Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , A volcano plot of lysosomal proteomics showing enrichment of Ragulator/LAMTOR–RagGTPase components in MTM1 -KO Cas9 myotubes. Data from three biologically independent experiments; two-sided Welch’s t -test; P < 0.01. b , An immunoblot analysis of mTOR complex components and RagGTPases in CTRL and MTM1 -KO Cas9 myotubes after 9 days in 2% HS. Data are shown as mean ± s.d. from three biologically independent experiments; two-sided Mann–Whitney test. c , The LAMTOR–RagA/C enrichment in lysosomal immunoprecipitates from CTRL and MTM1 -KO Cas9 myotubes under amino acid starvation (HBSS) after 9 days. Inputs are normalized to LAMTOR levels. Three biologically independent experiments. d , A volcano plot comparing lysosomal and total cellular metabolites between CTRL and MTM1 -KO Cas9 myotubes. Data from six independent Lyso-IP preparations and three total cell extracts; two-sided Welch’s t- test; P < 0.01. e , The validation of RagA knockdown and expression of RagA WT, constitutively active (Q66L) or dominant-negative (R37P, T21N) mutants. Three biologically independent experiments; one-way ANOVA with Dunnett’s test. f , The effects of RagA manipulation on MTM1 -KO Cas9 myotube differentiation. Data are shown as mean ± s.d.; n = 25 myotubes from three technical replicates; one-way ANOVA with Dunnett’s test. Scale bar, 100 µm. g , h , Lamtor1 knockdown reduces mTORC1 signalling ( g ) and restores differentiation ( h ) in MTM1 -KO Cas9 myotubes (percentage of multinucleated myotubes and myotube area). Data are shown as mean ± s.d.; n = 48 myotubes per condition from three biologically independent experiments; one-way ANOVA with Dunnett’s test. Scale bar, 100 µm. Protein molecular weight in kDa. Illustration in c created in BioRender; Karim, H. https://biorender.com/n6ucxpz (2026).

Article Snippet: For LAMTOR1 and LAMTOR3 constructs, the pRK5-LAMTOR1/p18-FLAG (Addgene no. 42331) and pRK5-HA-LAMTOR3/HBXIP (Addgene no. 42328) plasmids were used to generate mutants (LAMTOR1-K103A/K104A, LAMTOR3-K6A, LAMTOR3-K34 and the LAMTOR3 double mutation K6A/K34A).

Techniques: Western Blot, MANN-WHITNEY, Biomarker Discovery, Knockdown, Expressing, Dominant Negative Mutation, Molecular Weight

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , Representative images showing colocalization of RagA with HA–Lyso-Tag in myotubes. Scale bar, 100 µm. b , c , Colocalization of RagA with LAMP2 ( b ) and LAMTOR1 with LAMP2 ( c ) in isolated muscle fiber bundles from Mtm1-KO mice. Representative images are shown. Scale bar, 100 µm. d , GTP pull-down assay performed in HEK293 cells expressing RagA mutants. The Q66L and T21N mutants correspond to constitutively active (GTP-bound) and dominant-negative (GDP-bound) forms, respectively. GTP-loaded RagA (WT and Q66L) was captured by the GTP affinity matrix, whereas the T21N mutant was not retained. RagC displayed an inverse binding profile. Representative immunoblots from two biologically independent experiments are shown. e , GTP pull-down assay performed on myotube lysates showing increased levels of GTP-bound RagA in MTM1-KO cas9 compared with CTRL myotubes, with an inverse pattern observed for RagC. f , Representative images showing TFEB localization and colocalization in CTRL and MTM1-KO cas9 myotubes at day 7 of differentiation following nutrient deprivation (HBSS), Torin treatment, or combined treatment. Under all conditions, including differentiation medium (2% HS), MTM1-KO cas9 myotubes exhibit reduced nuclear translocation of TFEB and increased lysosomal retention. Scale bar, 20 µm. All graphs show mean ± s.d. Individual data points represent individual myotube areas, fibers, or cells analysed across biologically independent samples. Two-sided Mann–Whitney U tests were used ( a – c , e ; n = 19 myotube areas per condition in a, n = 69 fibers for RagA/LAMP2 and n = 54 fibers for LAMTOR1/LAMP2 from three mice in b,c, and three biologically independent experiments in e). For f, Šidák’s multiple-comparisons test was used following ANOVA (n = 40 myotubes per condition, three biologically independent experiments). No statistical analysis was performed for representative immunoblots ( d ). Exact P values are indicated in the graphs, except when P < 0.0001. Molecular weights in d and e are indicated in kDa.

