Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a C4a and C4b restore the astrocytic engulfment of microglial debris in vitro in serum-free culture medium. b Quantifications of the phagocytic influence by complement supplementation and preopsonization in serum-free culture medium. N = 11 independent biological replicates of each group. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). c Scheme of in vivo microglial depletion and time points for analysis. d Reanalysis of RNA-seq data from whole-brain homogenate (GSE108269 ) showing that Gfap and C4b are upregulated and C1qa is downregulated during microglial ablation, whereas C2 , C3 and C4a remain at low levels and are unaffected. N = 5 mice at D0 and N = 4 mice at D2 to D21. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). e qPCR further confirmed the upregulation of C4b in sorted astrocytes upon microglial depletion. N = 5 in each group. Two-tailed independent t test. f Scheme of the in vivo examination of astrocytic engulfment using AAV PHP.eB-based astrocyte labeling and microglial depletion. g Confocal orthogonal colocalization and 3D reconstruction show that C4b −/− impairs the astrocytic engulfment of microglial debris under physiological condition (D21) and upon CSF1R inhibition (D23). h Quantification of microglial debris engulfment by astrocytes. N = 7 (D21) and 8 (D23) WT mice, and N = 3 (D21) and 5 (D23) C4b −/− mice. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). PLX5622 PLX5622-formulated AIN-76A diet, CD control AIN-76A diet, IV intravenous, MFI mean fluorescence intensity, Ctx cortex, Hipp hippocampus, OB olfactory bulb. Data are presented as mean ± SD. The source data are provided as a Source Data file.

Article Snippet: Recombinant mouse complement component C2 protein CF (C2) was acquired from R&D Systems (Cat#: 6725-SE-010).

Techniques: In Vitro, In Vivo, RNA Sequencing, Two Tailed Test, Labeling, Inhibition, Control, Fluorescence

Journal: iScience

Article Title: NPC2 suppresses osteolytic metastasis in lung adenocarcinoma via the AKT/mTOR pathway and tumor-osteoclast crosstalk

doi: 10.1016/j.isci.2026.115017

Figure Lengend Snippet: Screening for target gene NPC2 with scRNA-seq analysis (A) Heatmap of TOP 10 differential genes in each subset of epithelial cells. (B) GO enrichment analysis for differentially expressed genes in epithelial cells of PT, MPT and MT groups. Compared with PT group, these genes enriched in lysosomal membrane were significantly declined in epithelial cells of MPT group. (C) Contrast to PT group, the epithelial cells in MT group presented upregulated genes that were enriched in lysosomal lumen. (D) Uniform manifold approximation and projection (UMAP) was utilized for unsupervised clustering and unbiasedly visualizing cell subsets of epithelial cells by groups on a two-dimensional map. (E) Unsupervised pseudotime analysis was conducted for epithelial cells across all three groups. (F) Copy number variation (CNV) analysis of epithelial cells was performed using macrophages as a reference. A hierarchical heatmap displays CNVs for individual cells (rows) from NPC2 expressing tumor cells, inferred from the average expression of 101 genes around each chromosomal position (columns). Red: amplifications; Blue: deletions. (G) Boxplot of CNV scores for each cell group. Data are presented as the mean ± SEM, ∗∗∗∗ p < 0.0001.

Article Snippet: The primary antibodies used were as follows: NPC2, Cathepsin B and D Polyclonal antibodies GAPDH Monoclonal antibody (Proteintech); Anti-NF-kB P65 and Anti-NF-kB P65 (phospho S536) antibodies (Abcam); mTOR(7C10) and Phospho-mTOR Rabbit (ser2448) mAb, AKT Rabbit mAb and Phospho-Akt (Ser473) Rabbit mAb (CST, Danvers, USA).

Techniques: Membrane, Expressing

Journal: iScience

Article Title: NPC2 suppresses osteolytic metastasis in lung adenocarcinoma via the AKT/mTOR pathway and tumor-osteoclast crosstalk

doi: 10.1016/j.isci.2026.115017

Figure Lengend Snippet: Clinical research about NPC2 and bone metastasis in LUAD (A) H&E and NPC2 staining of pathological sections from patients with LUAD at various sites. For NPC2 staining, the brown-yellow color resided in the cytoplasm is the positive result; scale bars, 200 μm. (B) IHC scores for NPC2 staining categorized by pathological sections from different sites; Data are expressed as the mean ± SEM ( n = 5); ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01. (C) KM survival curves for overall survival in patients with LUAD with varying NPC2 expression levels obtained from public databases (left: GEPIA2; right: LinkedOmics). (D) KM curves of metastatic LUAD (left) and LUAD bone metastasis (right) based on different serum NPC2 levels. Patients were categorized into high-expression (NPC2 concentration >32.27 ng/mL) and low-expression groups (NPC2 concentration ≤32.27 ng/mL), based on the median concentration of 32.27 ng/mL (range: 8.29 to 152.28 ng/mL), with 20 patients in each group.

