Fig 2: Regulation of the expression of sCLU and Gadd45a increases the sensitivity of HCC cells to OXA. (A) Parental SMMC7721 and Bel7402 cells (Control), or cells transfected with Sc or pcDNA3.1-Gadd45a-His vectors were subjected to Western blot analysis. (B) The density of each band in (A) was measured and normalized to that of GAPDH. (C and D) Parental Bel7402 or SMMC7721 cells were seeded in a 96-well plate and were allowed to grow overnight. The next day, the media were replaced with the same media containing various concentrations of oxaliplatin. The cell viability was measured using the CCK-8 assay after incubation for 24 and 48h. (E, F) Parental Bel7402 or SMMC7721 cells, cells transfected with CLU-4 shRNA vector, or cells transfected with pcDNA3.1-Gadd45a-His vectors were cultured in medium with 16nM OXA for 48 h, and then the cell viability was measured. (G, H) Parental Bel7402 or SMMC7721 cells (control), cells transfected with CLU-4 or pcDNA3.1-Gadd45a-His vectors were incubated in the absence or presence of OXA (16 nM) for 48 h, and then the cell apoptosis rates(%) were measured. A significant (P<0.05) difference in the Gadd45a expression from the respective control is denoted by “*”. A significant (P<0.05) reduction from OXA-treated parental cells is denoted by “**”, and a significant (P<0.001) difference is denoted by “†”.

Fig 3: Secretory ApoJ from KLF6PODTA 1° mouse podocytes restores mitochondrial respiration under high glucose conditions.a, b OCR and quantifications in 1° PT treated with conditioned media (CM) from 1° KLF6PODTA (OE) versus NPHS2-rtTA (WT) podocytes in normal glucose (NG) and high glucose (HG). n (biological replicates) = 7; basal respiration: p = 0.0013:OE CM (NG) vs WT CM (HG), p = 0.0008:OE CM (NG vs HG), p = 0.0002:WT CM vs OE CM (HG); ATP production: p = 0.0006:OE CM (NG) vs WT CM (HG), p = 0.0004:WT CM (NG vs HG), p = < 0.0001:WT CM vs OE CM (HG); maximal respiration: p = 0.0343:WT CM (NG vs HG), p = 0.0389:OE CM (NG) vs WT CM (HG); spare respiratory capacity: p = 0.0143:OE CM (NG) vs WT CM (HG), p = 0.0117:WT CM (NG vs HG). c, d Volcano plot showing podocyte secretome and urine proteome from mice (n = 3). e Circos plot highlighting ligand-receptor interactions for upregulated proteins in podocyte secretome and DEGs in preconditioned-PT from snRNA-seq. f Representative images of ApoJ immunohistochemistry. Arrows-ApoJ staining (n = 3 mice/group, scale bar = 25 µm). g, h OCR and quantifications in 1° PT cells treated with CM + anti-ApoJ blocking antibody or CM + anti-IgG in HG (n (biological replicates) = 7); WT CM vs OE CM: p = 0.0259(basal respiration), p = 0.0026(maximal respiration), p = 0.0004(spare respiratory capacity). i, j OCR and quantifications in 1° PT treated with CM from differentiated human podocytes in HG (n (biological replicates) = 6, p = 0.0001(basal respiration), p = 0.0009(ATP production), p = 0.005(maximal respiration), p = 0.0156(spare respiratory capacity)). k Western blot for immunoprecipitation of ApoJ in 1° PT treated with CM in NG/HG. l, m Representative images from co-immunostaining for ApoJ, CaMK1D, Lrp2, and Hoechst. Arrows-indicate ApoJ/CamK1D and ApoJ/Lrp2 colocalization. (scale bar = 50 µm, 5 µm for inset). n, o OCR and quantification in 1° PT treated with recombinant ApoJ ± cilastatin/VEH (n (biological replicates) = 7 for control), (n (biological replicates)=5) for ApoJ+VEH, (n(biological replicates) = 5) for ± cilastatin; basal respiration: p = 0.0003(control vs ApoJ + VEH), p = 0.0157(control vs cilastatin), p = 0.0329 (ApoJ + VEH vs ApoJ + cilastatin); ATP production:p = 0.0001 (control vs ApoJ + VEH), p = 0.0086 (ApoJ + VEH vs ApoJ + cilastatin); maximal respiration:p = 0.0151 (control vs ApoJ + VEH). p Proposed schematic of potential KLF6-ApoJ-CaMK1D signaling between podocytes and PT cells. For all data: *p < 0.05, **p < 0.01 ***p < 0.001, Kruskal–Wallis test with Dunn’s posttest; data presented as mean ± SEM. Source data are provided as a Source Data file. p Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.

