Fig 1: Mac-2-Deficient Bone Marrow (Mac-2-/-) Protects Wild-Type Mice (Mac-2⁺/⁺) Against DOX-Induced Cardiotoxicity (a) Experimental Design: Myeloablated recipient mice underwent bone marrow transplantation using either Mac-2-deficient (KO) or wild-type (WT) donor cells. Successful engraftment, achieving 96% donor CD45⁺ chimerism by 6 weeks, was confirmed by flow cytometry. Mice were then treated with DOX to evaluate cardiotoxic effects. b Survival Analysis: Survival rates were comparable across groups (Log-rank test, p = 0.08, n = 9–13). c Cardiac tissue weights did not differ among the transplant groups (p > 0.05; n = 9–13). d-g Cardiac Function: Mice transplanted with Mac-2-deficient bone marrow (DOX WT [KO BM], green bars) maintained significantly better cardiac function, as indicated by improved stroke volume (d), cardiac output (e), left ventricular ejection fraction (f), and left ventricular fractional shortening (g) compared to mice receiving WT (Mac-2+) bone marrow from either (DOX KO [WT BM], yellow bars) or (DOX WT [WT BM], brown bars), while control WT mice (blue bars) showed normal cardiac function. Numerical p-values denote comparisons among BM transplanted groups (p = 0.05; n = 9–13), while stars represent significance relative to Control WT (*p = 0.05, **p < 0.001, ***p < 0.0001). h BNP levels: Plasma BNP concentrations were significantly elevated in DOX WT (WT BM) and DOX KO (WT BM) groups but lower in DOX WT (KO BM), suggesting improved cardiac stress response. i-o Inflammatory and Fibrotic Markers: Mice transplanted with WT bone marrow [DOX KO (WT BM), yellow bars] had elevated expression of Il6 (i),Tnf (j),Sqstm1 (k),Tfeb (l),Casp3 (m),Tgfb1 (n) and collagen-1 (o), markers of inflammation, apoptosis, and fibrosis. In contrast, these markers were significantly reduced in mice transplanted with Mac-2-deficient bone marrow [DOX WT (KO BM), green bars]. p Histological analysis showed comparable fibrosis levels across groups, with representative images shown for each group (scale bar, 20 μm). Numerical p-values denote comparisons among BM transplanted groups (p = 0.05; n = 9–13), while stars represent significance relative to Control WT (*p = 0.05, **p < 0.001, ***p < 0.0001). WT, wild-type; KO, Mac-2 knockout; BM, bone marrow; DOX, doxorubicin
Fig 2: DOX does not alter cardiac morphology, function, or inflammatory gene expression in Mac-2 knockout mice. a-b Timeline of DOX administration with no unexpected mortality. c Heart weight/body weight ratio was unchanged between saline and DOX-treated KO mice (n = 9–12). d-g Echocardiography revealed no significant changes in stroke volume (d), cardiac output (e), left ventricular ejection fraction (f), left ventricular fractional shortening (g), between groups (n = 9–13). h-k Cardiac gene expression shows no significant DOX-associated changes in Il6 (h), Tnf (i), Sqstm1 (j), Tfeb (k), Casp3 (l), Tgfb1 (m) and collagen-1(n), markers indicate minor, non-significant trends toward elevation in DOX KO hearts (n = 9–12). o Histological analysis of fibrosis, defined as percent fibrotic area in heart sections, did not differ between groups. (n = 9–12). Scale bar, 20 μm. KO, Mac-2 knockout; DOX, doxorubicin
Fig 3: Galectin-3 (Mac-2) regulates DOX uptake, macrophage activation, and downstream stress signaling in vitro. a Flow cytometry showing increased percentages of Mac-2+ RAW 264.7 macrophages after 24-hour treatment with 0.1 µM, 0.5 µM, and 1 µM DOX (p < 0.01; n = 6–9). b Schematic of CRISPR/Cas9-mediated Lgals3 (Mac-2) knockout in RAW 264.7 macrophages targeting exon 3 with specific guide RNAs. Fluorescence microscopy and flow cytometry confirming efficient transfection with 86% (8600/10000) co-expression of RFP and GFP in knockout cells, scale bars: 50 μm. c Flow cytometry histograms quantifying % DOX⁺ macrophages in WT and Mac-2 KO cells after 24 h DOX treatment (0.1–1 µM). Mac-2 KO macrophages show significantly reduced DOX uptake at every concentration tested (p < 0.001; n = 4–8). d Flow cytometry quantification of % marker⁺ macrophages (CCR2, F4/80, MHC-II) after 24 h of 0.1 µM DOX. Mac-2 KO cells display significantly lower CCR2⁺, F4/80⁺, and MHC-II⁺ populations compared with WT (p < 0.001; n = 8). e IL-6-induced chemotactic migration is significantly increased in WT macrophages compared to KO cells (p < 0.001; n = 8). f Relative mRNA levels of pro-inflammatory (M1) genes (Ccl2, Il6, Tnf) increase with DOX in WT macrophages but are attenuated in KO; n = 6–9. (* represents p = 0.05; n = 6–9). g Anti-inflammatory (M2) markers (Il10, Cd206, Arg1) are reduced in WT macrophages after DOX but partially restored in KO. h Lysosomal/Autophagy signaling genes (Tfeb, Lamp1, Map1lc3b, Sqstm1 were altered in WT macrophages following DOX, but normalized or rescued in KO (p = 0.05). i Macrophage-mediated caspase-3/7 activation in cardiomyocytes (CMs): CMs were either treated directly with DOX or co-cultured with WT or KO macrophages (± DOX). The “indirect” condition refers to CMs cultured with pre-DOX-treated macrophages (0.1 µM, 24 h), without direct drug exposure. Caspase-3/7 activation is increased in CMs co-cultured with DOX-treated WT macrophages but not KO macrophages (p < 0.0001). j Phospho-TFEB (Ser211) ELISA in cardiac fibroblasts co-cultured with macrophages shows increased activation with DOX-treated WT macrophages, suppressed with KO macrophages (p < 0.01). DOX, doxorubicin; KO, Mac-2 knockout; WT-C, wild-type controls; KO-C, knockout controls, CMC, cardiomyocytes; CFC, cardiac fibroblasts
Fig 4: DOX alters cardiac morphology, function, and inflammatory gene expression in mice. a-b Timeline of DOX administration with no unexpected mortality. c DOX-treated mice showed cardiac enlargement (Heart weight/Body weight ratio) compared with saline controls (p = 0.05; n = 10–13). Cardiac functional analysis revealed significant decreases in stroke volume (d), cardiac output (e), left ventricular ejection fraction (f), left ventricular fractional shortening (g), in the DOX group compared to that in saline-administered controls (p = 0.05; n = 10–13). Transcript levels of Lgals3 (h), Il6 (i), Tnf (j), Sqstm1 (k), Tfeb (l), Casp3 (m), Tgfb1 (n) and Col1a1 (o) were higher in DOX-treated mice than in saline-administered controls (p = 0.05; n = 10–13). p Increased interstitial collagen deposition indicating enhanced fibrosis in the treated group (p < 0.01; n = 9–13); scale bar, 20 μm. WT, wild-type, DOX, doxorubicin
from Cell Signaling Technology for PathScan ® RP Phospho-TFEB (Ser211) Sandwich ELISA Kit