Figure 6. Loss of Plekhm1 has no effects on TFEB expression, lysosome biogenesis, autophagic, and endocytic degradation pathways in mature osteoclasts.

(A) qPCR detection of mRNA expression of Tfeb and its downstream target genes, Clcn7, Dpp7, Ostm1, and Tcirg1, during osteoclast differentiation in wild-type and Plekhm1^–/– (KO) cultures (mean ± SD, n = 3). m, bone marrow monocytes; pOC, preosteoclasts; OC, mature osteoclasts. (B) Western blot detection of protein expression of TFEB during osteoclast differentiation in WT and KO cultures. The lysate of 293-T cells served as a positive control. Actin served as a loading control. The data are representatives of 3 independent experiments. (C) Western blot detection of LAMP-2 expression during osteoclast differentiation. Tubulin served as a loading control. The experiment was repeated 3 times. (D) Mature osteoclasts were either untreated (FBS) or underwent serum and amino acid starvation in HBSS buffer for 2 hours. In another set of experiment, osteoclasts were treated with DMSO, 250 nm of mTOR inhibitor Torin1, and 50 μM of lysosome inhibitor chloroquin (CQ) for 2 hours. The protein levels of phospho-mTOR, LC3-I, and LC3-II were detected by Western blots. The ratio of LC3-II/LC3-I was measured by densitometry of Western blots using NIH ImageJ software. The experiment was repeated twice. (E and F) Receptor degradation assays of c-Fms (E) and EGF receptor (EGF-R) (F) in osteoclasts stimulated with cytokines for the indicated times, as detected by Western blotting and densitometry quantification using NIH ImageJ software. Actin served as loading controls. The experiments were repeated twice.