Article Snippet: The primary antibodies used were as follows: NPC2, Cathepsin B and D Polyclonal antibodies GAPDH Monoclonal antibody (Proteintech); Anti-NF-kB P65 and Anti-NF-kB P65 (phospho S536) antibodies (Abcam); mTOR(7C10) and Phospho-mTOR Rabbit (ser2448) mAb, AKT Rabbit mAb and Phospho-Akt (Ser473) Rabbit mAb (CST, Danvers, USA).

Techniques: Staining, Expressing, Concentration Assay

Journal: iScience

Article Title: NPC2 suppresses osteolytic metastasis in lung adenocarcinoma via the AKT/mTOR pathway and tumor-osteoclast crosstalk

doi: 10.1016/j.isci.2026.115017

Figure Lengend Snippet: The malignant biological behaviors of LUAD cells affected by NPC2 (A) The level of NPC2 mRNA relative to GAPDH declined in shR groups ( n = 3). (B) NPC2 expression was reduced in the shR groups, as detected by Western blot. (C–E) CCK-8 assay examined the effect of NPC2 knockdown on LUAD cell growth (left: A549 cells; middle: PC9 cells; right: H1975 cells), OD values were significantly higher in the shR group of PC9 cells after 24 h compared to the NC group. n = 5. (F) The colony-forming efficiency of each group is shown by the percentage of colony numbers. n = 3. (G) Representative images of colony formation assay of A549 and PC9 cells, cells were seeded in six-well plate and photographed after 7–10 days. (H) Micrographs were acquired immediately after wounding and at 24 and 48 h post wounding. Scale bars, 200 μm. (I) Bar graph shows the wound closure distance at 48 h n = 3. (J and K) Representative images from Transwell assays assessing migration and invasion. Scale bars,100 μm (L and M) Bar graphs show that the migration and invasion abilities of LUAD cells were increased in the shR groups. n = 3. Data are presented as the mean ± SEM; ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The primary antibodies used were as follows: NPC2, Cathepsin B and D Polyclonal antibodies GAPDH Monoclonal antibody (Proteintech); Anti-NF-kB P65 and Anti-NF-kB P65 (phospho S536) antibodies (Abcam); mTOR(7C10) and Phospho-mTOR Rabbit (ser2448) mAb, AKT Rabbit mAb and Phospho-Akt (Ser473) Rabbit mAb (CST, Danvers, USA).

Techniques: Expressing, Western Blot, CCK-8 Assay, Knockdown, Colony Assay, Migration

Journal: iScience

Article Title: NPC2 suppresses osteolytic metastasis in lung adenocarcinoma via the AKT/mTOR pathway and tumor-osteoclast crosstalk

doi: 10.1016/j.isci.2026.115017

Figure Lengend Snippet: NPC2 knockdown in LUAD cells promoted osteoclast-mediated bone destruction (A) The micro-environment constituent of the PT, MPT and MT groups showed by bar graph. (B) TRAP staining on day 10 during osteoclast induction. All experimental groups had mature TRAP-positive osteoclasts except the negative control group. TRAP-positive osteoclast: Red violet cell containing >3 nuclei. Scale bars, 50 μm. (C) The proportions of TRAP-positive osteoclast area were quantified in each well (12-well plate), n = 3. (D and E) qRT-PCR analysis of the relative expression level of specific osteoclastogenic genes after 10 days of osteoclast induction. n = 3. (F) Fluorescence microscopy of F-actin ring (green) stain. Scale bars, 50 μm. (G) F-actin ring counting in each well (12-well plate), n = 3. (H and I) The levels of phosphorylated NF-kB and NF-kB were determined by immunoblotting analysis ( n = 3). (J) Mouse models with osteolytic lesions of LUAD were detected by X-ray at the second week after being injected with PC9 cells. Number of lesions (marked by red arrows) in the shR group was more than that in the NC group. Scale bars, 2 mm. (K) The increase in osteolytic lesion area grew faster in the shR group compared to the NC group ( n = 5). (L) Mouse models with osteolytic lesions of LUAD were detected by X-ray at the third week after being injected with PC9 cells. Lesions were marked by a red arrow. Scale bars, 2 mm; H&E, NPC2, and TRAP staining of the osteolytic lesions. Scale bars, 200 μm. (M) The area of osteolytic lesions was larger in the shR than the shR-NC group ( n = 5), smaller in OE than the OE-NC group ( n = 4). (N) The percentages of TRAP-positive staining area are shown by a bar graph. Data are presented as the mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The primary antibodies used were as follows: NPC2, Cathepsin B and D Polyclonal antibodies GAPDH Monoclonal antibody (Proteintech); Anti-NF-kB P65 and Anti-NF-kB P65 (phospho S536) antibodies (Abcam); mTOR(7C10) and Phospho-mTOR Rabbit (ser2448) mAb, AKT Rabbit mAb and Phospho-Akt (Ser473) Rabbit mAb (CST, Danvers, USA).