Article Snippet: For LAMTOR1 and LAMTOR3 constructs, the pRK5-LAMTOR1/p18-FLAG (Addgene no. 42331) and pRK5-HA-LAMTOR3/HBXIP (Addgene no. 42328) plasmids were used to generate mutants (LAMTOR1-K103A/K104A, LAMTOR3-K6A, LAMTOR3-K34 and the LAMTOR3 double mutation K6A/K34A).

Techniques: Isolation, Pull Down Assay, Expressing, Dominant Negative Mutation, Mutagenesis, Binding Assay, Western Blot, Translocation Assay, MANN-WHITNEY

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , Representative immunoblots of purified recombinant LAMTOR–RagA/C protein complexes and phosphoinositide-binding domain probes, including GST–HRS (PI3P binding) and GST–PH-PLCδ (PI(4,5)P 2 -binding), used as specificity controls (three independent protein preparations). b , Representative immunoblots of phosphoinositide (PIP)–bead binding assays performed with recombinant RAGULATOR complexes. LAMTOR1 (LT1) and LAMTOR3 (LT3) bind selectively to PI3P- and PI(3,5)P 2 -containing beads. GST–HRS and GST–PH-PLCδ served as experimental controls (three independent protein preparations). c , Quantitative confirmation of LAMTOR complex (LT1–LT5) binding to PI3P and PI(3,5)P 2 using Cova PIP ELISA. GST–HRS (PI3P binding) and GST–SnxA (PI(3,5)P 2 -binding) were used as positive controls. Data are shown as mean ± s.d. from three independent protein preparations. d , Coomassie-stained SDS–PAGE of purified wild-type and mutant LAMTOR complexes (point mutations in LT1 and LT3 are indicated) (three independent protein preparations) Sequence alignments highlight conserved lysine (K103/K104 in LT1; K34 in LT3) and arginine (R6/R7) residues implicated in PI3P/PI(3,5)P 2 binding across species. e , Binding of pentameric wild-type and mutant LAMTOR complexes to PI3P and PI(3,5)P 2 assessed by Cova PIP ELISA and detected using an anti-LAMTOR1 antibody. Data are shown as mean ± s.d. from three independent protein preparations. f , Representative immunoblots showing that disruption of LT1 and LT3 phosphoinositide binding does not impair LAMTOR complex assembly in HEK293 cells but reduces RagA recruitment (two independent experiments). g , Inhibition of protein ubiquitination by MLN4924 promotes lysosomal accumulation of LT1 and LT3. Representative images showing colocalization of endogenous LT1 and LT3 with Lyso-Tag (HA) in CTRL and MTM1-KO cas9 myotubes at day 7 of differentiation following MLN4924 treatment. Data are shown as mean ± s.d., n = 39 myotubes per condition from three biologically independent experiments. One-way ANOVA followed by Dunnett’s multiple-comparisons test. Scale bar, 20 µm. Protein molecular weights in a , b , d , and f are indicated in kDa.

Article Snippet: For LAMTOR1 and LAMTOR3 constructs, the pRK5-LAMTOR1/p18-FLAG (Addgene no. 42331) and pRK5-HA-LAMTOR3/HBXIP (Addgene no. 42328) plasmids were used to generate mutants (LAMTOR1-K103A/K104A, LAMTOR3-K6A, LAMTOR3-K34 and the LAMTOR3 double mutation K6A/K34A).

Techniques: Western Blot, Purification, Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, SDS Page, Mutagenesis, Sequencing, Disruption, Inhibition, Ubiquitin Proteomics

