Fig 2: Pre-OCs are more susceptible to TXNRD1 inhibitors than BMDMs, and safe doses of TXNRD1 inhibitors for pre-OCs do not impair osteoclast differentiation and bone resorption.a, schema for osteoclast differentiation stages in vitro. Analysis was conducted at D0 (BMDMs), D3 (pre-OCs), and D5 (ma-OCs). (b to c), the viability of BMDMs and pre-OCs was measured after 24 h of incubation with AF (b) or TRi-1 (c). n = 3 per group. d, PI staining of BMDMs and pre-OCs was measured after 24 h of incubation with AF (1.0 µM, 2.0 µM) or TRi-1 (2.0 µM, 5.0 µM). n = 3 per group. (e to g), BMDMs incubated with mediums containing M-CSF, RANKL, and maximum safe doses of AF or TRi-1 for pre-OCs. TRAP staining and F-actin formation assay of ma-OCs on day 5 with quantification of TRAP-positive cell area (%) (f) and osteoclast size (mm2) (g). Scale bar, 50 µm. n = 3 per group. (h to i), bone resorption assay was conducted on bovine cortical bone slices and the bone slices were scanned on day 7 (h). Scale bar, 50 µm. n = 3 per group. Bone resorption area (%) (i) were used to evaluate the effects of different dose of AF and TRi-1 on bone resorption capacity. All data in this figure are represented as mean ± SD. ns, no significance, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: Treatments that prevent disulphide accumulation or pharmacological SLC7A11 inhibition rescued the cell death induced by TXNRD1 inhibitors in pre-OCs. NFATc1 knockdown in pre-OCs attenuated TXNRD1 inhibitor-induced cystine accumulation and decreased the sensitivity to TXNRD1 inhibitors. SLC7A11 knockout eliminated the difference in sensitivity of BMDMs and pre-OCs to TXNRD1 inhibitors, and overexpression of SLC7A11 or NFATc1 in BMDMs increased cell sensitivity to TXNRD1 inhibitorsa, the HPLC-MS/MS results of intracellular cystine concentrations in BMDMs, BMDMs treated with TXNRD1 inhibitors, pre-OCs, and pre-OCs treated with TXNRD1 inhibitors. n = 3 per group. b, the viability of pre-OCs was measured after 24 h of incubation with a medium containing 2 µM AF or 5 µM TRi-1 with or without 5 mM NAC. n = 3 per group. c, the HPLC-MS/MS results of intracellular cystine concentrations in pre-OCs treated with TXNRD1 inhibitors and pre-OCs treated with both TXNRD1 inhibitors and NAC. n = 3 per group. d, the viability of BMDMs and pre-OCs was measured after 24 h of incubation with a medium containing 2 µM AF or 5 µM TRi-1 with or without 500 µM l-Penicillamine (L-Pen). n = 3 per group. e, the HPLC-MS/MS results of intracellular cystine concentrations in pre-OCs treated with TXNRD1 inhibitors and pre-OCs treated with both TXNRD1 inhibitors and L-pen. n = 3 per group. f, the viability of BMDMs and pre-OCs was measured after 24 h of incubation with a medium containing 2 µM AF or 5 µM TRi-1 with or without 2 mM 2-Mercaptoethanol (2 ME). n = 3 per group. g, The HPLC-MS/MS results of intracellular cystine concentrations in pre-OCs treated with TXNRD1 inhibitors and pre-OCs treated with both TXNRD1 inhibitors and 2 ME. n = 3 per group. h, the viability of BMDMs and pre-OCs was measured after 24 h of incubation with medium containing 2 µM AF or 5 µM TRi-1 with or without Sulfasalazine (SSZ) (low dose 100 µM, high dose 400 µM). n = 3 per group. i, the HPLC-MS/MS results of intracellular cystine concentrations in pre-OCs treated with TXNRD1 inhibitors and pre-OCs treated with both TXNRD1 inhibitors and SSZ. n = 3 per group. (j and k), the viability of siCTL and siNFATc1 