Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: The mitochondrial protein SH3GLB1 was highly expressed in alveolar macrophages in drowning-ALI. (A) Representative TEM images of AM mitochondria in the lung (the red arrow indicates injured and swollen mitochondria; scale bars, 2 µ m/500 nm). (B) Representative analysis of single-cell sequencing data and identification of the top 15 mitochondrial proteins. (C) Representative macrophage subcluster-specific expression patterns of SH3GLB1 between the sham and sea water groups. (D) UMAP analysis and (E) percentages of high and low SH3GLB1 expression in AMs treated with sham or sea water; colored unsupervised clustering results with cell type annotations are shown on the right. (F) Relative SH3GLB1 mRNA levels in alveolar macrophages from shamor sea water-induced ALI model mice (n=7). (G) Representative western blots of SH3GLB1 protein levels in shamor sea water-induced ALI model mice. The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; ALI, acute lung injury; UMAP, uniform manifold approximation and projection; TEM, transmission electron microscopy; AMs, alveolar macrophages.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Single Cell, Sequencing, Expressing, Western Blot, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: SH3GLB1 deficiency protects against inflammation in ALI. (A) Gene expression of SH3GLB1 high AMs in cohort 1 (n=3) represented in a volcano plot, with the fold change (log2) expressed as the level in sham versus sea water-ALI subjects on the x-axis and the P-value on the y-axis (log10). Significantly (P<0.05) regulated genes are marked in colors: red indicates upregulated genes and blue indicates downregulated genes in ALI. (B) Representative GO analysis of SH3GLB1 high AMs. (C) Representative KEGG analysis of SH3GLB1 high AMs. (D) Representative hematoxylin and eosin staining of lung tissues (scale bars, 200 µ m). (E) Representative severity scores of lung injury (n=3). Representative ELISA results showing the levels of inflammatory factors, including (F) TNFα, (G) IL-6 and (H) IL-1β, in the BALF (n=6). (I-K) Representative ELISA analysis of the levels of inflammatory factors, including (I) IL-1β, (J) IL-6 and (K) TNFα, in the serum (n=6). (L) Representative western blots of SH3GLB1 protein levels in NC or SH3GLB1-KD BMDMs. (M) Relative SH3GLB1 mRNA levels in (NC or SH3GLB1-KD BMDMs. n=6/group. Representative ELISA analysis of the levels of inflammatory factors, including IL-1β, IL-6 and TNFα, in the supernatants of cells treated with (N) LPS or (O) seawater, (n=3).The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; AMs, alveolar macrophages, ALI, acute lung injury; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BALF, bronchoalveolar lavage fluid; BMDMs, bone marrow-derived macrophages; KD, knockdown; WT, wild-type; KO, knockout; NC, negative control; LPS, lipopolysaccharide.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Gene Expression, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Knockdown, Knock-Out, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: Reduced SH3GLB1 ameliorates LPS-induced mitochondrial dysfunction in macrophages. (A) Representative TEM images of macrophage mitochondria in the lung (scale bars, 2 µ m/500 nm). Representative (B) confocal microscopy and (C) flow cytometry analysis of the mitochondrial membrane potential by JC-1 staining in BMDMs. A high level of green fluorescence (x-axis) represents a reduced mitochondrial membrane potential (ΔΨm) and a high level of red fluorescence (y-axis) represents a normal ΔΨm. The red fluorescence rate was analyzed (n=3). (D) Representative confocal microscopy images of mitochondrial ROS production (MitoSOX fluorescence; red) in NC or SH3GLB1-KD BMDMs under normal or LPS conditions (scale bar, 50 µ m). (E) Analysis of the OCR of mitochondrial respiratory capacity, including basal respiration, ATP production, maximal respiration and spare respiration, in NC or SH3GLB1-KD BMDMs under normal or LPS conditions. The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; LPS, lipopolysaccharide; TEM, transmission electron microscopy; BMDMs, bone marrow-derived macrophages; KD, knockdown; OCR, oxygen consumption rate; NC, negative control.