Fig 2: Bone resorption and LIF expression in Rankl+/- mice. (A) ELISA analysis of RANKL in serum from WT and Rankl+/- male mice at 12 weeks of age (n = 5). (B) Histological analysis of toluidine blue staining in the trabecular area of the tibiae from WT mice (left panel) and Rankl+/- mice (right panel). (C) Trabecular bone volume of tibiae from WT mice and Rankl+/- mice (n = 5). (D) Levels of CTX in serum from WT and Rankl+/- mice (n = 5). (E) Histochemical analysis of TRAP in the trabecular area of the tibiae from WT mice (left panel) and Rankl+/- mice (right panel). TRAP (red). (F) Number of osteoclasts/bone surface (N/mm) (n = 5). (G) Osteoclast surface/bone surface (%) (n = 5). (H) Immunohistochemical analysis of LIF in the trabecular area of the femur from WT mice (top left panels) and Rankl+/- mice (top right panels). The lower two panels show double-staining of LIF and TRAP. LIF-positive cells were marked by red arrows. TRAP-positive cells were marked by blue arrows. (I) LIF-positive cells/bone surface (N/mm). (n = 5). **p < 0.01. Scale bar, 50 µm.

Fig 3: Impacts of the elimination of on bone resorption and LIF expression in C57BL/6 mice. (A) Histochemical analysis of the trabecular area of tibiae from C57BL/6 male mice at 12 weeks of age injected with vehicle (left panel) or the anti-RANKL antibody (right panel). (B) Trabecular bone volume of the tibiae (n = 6). (C) Levels of CTX in serum from C57BL/6 mice injected with vehicle or the anti-RANKL antibody (n = 6). (D) Histochemical studies of TRAP in the trabecular area of the tibiae from C57BL/6 mice injected with vehicle (left panel) or the anti-RANKL antibody (right panel). TRAP (red). (E, F) Number of osteoclasts/bone surface (N/mm) (n = 6). Osteoclast surface/bone surface (%) (n = 6). (G) Immunohistochemical analysis of LIF in the trabecular area of the femur from C57BL/6 mice injected with vehicle (left panels) or the anti-RANKL antibody (right panels). (H) LIF-positive cells/bone surface (N/mm). (n = 6). **p < 0.01. Scale bar, 50 µm.

Fig 4: Knocking down LIFR ameliorated renal fibrotic lesions induced by UUO. (a) Schematic diagram of the experimental design. (b) Real-time PCR showed the mRNA levels of Lifr at day 10 after UUO. (c and d) Representative Western blot (c) and quantitative data (d) showed the protein level of LIFR at day 10 after UUO. (e and f) Representative images of H&E, Masson trichrome and Sirius red staining of renal cortex at day 10 after UUO in three groups (e) and quantitative analyses of fibrotic area (f). Scale bar, 50 µm. (g and h) Representative Western blot (g) and quantitative data (h) showed the protein levels of TNC, Fibronectin, COL1A1, a-SMA, EGR1, SHH at day 10 after UUO. (i and j) Representative Western blot (i) and quantitative data (j) showed the protein levels of p-ERK, ERK, p-STAT3 and STAT3 at day 10 after UUO. (k) Real-time PCR showed the mRNA levels of Il1ß, Il11, Ccl2, Tnf and Lif at day 10 after UUO. Data were expressed as means ± SD. Student's t-test in (f) or one-way ANOVAs (Least-Significant Difference test or Dunnett's T3 test) in (b), (d), (h), (j) and (k) was used for determining the P values while comparing between each pair of groups, respectively, #P < 0.05, *P < 0.01 versus UUO10day+Lenti-Ctrl-shR (n = 6).

Fig 5: LIF promotes pro-fibrotic response in renal tubular cells. (a) Top enriched hallmark pathways identified by GSVA. The R package limma (3.46.0) was used for pathways differential expression analysis between two groups. (b and c) Representative Western blot (b) and quantitative data (c) showed LIF upregulated the expression of Fibronectin, TNC, COL1A1 and a-SMA in primary renal tubular epithelial cells in a dose-dependent manner. #P < 0.05, *P < 0.01 versus controls (n = 4). One-way ANOVAs (Least-Significant Difference test or Dunnett's T3 test) in (c) was used for determining the P values while comparing between each pair of groups, respectively.