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , Representative immunoblots showing AKT (p-S473 and p-T308) and mTORC1 signalling (p-S6K/S6K and p-4E-BP1/4E-BP1) in skeletal muscle from 2- and 6-week-old Mtm1 -KO mice. Data are shown as mean ± s.e.m., n = 8 mice per group. Two-sided Mann–Whitney test. b , c , Two AZD8055 treatment regimens were used ( b ) to assess survival in Mtm1 -KO mice ( c ). n = 8 mice per group (biological replicates). d , e , The effects of mTORC1 inhibition on body weight ( d ), TA muscle mass and specific force in Mtm1 -KO mice ( e ). Data are shown as mean ± s.e.m, n = 8 mice per group. One-way ANOVA with Tukey’s multiple-comparisons test. f , Representative immunoblots showing reduced mTORC1 activity (p-S6K/S6K) in TA muscle following AZD8055 treatment. Data are shown as mean ± s.e.m, n = 8 mice per group. One-way ANOVA with Šídák’s multiple-comparisons test. g , Representative images from three mice showing improved TA muscle morphology and ultrastructure following AZD8055 treatment (H&E, SDH and TEM). Arrows indicate z-lines misalignment/interruption in Mtm1 -KO muscle which was improved by AZD8055 treatment. Scale bars: white, 500 µm; black, 2 µm. h , Fibre diameter distributions of TA muscle after 30 days of AZD8055 treatment. Data are shown as mean ± s.d., n = 6 mice per group. One-way ANOVA with Dunnett’s multiple-comparisons test. i , A model summarizing phosphoinositide-dependent regulation of mTORC1 during muscle differentiation and maintenance. Physiological ER stress restrains mTORC1 via MTM1 at ER–lysosome contact sites by controlling PI3P and PI(3,5)P 2 levels that bind LAMTOR1 and LAMTOR3. Loss of MTM1 in XLCNM leads to phosphoinositide accumulation, persistent LAMTOR–Rag–mTORC1 activation and disrupted anabolic–catabolic balance. For a and f , protein molecular weight is indicated in kDa. Illustrations in b and i created in BioRender; Karim, H. https://biorender.com/n6ucxpz (2026).

Article Snippet: For LAMTOR1 and LAMTOR3 constructs, the pRK5-LAMTOR1/p18-FLAG (Addgene no. 42331) and pRK5-HA-LAMTOR3/HBXIP (Addgene no. 42328) plasmids were used to generate mutants (LAMTOR1-K103A/K104A, LAMTOR3-K6A, LAMTOR3-K34 and the LAMTOR3 double mutation K6A/K34A).

Techniques: Western Blot, MANN-WHITNEY, Inhibition, Activity Assay, Activation Assay, Molecular Weight