Techniques: Knockdown, Staining, Negative Control, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Western Blot, Injection

Journal: iScience

Article Title: NPC2 suppresses osteolytic metastasis in lung adenocarcinoma via the AKT/mTOR pathway and tumor-osteoclast crosstalk

doi: 10.1016/j.isci.2026.115017

Figure Lengend Snippet: Potential biological mechanisms implicated in NPC2-mediated LUAD osteolytic metastasis (A) GO enrichment analysis for the highly expressed genes in NPC2 low-expression tumor cells. (B) KEEG pathway analysis for the highly expressed genes in NPC2 low-expression tumor cells. (C) The heatmap of hallmark gene set enrichment score between NPC2 low-expression and high-expression tumor cell populations. (D) Immunoblotting analysis to detect the phosphorylation levels of AKT and mTOR in shR and OE groups. (E and F) Cell-free cholesterol (FC) was measured by ELISA. n = 3. (G) Mitochondrial membrane potential (Δψm) was detected by the orange-red cationic fluorescent probe TMRE. Cells were treated with Carbonyl Cyanide m -Chlorophenylhydrazone (CCCP) as Positive Control. Scale bars, 20 μm (H) Changes in Δψm for each group were analyzed by flow cytometry. (I and J) Colocalization of LAMP1 and CTSB. Yellow or orange fluorescence indicates colocalization of CTSB (red) and LAMP1 (green). Representative images are shown, and the degree of colocalization was quantified using the correlation coefficient (Rr) (For A549 cells, the biological replicates was n = 3, and for PC9 cells n = 5). Colocalization of CTSB and LAMP1 was significantly increased in shR groups. Scale bars, 20 μm. (K) Western blot analysis showing reduced expression levels of lysosomal enzymes CTSB and CTSD in shR groups. (L) Uniform manifold approximation and projection (UMAP) was utilized after principal component analysis (PCA) for unsupervised clustering and unbiasedly visualizing cell subsets of mononuclear/macrophage on a two-dimensional map. (M) The histogram of mononuclear/macrophage cell composition in each sample. “Osteoclasts-like cell” were found in samples L-00、 L-03, and L-09 from primary lung tumors. (N) Ligand-receptor interactions between NPC2 Low-expression tumor cells and “osteoclast-like cell.” Data are presented as the mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The primary antibodies used were as follows: NPC2, Cathepsin B and D Polyclonal antibodies GAPDH Monoclonal antibody (Proteintech); Anti-NF-kB P65 and Anti-NF-kB P65 (phospho S536) antibodies (Abcam); mTOR(7C10) and Phospho-mTOR Rabbit (ser2448) mAb, AKT Rabbit mAb and Phospho-Akt (Ser473) Rabbit mAb (CST, Danvers, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Membrane, Positive Control, Flow Cytometry, Fluorescence

Journal: iScience

Article Title: NPC2 suppresses osteolytic metastasis in lung adenocarcinoma via the AKT/mTOR pathway and tumor-osteoclast crosstalk

doi: 10.1016/j.isci.2026.115017

Figure Lengend Snippet: Proposed model of NPC2 deficiency-induced LUAD bone metastasis NPC2 low-expression caused accumulation of FC in the lysosomal lumen, promoting the recruitment and activation of mTORC1 via SLC38A9. AKT kinase and mTORC1 are activated by phosphorylation, accelerating the cell cycle by up-regulation of transcription factors such as MYC, E2F …, and elevating the migration and invasion of LUAD cells. RANKL, a critical cytokine that promotes the maturation of osteoclasts, can also be upregulated by mTORC1. Besides, NPC2 low-expression inhibits lysosomal cathepsins and weakens the mitochondrial membrane potential (Δψm). The abnormal accumulation of metabolic products of tumor cells failing to be promptly degraded by lysosome enzymes leads to the apoptosis of NPC2 knockdown LUAD cells. Meanwhile, mitochondrial damage and mitophagy deficiency generate the accumulation of abnormal mitochondria leads to the release of inflammation-associated cytokines such as ANXA1 and the expression of APP on the cell membrane. Then ANXA1 and proteolytic fragments of APP interact with FPR1/3 and CD74 of pre-osteoclast and promote the differentiation of osteoclast. Together, these render NPC2 knockdown LUAD cells susceptible to osteolytic bone metastasis. Red arrows denote stimulatory effects, while blue arrows indicate inhibitory effects. Abbreviations: LUAD, lung adenocarcinoma; Aβ, amyloid beta peptide; APP, amyloid precursor protein; FC, free cholesterol.

Article Snippet: The primary antibodies used were as follows: NPC2, Cathepsin B and D Polyclonal antibodies GAPDH Monoclonal antibody (Proteintech); Anti-NF-kB P65 and Anti-NF-kB P65 (phospho S536) antibodies (Abcam); mTOR(7C10) and Phospho-mTOR Rabbit (ser2448) mAb, AKT Rabbit mAb and Phospho-Akt (Ser473) Rabbit mAb (CST, Danvers, USA).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Migration, Membrane, Knockdown