Fig 2: Pip5k1c loss impairs osteoclast formation and bone resorption.A, serum levels of collagen type I cross-linked C-telopeptide (CTX-1) from 4-month-old control and cKO male mice. Results were expressed as mean ± SD; N = 6 mice for control, N = 7 mice for cKO. *p < 0.05 versus control, Unpaired two-tailed t test; B, TRAP staining of tibial sections from 4-month-old male mice. Scale bar, 50 µm. C and D, quantitative analyses of the osteoclast surface per bone surface (C) and osteoclast numbers per bone perimeter (D) of primary spongiosa (PS) and secondary spongiosa (SS) from (B). Results were expressed as mean ± SD; N = 6 mice per group from 4-month-old male mice, *p < 0.05 versus control, unpaired two-tailed t test. E, bone marrow monocytes (BMMs) from 4-month-old male control (WT) and cKO mice were cultured in osteoclast proliferation medium (a-MEM, 20% FBS +10 ng/ml M-CSF) for 3 days and osteoclast differentiation medium (a-MEM + 20% FBS, 10 ng/ml M-CSF, 50 ng/ml RANKL) for 6 days, followed by TRAP staining. Scale bar, 100 µm. F, quantitative analyses of TRAP+ multinucleated cells (MNC, defined as having three or more nuclei per cell) generated by each group. Results were expressed as mean ± SD; **p < 0.01 versus control, unpaired two-tailed t test. G and H, flow cytometry. Bone marrow cells from 4-month-old control and cKO male mice were stained with CD3-FITC, CD45R-PE, CD11b-APC-Cy7, CD117-Percp-Cy5.5, and CD115-APC antibodies and analyzed with flow cytometry as described in Methods. The CD3- CD45R- CD11b-/low CD117+ CD115hi cells in bone marrow cells were gated (G) and calculated (H). Results were expressed as mean ± SD; N = 3 male mice per group. **p < 0.01 versus WT, unpaired two-tailed t test. I and J, BMSC (bone marrow stromal cell)-BMM (bone marrow cell) co-cultures. Primary BMSC and BMM from 4-month-old control and cKO male mice were co-cultured for 14 days in the absence of M-CSF and RANKL, followed by TRAP staining (I) and quantitative analyses of TRAP+ cells (J). Results were expressed as mean ± SD; N = five mice per group. **p < 0.01; two-way ANOVA (interaction: F (1,16) =1.744, p = 0.2053; Row factor: F (1,16) = 23.07, p = 0.0002; column factor: F (1,16) = 54.28, p < 0.0001). K–M, ELISA assay. Serum levels of Rankl (K) and Opg (L) from 4-month-old control and cKO male mice were determined. The Rankl/Opg ratio was calculated (M). Results were expressed as mean ± SD; N = 7 mice for control, N = 8 for cKO. *p < 0.05 versus control, n.s., not significant; unpaired two-tailed t test. N and O, IHC staining. Tibial sections of 4-month-old control and cKO male mice were subjected to IHC staining for expression of Rankl. Scale bar, 50 µm. Quantification data (O). Results were expressed as mean ± SD; N = 6 mice per group. **p < 0.01 versus control, Unpaired two-tailed t test. TRAP, tartrate-resistant acid phosphatase; IHC, immunohistochemical; OC.S/BS, osteoclast surface per bone surface; OC.N/BPm, osteoclast numbers per bone perimeter; Rankl, receptor activator of nuclear factor-?B ligand; Opg, osteoprotegerin.

Fig 3: Confocal micrographs of DPP4 and actin immunofluorescence staining from wildtype (WT) and respective knockout (KO) cell lines.(A) DPP4 localization is restricted to the actin-rich microvillus brush-border in WT cells. (B, C) DPP4 can still be targeted to the apical plasma membrane, but also mislocalizes to subapical compartments in TM9SF4- (B) and ANO8-KO (C) cells (white arrowheads). (D) PIP5K1C-KO cell lines display large, DPP4- and actin-positive, basolateral compartments (white arrowheads). (E) DPP4 mislocalizes to subapical compartments in ARHGAP33-KO cells (white arrowheads). (F) MTMR2-KO cell lines display large, actin-rich basolateral compartments, that also show DPP4 (white arrowheads). (G, H) In GALNT2- and TMEM110-KO cells, DPP4 localizes strictly to the apical plasma membrane. (I) Delocalized DPP4 in polarized CaCo2 cells was quantified (dot box plot, Mann–Whitney U test. **p<0.01, ***p<0.005, n.s., not significant, n = 100 cells per condition). XY = top view of polarized monolayer; XZ/YZ = lateral view of polarized monolayer. Scale = 5 µm.

Fig 4: Electron micrographs with various configurations of abundant ectopic microvilli reflect polarity defects in 18-day-old 2D cultures of selected CaCo2 knockout (KO) cell lines.(A) Distinct intracellular lumen, lined by ectopic brush-border and adjacent terminal web, thus, a typical microvillus inclusion (arrow) inside a PIP5K1C-KO cell. Arrowheads mark lysosomes. (B) Spherical intracellular mass of tangled microvilli associated with ectopic terminal web (arrow) inside a GALNT2-KO cell. Double arrowheads mark enlarged late endocytic organelles. (C) Paracellular, basolateral spot of densely packed microvilli plus associated ectopic terminal web (arrow) in polarized TM9SF4-KO-cell culture. (D) Numerous, slightly bent microvilli facing widened intercellular space (arrows) in polarized ANO8-KO cell culture. (E) Paracellular microvillar spot with adjacent ectopic terminal web (arrow) and associated cell junctions (double arrows) in polarized TMEM110-KO cell culture. Arrowheads mark lysosomes, double arrowhead marks poorly structured late endocytic organelles. (A–E) Scale = 1 µm.

Fig 5: Ultrastructure of late endocytic and lysosomal organelles in CaCo2 wildtype (WT) cells versus selected knockout (KO) cell lines of cryo-fixed 18-day-old polarized filter cultures under steady-state conditions.(A, B) WT CaCo2 cells with normal endosomes and lysosomes: multivesicular bodies (MVBs) with varying contents (regarding intraluminal vesicle [ILV] number, size, and staining patterns), different types of (autophago)lysosomes (arrowheads), all filled with clearly stained, finely granular material plus membrane remnants (i.e., multilamellar bodies), and/or opaque, amorphous residues (i.e., dense-core lysosomes). Double arrowheads mark examples of inconspicuous spherical organelles with weakly stained, quite homogeneous granular contents harboring only sporadic ILVs and/or other structured components, interpreted as kind of endolysosome (Bright et al., 2016). (C) Large, poorly structured putative endolysosome (double arrowhead), virtually the predominant type of late endocytic and/or catabolic organelles in ANO8 KO at steady state. (D) Moderately sized and enlarged putative endolysosomes (double arrowheads) in PIP5K1C KO cells. (E) Enlarged putative endolysosome (arrowhead), the predominant type of late endocytic/catabolic organelles in TM9SF4 KO cells. (F) Moderately enlarged putative endolysosomes (double arrowheads) occurring together with normal lysosomes in ARHGAP33 KO cells. (G) Enlarged putative endolysosomes (double arrowheads) occurring together with slightly smaller lysosomes in MTMR2 KO cells. Arrows mark lateral microvilli. (A–G) Scale = 500 nm.