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Pyroptosis, apoptosis and necroptosis in macrophages concurrently exist in human atherosclerotic lesions. (A–C) Necrotic core formation, lipid deposition and extracellular fibrosis are observed in lower extremity and carotid atherosclerotic lesions, but not in histologically normal artery by means of HE, Oil Red O, and Movat's staining. Scale bar: 1000 µm. (D) Immunohistochemical staining for CD68, a marker for macrophages, is conducted on histologically normal arteries as well as on lower extremity and carotid atherosclerotic lesions. Scale bar: 100 µm. (E–G) Compared with histologically normal artery (E), lower extremity (F) and carotid atherosclerotic lesions (G) show the pyroptotic, apoptotic and necroptotic characteristics of macrophages, including discontinuity of plasma membrane (red arrows), chromatin condensation, and electron‐light zones in transmission electron microscopy images. Scale bar: 2.5 µm. (H) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in human atherosclerotic lesions and peripheral normal artery. (I) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in human atherosclerotic lesions and peripheral normal artery. (J) The capacity of caspase‐3 in human atherosclerotic lesions and peripheral normal artery. (K and L) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in human atherosclerotic lesions and peripheral normal artery. (M) Representative Western blots and relative quantitative analysis of TLR4, MyD88 and phospho‐NF‐kB in human atherosclerotic lesions and peripheral normal artery. (N) Human atherosclerotic lesions release a significant amount of TNF‐1α, IL‐1β, IL‐18 and IL‐6, whereas the peripheral normal artery releases less. (O) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (yellow) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the co‐existence of pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Triple‐positive cells are shown by the arrows. Scale bar: 50 µm. (P–R) Double immunofluorescence staining for GSDMD (M)/caspase‐3 (N)/RIPK3 (O) (red), CD68 (green), and DAPI (blue) in human atherosclerosis and peripheral normal artery demonstrates the presence of pyroptotic, apoptotic and necroptotic markers in macrophages, as indicated by the colocalisation of caspase‐3/GSDMD/RIPK3 and CD68 (a macrophage marker). Double‐positive cells are shown by the arrows. Scale bar: 50 µm. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Staining, Immunohistochemical staining, Marker, Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Double Immunofluorescence Staining, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Silencing galectin‐3 downregulated TLR4/MyD88/NF‐kB expression and attenuated ox‐LDL induced pyroptotic, apoptotic, and necroptotic cell death in macrophages. (A) Electron microscopy ultrastructural analysis of control and ox‐LDL‐induced macrophages. Control macrophages have a normal‐looking cellular structure, whereas ox‐LDL‐induced macrophages show loss of cell plasma integrity, chromatin condensation or fragmentation, and electron‐light zone. Scale bar: 2.5 µm. (B) Confocal microscopy with double immunofluorescence staining for caspase‐3 (red) and RIPK3 (green) in macrophages show the colocalisation of apoptotic and necroptotic components. Confocal microscopy analysis of double immunofluorescence labelling is indicative of overlapping expression of caspase‐3 (red) and GSDMD (green) in macrophages. Scale bar: 25 µm. (C) Representative Western blots and relative quantitative analysis of galectin‐3 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (D and E) Flow cytometry (E) and quantification analysis (F) with annexin V/PI double staining show that ox‐LDL increased the percentage of apoptotic cells in macrophages, which is alleviated by silencing galectin‐3. (F–H) Flow cytometry (F) and quantification analysis (G) with PI/Hoechst staining (H) show that ox‐LDL enhanced PI uptake in macrophages, which is markedly blocked by silencing galectin‐3. Scale bar: 50 µm. (I) Silencing galectin‐3 abrogated LDH release in macrophages ignited by ox‐LDL. (J) Ox‐LDL induced the accumulation of intracellular lipid droplets in macrophages, which are potently reversed by silencing galectin‐3. Scale bar: 50 µm. (K) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (L) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (M and N) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (O) Ox‐LDL treatment promotes the release of proinflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) from macrophages, which is markedly rescued by silencing galectin‐3. (P and Q) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐kB and phospho‐NF‐kB in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (R) Cell lysates from ox‐LDL‐treated macrophages are immunoprecipitated with anti‐TLR4 or anti‐MyD88 antibodies, and blotted with anti‐TLR4 or anti‐MyD88 antibodies. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Expressing, Electron Microscopy, Control, Clinical Proteomics, Confocal Microscopy, Double Immunofluorescence Staining, Immunofluorescence, Western Blot, Flow Cytometry, Double Staining, Staining, Immunoprecipitation, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: NLRP3 agonist nigericin counteracted the inhibitory effect of silencing galectin‐3 on pyroptosis, apoptosis and necroptosis in macrophages. (A) Confocal microscopy with double immunofluorescence staining for galectin‐3 (red) and NLRP3 (green) in macrophages reveals the colocalisation of galectin‐3 with NLRP3. Scale bar: 25 µm. (B and C) Flow cytometry (B) and quantification analysis (C) with annexin V/PI double staining show that silencing galectin‐3 decreases the percentage of apoptotic cells in ox‐LDL‐induced macrophages, and nigericin robustly blunts the inhibitory effect of siGalectin‐3. (D–F) Flow cytometry (D) and quantification analysis (E) with PI/Hoechst staining (F) show that silencing galectin‐3 diminishes the percentage of PI‐positive cells in ox‐LDL‐induced macrophages, and nigericin mostly abolishes the protective effect of siGalectin‐3. Scale bar: 50 µm. (G) Silencing galectin‐3 suppresses the LDH release in ox‐LDL‐induced macrophages, which is largely abrogated by nigericin. (H) Silencing galectin‐3 lessens the intracellular lipid droplet in ox‐LDL‐induced macrophages, while nigericin exerts the opposite effect. Scale bar: 50 µm. (I) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (J) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (K) The activity of caspase‐3 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (L and M) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (N) Silencing galectin‐3 inhibits the release of inflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) in ox‐LDL‐induced macrophages, and nigericin effectively blocks the role of siGalectin‐3. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

