (10 μM) were sprayed in 200 mM ammonium acetate (pH 6.8). “ox” subscript refers to oxidized (two intramolecular disulfides) MT3. Peak series corresponding to Cu4NEM0−13MT3ox are colored in orange, while peak series corresponding to Cu5NEM0−12MT3ox are colored in violet. The asterisks indicate m/z signals that correspond to 4+ ions.

Article Snippet: ■ EXPERIMENTAL SECTION Metallothionein-3 (MT3) (Addgene plasmid ID 105710) was overexpressed and purified in a bacterial system as described in the Supporting Information.

Techniques: Incubation, Concentration Assay

Journal: Journal of proteome research

Article Title: Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

doi: 10.1021/acs.jproteome.4c00271

Figure Lengend Snippet: Figure 4. Native mass spectra of NEM- and IAM-Cys labeled residues of the Cu(I)/Zn(II)-MT3 complexes obtained after incubation of Zn7MT3 with 4 CuCl2 mol eq (A). Native top-down CID MS for quadrupole-selected 1730 m/z ions (B). CID fragmentation map for the 1730 m/z ions (C).

Article Snippet: ■ EXPERIMENTAL SECTION Metallothionein-3 (MT3) (Addgene plasmid ID 105710) was overexpressed and purified in a bacterial system as described in the Supporting Information.

Techniques: Labeling, Incubation

Journal: Scientific Reports

Article Title: Single-cell RNA sequencing analysis revealed the immunosuppressive remodeling of tumor-associated macrophages mediated by the MIF-CD74 axis in gastric cancer

doi: 10.1038/s41598-025-10301-w

Figure Lengend Snippet: Expression of MIF and CD74 in gastric cancer. ( A ) Expression of MIF mRNA in GEPIA and TCGA-GC databases. ( B–D ) mRNA levels of MIF in the GSE54129 、GSE65801and GSE29272 dataset from the GEO database. ( E , F ) Kaplan-Meier survival analysis of GC patients based on MIF expression levels. ( G ) Representative IHC images of MIF protein expression in 71 paired GC tumor tissues and adjacent nontumor tissues. Statistical analysis of immunohistochemical score of MIF expression in GC tumor tissues and paired adjacent tissues is shown on the right. ( H ) Expression of CD74 in gastric cancer. ( I ) Representative immunofluorescence staining of CD74 (red), CD68 (green), and nuclei (blue) in paired GC samples.* P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We used the following antibodies: CD74 (Santa Cruz, ln2), MIF (Proteintech, 20415-1-AP), Goat anti-rabbit antibody (Servicebio, G1213) and goat anti-mouse antibody (Servicebio, G1214).

Techniques: Expressing, Immunohistochemical staining, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Single-cell RNA sequencing analysis revealed the immunosuppressive remodeling of tumor-associated macrophages mediated by the MIF-CD74 axis in gastric cancer

doi: 10.1038/s41598-025-10301-w

Figure Lengend Snippet: The MIF-CD74 axis remodeled the TME. ( A ) Expression of MIF in normal gastric epithelial cells and 4 different GC cell lines by Western blot. ( B ) Western blot assays were performed to verify the successful construction of MIF overexpression and knockdown GC cells. ( C ) Colony formation assays were performed to determine the effects of MIF on the growth of GC cells. The number of colonies (> 50 cells) was scored (mean ± SD, n = 3). ( D ) CCK-8 assays were performed to determine the effects of MIF on the proliferation of GC cells (mean ± SD, n = 3). ( E ) Control and MIF overexpressed HGC27 cells formed subcutaneous tumors in nude mice. The image on the right shows the weight of the subcutaneous tumors. ( F ) Control and MIF silenced AGS cells formed tumors subcutaneously in nude mice. The image on the right shows the weight of the subcutaneous tumors. ( G ) Representative immunofluorescence staining of CD74 (red), CD68 (green) and nucleus (blue) in subcutaneous tumor tissue sections. The numbers of CD74 + CD68 + cells per FOV are represented. ( H ) The effect of MIF on macrophages was analyzed by qRT-PCR. Differences were analyzed using the unpaired test two-sided t test ( n = 3). ( I ) Transwell was used to analyze MIF and its influence on macrophage migration. Differences were analyzed using the unpaired test two-sided t test ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, ns indicates P > 0.05.

Article Snippet: We used the following antibodies: CD74 (Santa Cruz, ln2), MIF (Proteintech, 20415-1-AP), Goat anti-rabbit antibody (Servicebio, G1213) and goat anti-mouse antibody (Servicebio, G1214).

Techniques: Expressing, Western Blot, Over Expression, Knockdown, CCK-8 Assay, Control, Immunofluorescence, Staining, Quantitative RT-PCR, Migration