Fig 4: Disruption of WAT’s circadian rhythm by tumor PKP3-CLU axis for cachectic fat mass loss.(A–I) MDA-MB-231 tumor-bearing mice with PKP3 knockdown, CLU knockdown, or PKP3 knockdown combined with ectopic overexpression of CLU (n = 5 per group): (A) Body weight (shCTRL vs shPKP3, P = 0.0015; shCTRL vs shCLU, P = 0.0012; shCTRL vs shPKP3+CLU, P = 0.0260; n = 5 per group); (B) Weight change of iWAT, gWAT, BAT (iWAT: shCTRL vs shPKP3, P < 0.0001; shCTRL vs shCLU, P < 0.0001; shCTRL vs shPKP3+CLU, P = 0.0003; gWAT: shCTRL vs shPKP3, P < 0.0001; shCTRL vs shCLU, P < 0.0001; shCTRL vs shPKP3+CLU, P < 0.0001; BAT: shCTRL vs shPKP3, P = 0.1656; shCTRL vs shCLU, P = 0.1322; shCTRL vs shPKP3+CLU, P = 0.7618; n = 5 per group), in a box-plot, the center line represents the median of the data, while the lower and upper limits of the box correspond to the first quartile and third quartile, respectively; (C) Representative iWAT, gWAT, BAT photos, Scale bar: 1 cm; (D) Statistical analysis of H&E staining of iWAT, gWAT, BAT in Fig. 4D (iWAT: shCTRL vs shPKP3, P < 0.0001; shCTRL vs shCLU, P < 0.0001; shCTRL vs shPKP3+CLU, P = 0.7042; gWAT: shCTRL vs shPKP3, P < 0.0001; shCTRL vs shCLU, P < 0.0001; shCTRL vs shPKP3+CLU, P = 0.0009; BAT: shCTRL vs shPKP3, P = 0.2816; shCTRL vs shCLU, P = 0.1173; shCTRL vs shPKP3+CLU, P = 0.0980; n = 10 per group). (E) Tumor-secreted CLU levels in iWAT by ELISA assay (shCTRL vs shPKP3, P < 0.0001; shCTRL vs shCLU, P < 0.0001; shCTRL vs shPKP3+CLU, P = 0.0277; n = 5 per group); (F–I) CLOCK, BMAL1, PER3 expression in WAT in a 48-hour cycle by (F-H) RT-qPCR (n = 4 per group) and (I) Western blot assay. (J) Statistical analysis of H&E staining of iWAT, gWAT, BAT in Fig. 6G (iWAT: shCTRL vs shPKP3, P < 0.0001; shCTRL vs shCLU, P < 0.0001; gWAT: shCTRL vs shPKP3, P = 0.0002; shCTRL vs shCLU, P = 0.0068; BAT: shCTRL vs shPKP3, P = 0.0745; shCTRL vs shCLU, P = 0.1963; n = 3 per group). The data are presented as mean ± SEM (n = [X] biologically independent samples). *p < 0.05, **p < 0.01, ***p < 0.001, ns for not significant. Statistical comparisons between groups were performed using unpaired two-tailed Student’s t tests. Source data are available online for this figure.

Fig 5: Identification of tumor-secreted CLU as a causal factor of cachectic fat mass loss in TNBC models.(A) A volcano plot represents the expression of all proteins: significantly downregulated and upregulated proteins (|log2 FC| >1 and P < 0.05) are colored blue and red using unpaired two-tailed Student’s t tests, respectively; proteins that do not show significant expression changes are colored gray (Healthy, n = 22; Cachexia, n = 20). (B) Secreted CLU levels in serum samples from cachectic TNBC patients compared to healthy women, slim healthy women, and non-cachectic TNBC patients by ELISA assay. (CTRL, n = 22; Slim CTRL, n = 21; Non-cachectic TNBC, n = 21; Cachectic TNBC, n = 20; CTRL vs Non-cachectic TNBC, P = 0.0009; CTRL vs Cachectic TNBC, P < 0.0001; Slim CTRL vs Non-cachectic TNBC, P = 0.0007; Slim CTRL vs Cachectic TNBC, P < 0.0001; Non-cachectic TNBC vs Cachectic TNBC, P = 0.0055). (C–H) MDA-MB-468 tumor-bearing mouse model with and without CLU knockdown (n = 5 per group): (C) Representative Western blotting images showing serum CLU levels at 35 days, 42 days after inoculation of cancer cells into mouse models; (D) Body weight of mice over the time after cancer cell inoculation (35 day, P = 0.0023; 42 day, P = 0.0008); (E) Change of iWAT, gWAT, BAT weight (shCTRL vs shCLU: iWAT, P < 0.0001; gWAT, P = 0.0005; BAT, P = 0.1135; n = 5 per group), in a box-plot, the center line represents the median of the data, while the lower and upper limits of the box correspond to the first quartile and third quartile, respectively; (F) Representative iWAT, gWAT, BAT photos, Scale bar: 1 cm; (G) Representative H&E staining images of iWAT, gWAT, BAT, Scale bar: 50 μM; (H) Secreted CLU levels in iWAT by ELISA assay (shCTRL vs shCLU, P < 0.0001; n = 5 per group), in a box-plot, the center line represents the median of the data, while the lower and upper limits of the box correspond to the first quartile and third quartile, respectively. The data are presented as mean ± SEM (n = [X] biologically independent samples). **p < 0.01, ***p < 0.001, ns for not significant. Statistical comparisons between groups were performed using unpaired two-tailed Student’s t tests. Source data are available online for this figure.