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

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Pyroptosis, apoptosis and necroptosis in macrophages coordinately occurred in ApoE −/− mice fed an HFD, which are alleviated by galectin‐3 deficiency, and conversely are aggravated by NLRP3 agonist nigericin. (A) Pyroptosis, apoptosis and necroptosis of macrophages are identified in the aortas of ApoE −/− mice fed an HFD, as evidenced by plasma membrane pore (red arrows), chromatin condensation (red arrows), and electron‐light zone (red arrows) by transmission electron microscopy. Scale bar: 2.5 µm. (B) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (pink) and DAPI (blue) in the aortas of ApoE −/− mice fed HFD or normal diet reveals the potential crosstalk among pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Three‐positive cells are shown by the arrows. Scale bar: 50 µm. (C–E) Dual immunofluorescence staining for caspase‐3 (C)/GSDMD (D)/RIPK3 (E) (red), F4/80 (green), and DAPI (blue) in the aortas of ApoE −/− mice fed an HFD or normal diet demonstrate that GSDMD/caspase‐3/RIPK3 immunoreactivity colocalises with macrophage marker CD68. Scale bar: 50 µm. (F) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in the aortas of ApoE −/− mice fed with a normal diet or HFD, NLRP3 agonist nigericin‐treated ApoE −/− mice fed with an HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (G) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase 8 and cleaved caspase 8 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with HFD. (H) The activity of caspase‐3 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (I and J) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (K and L) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐κB and phospho‐NF‐κB in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. n = 4–8 mice per group. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Marker, Western Blot, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Pyroptosis, apoptosis and necroptosis in macrophages concurrently exist in human atherosclerotic lesions. (A–C) Necrotic core formation, lipid deposition and extracellular fibrosis are observed in lower extremity and carotid atherosclerotic lesions, but not in histologically normal artery by means of HE, Oil Red O, and Movat's staining. Scale bar: 1000 µm. (D) Immunohistochemical staining for CD68, a marker for macrophages, is conducted on histologically normal arteries as well as on lower extremity and carotid atherosclerotic lesions. Scale bar: 100 µm. (E–G) Compared with histologically normal artery (E), lower extremity (F) and carotid atherosclerotic lesions (G) show the pyroptotic, apoptotic and necroptotic characteristics of macrophages, including discontinuity of plasma membrane (red arrows), chromatin condensation, and electron‐light zones in transmission electron microscopy images. Scale bar: 2.5 µm. (H) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in human atherosclerotic lesions and peripheral normal artery. (I) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in human atherosclerotic lesions and peripheral normal artery. (J) The capacity of caspase‐3 in human atherosclerotic lesions and peripheral normal artery. (K and L) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in human atherosclerotic lesions and peripheral normal artery. (M) Representative Western blots and relative quantitative analysis of TLR4, MyD88 and phospho‐NF‐kB in human atherosclerotic lesions and peripheral normal artery. (N) Human atherosclerotic lesions release a significant amount of TNF‐1α, IL‐1β, IL‐18 and IL‐6, whereas the peripheral normal artery releases less. (O) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (yellow) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the co‐existence of pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Triple‐positive cells are shown by the arrows. Scale bar: 50 µm. (P–R) Double immunofluorescence staining for GSDMD (M)/caspase‐3 (N)/RIPK3 (O) (red), CD68 (green), and DAPI (blue) in human atherosclerosis and peripheral normal artery demonstrates the presence of pyroptotic, apoptotic and necroptotic markers in macrophages, as indicated by the colocalisation of caspase‐3/GSDMD/RIPK3 and CD68 (a macrophage marker). Double‐positive cells are shown by the arrows. Scale bar: 50 µm. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Staining, Immunohistochemical staining, Marker, Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Double Immunofluorescence Staining, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Silencing galectin‐3 downregulated TLR4/MyD88/NF‐kB expression and attenuated ox‐LDL induced pyroptotic, apoptotic, and necroptotic cell death in macrophages. (A) Electron microscopy ultrastructural analysis of control and ox‐LDL‐induced macrophages. Control macrophages have a normal‐looking cellular structure, whereas ox‐LDL‐induced macrophages show loss of cell plasma integrity, chromatin condensation or fragmentation, and electron‐light zone. Scale bar: 2.5 µm. (B) Confocal microscopy with double immunofluorescence staining for caspase‐3 (red) and RIPK3 (green) in macrophages show the colocalisation of apoptotic and necroptotic components. Confocal microscopy analysis of double immunofluorescence labelling is indicative of overlapping expression of caspase‐3 (red) and GSDMD (green) in macrophages. Scale bar: 25 µm. (C) Representative Western blots and relative quantitative analysis of galectin‐3 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (D and E) Flow cytometry (E) and quantification analysis (F) with annexin V/PI double staining show that ox‐LDL increased the percentage of apoptotic cells in macrophages, which is alleviated by silencing galectin‐3. (F–H) Flow cytometry (F) and quantification analysis (G) with PI/Hoechst staining (H) show that ox‐LDL enhanced PI uptake in macrophages, which is markedly blocked by silencing galectin‐3. Scale bar: 50 µm. (I) Silencing galectin‐3 abrogated LDH release in macrophages ignited by ox‐LDL. (J) Ox‐LDL induced the accumulation of intracellular lipid droplets in macrophages, which are potently reversed by silencing galectin‐3. Scale bar: 50 µm. (K) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (L) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (M and N) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (O) Ox‐LDL treatment promotes the release of proinflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) from macrophages, which is markedly rescued by silencing galectin‐3. (P and Q) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐kB and phospho‐NF‐kB in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (R) Cell lysates from ox‐LDL‐treated macrophages are immunoprecipitated with anti‐TLR4 or anti‐MyD88 antibodies, and blotted with anti‐TLR4 or anti‐MyD88 antibodies. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Expressing, Electron Microscopy, Control, Clinical Proteomics, Confocal Microscopy, Double Immunofluorescence Staining, Immunofluorescence, Western Blot, Flow Cytometry, Double Staining, Staining, Immunoprecipitation, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: NLRP3 agonist nigericin counteracted the inhibitory effect of silencing galectin‐3 on pyroptosis, apoptosis and necroptosis in macrophages. (A) Confocal microscopy with double immunofluorescence staining for galectin‐3 (red) and NLRP3 (green) in macrophages reveals the colocalisation of galectin‐3 with NLRP3. Scale bar: 25 µm. (B and C) Flow cytometry (B) and quantification analysis (C) with annexin V/PI double staining show that silencing galectin‐3 decreases the percentage of apoptotic cells in ox‐LDL‐induced macrophages, and nigericin robustly blunts the inhibitory effect of siGalectin‐3. (D–F) Flow cytometry (D) and quantification analysis (E) with PI/Hoechst staining (F) show that silencing galectin‐3 diminishes the percentage of PI‐positive cells in ox‐LDL‐induced macrophages, and nigericin mostly abolishes the protective effect of siGalectin‐3. Scale bar: 50 µm. (G) Silencing galectin‐3 suppresses the LDH release in ox‐LDL‐induced macrophages, which is largely abrogated by nigericin. (H) Silencing galectin‐3 lessens the intracellular lipid droplet in ox‐LDL‐induced macrophages, while nigericin exerts the opposite effect. Scale bar: 50 µm. (I) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (J) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (K) The activity of caspase‐3 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (L and M) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (N) Silencing galectin‐3 inhibits the release of inflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) in ox‐LDL‐induced macrophages, and nigericin effectively blocks the role of siGalectin‐3. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