pre-OCs was measured after 24 h of incubation with AF (j) or TRi-1 (k). n = 3 per group. l, The HPLC-MS/MS results of intracellular cystine concentrations in siCTL and siNFATc1 pre-OCs treated with TXNRD1 inhibitors, n = 3 per group (m and n), the viability of SLC7A11 KO BMDMs and pre-OCs was measured after 24 h of incubation with AF (m) or TRi-1 (n). n = 3 per group (o and p), western blot analysis of SLC7A11 protein levels in EV and SLC7A11 overexpression BMDMs. n = 3 per group. q, RT-qPCR analysis of Slc7a11 in EV and SLC7A11 overexpression BMDMs. n = 3 per group. (r and s), the viability of EV, NFATc1 overexpression, and SLC7A11 overexpression BMDMs was measured after 24 h of incubation with AF (r) or TRi-1 (s). n = 3 per group. All data in this figure are represented as mean ± SD. ns, no significance, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: SLC7A11-mediated cystine uptake and subsequent cystine reduction to cysteine in pre-OCs promote the stress of cellular disulphide metabolism.(a to b), the reductive abilities of TrxR with or without TXNRD1 inhibitors were detected by TRFS probes (a) and were quantified as MFI of TRFS (b). Scale bar, 20 µm. n = 5 per group. c, the relative quantitative results of intracellular GSH, GSSG, and GSH/GSSG in BMDMs and pre-OCs. n = 3 per group. d, the untargeted metabolome results (pre-OC versus BMDM, SLC7A11 KO pre-OC versus WT pre-OC). n = 5 per group. (e to f), western blot analysis of SLC7A11 protein levels during osteoclast differentiation. n = 3 per group. g, RT-qPCR analysis of Slc7a11 in BMDMs, pre-OCs, and ma-OCs. n = 3 per group. (h to j), cystine uptake of BMDMs, pre-OCs, and ma-OCs were detected by microscopy (h) and flow cytometry (i) after being incubated with cystine-FITC probes and were quantified as MFI of cystine-FITC (j). Scale bar, 20 µm. n = 3 per group. All data in this figure are represented as mean ± SD. ns, no significance, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: The ferroptosis inhibitors (DFO, Ferro-1), the ROS scavengers (Trolox, Tempol), the apoptosis inhibitor (Z-VAD), the necroptosis inhibitor (Nec-1), or the autophagy inhibitor (CQ) cannot rescue TXNRD1 inhibitor-induced cell death in pre-OCs(a and b), lipid peroxidation was detected by Liperfluo using flow cytometry. n = 3 per group. c, the viability of BMDMs and pre-OCs was measured after 24 h of incubation with RSL3. n = 3 per group. d, the viability of BMDMs and pre-OCs was measured after 24 h of incubation with a medium containing 2 µM AF with or without 2 µM Ferrostantin-1 or 50 µM DFO. n = 3 per group. e, the viability of BMDMs and pre-OCs was measured after 24 h of incubation with a medium containing 5 µM TRi-1 with or without 2 µM Ferrostantin-1 or 50 µM DFO. n = 3 per group. (f and g) ROS levels were detected by Brite 570 using flow cytometry. n = 3 per group. h, the viability of BMDMs and pre-OCs was measured after 5 h of incubation with a medium containing 2 µM AF or 5 µM TRi-1with or without 100 µM Trolox or 25 µM Tempol. n = 3 per group. (i and j) ROS levels were detected by Brite 570 using flow cytometry. n = 3 per group. (k and l) the viability of pre-OCs was measured after 12 h of incubation with a medium containing 2 µM AF or 5 µM TRi-1with or without Nec-1 (5 µM, 10 µM), Z-VAD (5 µM, 10 µM), or CQ (25 µM). n = 3 per group. All data in this figure are represented as mean ± SD. ns, no significance, *P < 0.01, **P < 0.01, ***P < 0.001, ****P < 0.0001.