Fig 1: Expression of known prolipolytic factors and predicted secreted proteins in three human cancer cell lines exposed to acidosis. (a-f) mRNA relative expression of Il6 (a), Tnfα (b), Adm (c), Zag (d), Pthlh (e) and Prl2c (f) in the human pH6.5/cancer cell lines, FaDu, SiHa and HCT116, and in murine pH6.5/C26 and LLC. Gene expression in pH6.5/cancer cells was expressed relative to the mean gene expression in pH7.4/cancer cells (pH7.4 = 1; dotted line). Data are presented as the mean ± SEM. n = 4–7. Each point corresponds to one n. (g, j) Venn diagrams were constructed from predicted secreted proteins differentially expressed between pH6.5/and pH7.4/FaDu, SiHa and HCT116 cancer cell lines. (h,i) Principal Component Analyses (PCA) was performed on predicted secreted proteins from human pH6.5/(pink) and pH7.4/FaDu (circle), SiHa (cross) and HCT116 (triangle) cancer cells (green) depicting PC1 and 2 (h) and PC1 and 3 (i). (k,l,m) Gene expression (in counts) of 24 predicted secreted proteins upregulated upon acidosis and shared between FaDu (k), SiHa (l) and HCT116 (m) cancer cells, ranked by the mean log2 fold change of 3 cancer cell lines. Data are presented as the mean ± SEM. (g–m). n = 5. Il6, interleukin 6; Tnfα, tumour necrosis factor alpha; Adm, adrenomedullin; Zag, zinc-alpha-2-glycoprotein; Pthlh, parathyroid hormone like hormone; Prl2c, prolactin family 2 subfamily c member 2. Lama1, laminin subunit alpha 1; Tnfrsf1B, tnf receptor superfamily member 1B; Igsf10, immunoglobulin superfamily member 10; Patl2, Pat1 homolog 2; C4B, complement c4b; C4A, complement c4a; Btn3A3, butyrophilin subfamily 3 member a3; Wdr90, wd repeat domain 90; Gusb, glucuronidase beta; Btn3A1, butyrophilin subfamily 3 member A1; Hla-A, major histocompatibility complex, class I, A; Btn3A2, butyrophilin subfamily 3 member A2; Sowaha, sosondowah ankyrin repeat domain family member A; Atad5, ATPase Family AAA domain containing 5; Ppox, protoporphyrinogen oxidase; Cfdp1, craniofacial development protein 1; Nptxr, neuronal pentraxin receptor; H6pd, hexose-6-phosphate dehydrogenase/glucose 1-dehydrogenase; Mrpl55, mitochondrial ribosomal protein L55; Sema3F, semaphorin 3F, SerpinE3, serpin family E member 3; Dpp7, dipeptidyl peptidase 7; Tmc06, transmembrane and coiled-coil domains 6; Cdc23, cell division cycle 23; FaDu, hypopharyngeal cancer cells; SiHa, cervix cancer cells; HCT116, colon cancer cells; C26, colon cancer cells; LLC, Lewis lung carcinoma cells; pH6.5, pH6.5-exposed cancer cells; pH7.4, pH7.4-maintained cancer cells. ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 2: Human GUSB protein induces adipose lipolysis in murine and human adipocytes. (a) 3T3-L1 adipocytes (n = 4, each point corresponds to one n) treated with control medium without (control) or with 1 μM isoproterenol (Iso), or human recombinant GUSB at concentration of 0.3–300 ng/ml, for 6 h or 24 h. (b) Human primary subcutaneous and visceral adipocytes (n = 3, each point corresponds to one patient) treated with control medium (control) or 150 ng/ml human recombinant GUSB, for 24 h or 48 h. The results of the two-way ANOVA are shown below the graphs. (c) Plasma human GUSB protein concentration in male CT mice (sham-injected) and HCT116 tumour-bearing mice on day 16 post-injection (necropsy). (d) Correlation of right anterior, left anterior, posterior subcutaneous (SAT), visceral epididymal (EAT) and mesenteric (MAT) adipose tissue weights with plasma human GUSB concentrations, in HCT116 tumour-bearing mice, on the day of necropsy. In bold p < 0.1, in green bold p < 0.05. (e) Human GUSB protein concentration in conditioned media from human pH6.5/and pH7.4/FaDu, SiHa and HCT116 cancer cell lines (n = 5–6, each point corresponds to one n). (f) Human plasma protein GUSB concentrations in male FaDu tumour-bearing mice on the day of necropsy (n = 12). (g–h) Gusb expression in HCT116 tumour (g) and GUSB circulating levels (h) in HCT116-tumour bearing mice treated with NaHCO3 (n = 8–12, each point corresponds to one n). FaDu, human hypopharyngeal cancer cells; SiHa, human cervix cancer cells; HCT116, human colon cancer cells; GUSB, glucuronidase beta. (c, e-f) Each point corresponds to one mouse. Data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ∗ shown in grey are t-test analysis results compared to the control condition.
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