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

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Pyroptosis, apoptosis and necroptosis in macrophages coordinately occurred in ApoE −/− mice fed an HFD, which are alleviated by galectin‐3 deficiency, and conversely are aggravated by NLRP3 agonist nigericin. (A) Pyroptosis, apoptosis and necroptosis of macrophages are identified in the aortas of ApoE −/− mice fed an HFD, as evidenced by plasma membrane pore (red arrows), chromatin condensation (red arrows), and electron‐light zone (red arrows) by transmission electron microscopy. Scale bar: 2.5 µm. (B) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (pink) and DAPI (blue) in the aortas of ApoE −/− mice fed HFD or normal diet reveals the potential crosstalk among pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Three‐positive cells are shown by the arrows. Scale bar: 50 µm. (C–E) Dual immunofluorescence staining for caspase‐3 (C)/GSDMD (D)/RIPK3 (E) (red), F4/80 (green), and DAPI (blue) in the aortas of ApoE −/− mice fed an HFD or normal diet demonstrate that GSDMD/caspase‐3/RIPK3 immunoreactivity colocalises with macrophage marker CD68. Scale bar: 50 µm. (F) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in the aortas of ApoE −/− mice fed with a normal diet or HFD, NLRP3 agonist nigericin‐treated ApoE −/− mice fed with an HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (G) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase 8 and cleaved caspase 8 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with HFD. (H) The activity of caspase‐3 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (I and J) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (K and L) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐κB and phospho‐NF‐κB in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. n = 4–8 mice per group. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Marker, Western Blot, Activity Assay