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Confocal Microscopy, Flow Cytometry, Membrane, Staining, Fluorescence, Transmission Assay, Electron Microscopy, Derivative Assay, Knockdown, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: Restoration of SH3GLB1 expression provokes ALI. (A) Representative hematoxylin and eosin staining of lung tissues (scale bars, 100 µ m). (B) Representative severity scores of lung injury (n=3). Representative ELISA results showing the levels of inflammatory factors, including (C) TNFα, (D) IL-1β and (E) IL-6, in the BALF (n=6). Representative ELISA analysis of the levels of inflammatory factors, including (F) IL-1β, (G) IL-6 and (H) TNFα, in the serum (n=6). (I) Western blots of SH3GLB1 expression. (J) Relative SH3GLB1 mRNA levels in vectoror SH3GLB1-overexpressing BMDMs. n=6/group. Representative ELISA analysis of the levels of inflammatory factors, including IL-1β, IL-6 and TNFα, in the supernatants of cells treated with (K) LPS or (L) sea water (n=3).The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; ALI, acute lung injury; BMDMs, bone marrow-derived macrophages; WT, wild-type; OV; overexpression.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: SH3GLB1 overexpression provoked mitochondrial dysfunction in macrophages. (A) Representative TEM images of macrophage mitochondria in the lung (scale bars, 2 µ m/500 nm). Representative (B) confocal microscopy and (C) flow cytometry analysis of the mitochondrial membrane potential by JC-1 staining in BMDMs (n=3). (D) Representative confocal microscopy images of mitochondrial ROS production in vectoror SH3GLB1-OV BMDMs under normal or LPS conditions (scale bar, 50 µ m). (E) Analysis of the OCR of mitochondrial respiratory capacity, including basal respiration, ATP production, maximal respiration and spare respiration, in vectoror SH3GLB1-overexpressing BMDMs under normal or LPS conditions. The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; TEM, transmission electron microscopy; OCR, oxygen consumption rate; BMDMs, bone marrow-derived macrophages.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Over Expression, Confocal Microscopy, Flow Cytometry, Membrane, Staining, Transmission Assay, Electron Microscopy, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: SH3GLB1 interacts with the lysosomal protein Rab7 to contribute to macrophage inflammation. (A) Representative top five proteins potentially binding to SH3GLB1 in mitochondria identified by mass spectrometry analysis. (B) Representative western blots of Rab7 protein levels in BMDMs treated with LPS or sea water. (C) Co-IP assay showing the interaction of SH3GLB1 with Rab7. (D) Representative images of immunofluorescence staining for SH3GLB1 (green) and Rab7 (red) in BMDMs. Scale bar, 20 µ m. (E) Representative molecular docking analysis of SH3GLB1 and Rab7. (F) Western blots of recombinant Rab7 binding to recombinant SH3GLB1. Representative ELISA results showing the levels of inflammatory factors, including IL-1β, IL-6 and TNFα, in the supernatants of cells treated with (G and H) LPS (n=3) or (I and J) sea water (n=3). The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; BMDMs, bone marrow-derived macrophages; LPS, lipopolysaccharide; Co-IP, coimmunoprecipitation.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Binding Assay, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Recombinant, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: SH3GLB1 facilitated adverse Rab7-mediated mitophagy. (A) Representative images of immunofluorescence staining for SH3GLB1 (green) and mitochondria (red) or lysosomes (red) in BMDMs. Scale bar, 20 µ m. Representative (B and F) western blot and (C-E and G-I) quantification of mitophagy-related protein levels in BMDMs treated with CCCP. (J) mKeima expressed in BMDMs was imaged by confocal microscopy (mKeima-561 nm: red; mKeima-488 nm: green; scale bar, 20 µ m); (K) the 561/488 nm ratio of mKeima was statistically quantified (n=3). (L) Representative flow cytometry analysis of mKeima in BMDMs. The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; BMDMs, bone marrow-derived macrophages; CCCP, carbonyl cyanide-chlorophenylhydrazone.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Immunofluorescence, Staining, Western Blot, Confocal Microscopy, Flow Cytometry, Derivative Assay