Journal: bioRxiv

Article Title: CK1δ-Dependent SNAPIN Dysregulation Drives Lysosomal Failure in HIV-1 Vpr–Exposed Neurons: A Targetable Mechanism in HAND

doi: 10.1101/2025.07.11.664248

Figure Lengend Snippet: HIV-1 Vpr upregulates CK1δ expression in neurons. (A) Immunofluorescence staining of CK1δ (green) in SH-SY5Y-derived neurons treated with vehicle (Control), Vpr, LH846, or Vpr + LH846 or DMSO. CK1δ signal intensity increased following Vpr exposure and was partially reduced by LH846 treatment. Nuclei are labeled with DAPI (blue); LAMP1 (red) marks lysosomes. Scale bars: 10 µm (B) Quantification of CK1δ fluorescence intensity per cell confirms a significant increase in Vpr- treated neurons, with partial normalization by CK1δ inhibition (mean ± SEM, n = 100 cells minimum per condition).

Article Snippet: CK1δ: Cells were fixed in 4% PFA and stained with antibodies against CK1δ (Proteintech, 14388- 1-AP).

Techniques: Expressing, Immunofluorescence, Staining, Derivative Assay, Control, Labeling, Fluorescence, Inhibition

Journal: bioRxiv

Article Title: CK1δ-Dependent SNAPIN Dysregulation Drives Lysosomal Failure in HIV-1 Vpr–Exposed Neurons: A Targetable Mechanism in HAND

doi: 10.1101/2025.07.11.664248

Figure Lengend Snippet: CK1δ inhibition restores lysosomal distribution disrupted by HIV-1 Vpr. SH-SY5Y cells stably expressing LAMP1-mCherry were transfected with SNAPIN WT or phosphomimetic SNAPIN S50D, differentiated into neurons for 4 days and treated 24h with Vpr ± CK1δ inhibitor LH846 (1.5μM). LAMP1⁺ lysosomes (red) and nuclei (blue, DAPI) were imaged by confocal microscopy. Lysosomal spatial distribution was quantified using radial intensity profiles from nuclear centers in ImageJ (ImageJ Radial Profile plugin), and data were presented as normalized histograms of different colors to facilitate visual comparison between groups.

Article Snippet: CK1δ: Cells were fixed in 4% PFA and stained with antibodies against CK1δ (Proteintech, 14388- 1-AP).

Techniques: Inhibition, Stable Transfection, Expressing, Transfection, Confocal Microscopy, Comparison

Journal: bioRxiv

Article Title: CK1δ-Dependent SNAPIN Dysregulation Drives Lysosomal Failure in HIV-1 Vpr–Exposed Neurons: A Targetable Mechanism in HAND

doi: 10.1101/2025.07.11.664248

Figure Lengend Snippet: SH-SY5Y cells stably expressing LAMP1-mCherry were transfected with SNAPIN-GFP, differentiated into neurons for 4 days and treated 24h with Vpr ± CK1δ inhibitor LH846 (1.5μM). LAMP1⁺ lysosomes (red), SNAPIN-GFP (Green) and nuclei (blue, DAPI) were imaged by confocal microscopy. Quantification (bottom panel) of Manders’ colocalization coefficient between SNAPIN and LAMP1 confirms a significant increase with Vpr that is reversed by CK1δ inhibition (mean ± SEM; n = 100 cells minimum). Scale bar: 10 μm.

Article Snippet: CK1δ: Cells were fixed in 4% PFA and stained with antibodies against CK1δ (Proteintech, 14388- 1-AP).

Techniques: Stable Transfection, Expressing, Transfection, Confocal Microscopy, Inhibition

Journal: bioRxiv

Article Title: CK1δ-Dependent SNAPIN Dysregulation Drives Lysosomal Failure in HIV-1 Vpr–Exposed Neurons: A Targetable Mechanism in HAND

doi: 10.1101/2025.07.11.664248

Figure Lengend Snippet: CK1δ inhibition restores lysosomal acidification and mitophagy in Vpr-exposed neurons. (A) Quantification of lysosomal pH using the pH-sensitive dual-emission LC3 reporter (pHluorin-mCherry-LC3). Vpr treatment significantly increased the green/red (G/R) fluorescence ratio, indicating lysosomal deacidification, which was reversed by the CK1δ inhibitor LH846 (mean ± SEM; n = 100 cells minimum). (B) Confocal images of SH-SY5Y-derived neurons stably expressing mCherry-LAMP1 and stained with MitoBright LT Green (Abbreviated as MitoGreen) (Dojindo) show that Vpr induces accumulation of mitochondria colocalizing with lysosomes, consistent with impaired mitophagy. LH846 treatment reduced mitochondrial buildup and restored spatial segregation between mitochondria and lysosomes. (C) Quantification of LAMP1–MitoGreen colocalization using Manders’ coefficient confirms elevated overlap in Vpr-exposed neurons and significant rescue by LH846. Scale bars: 10 μm.

Article Snippet: CK1δ: Cells were fixed in 4% PFA and stained with antibodies against CK1δ (Proteintech, 14388- 1-AP).

Techniques: Inhibition, Fluorescence, Derivative Assay, Stable Transfection, Expressing, Staining

Journal: Experimental Physiology

Article Title: Unveiling the role of melatonin‐related gene CSNK1D in osteoclastogenesis and its implications for osteoporosis treatment

doi: 10.1113/EP092189

Figure Lengend Snippet: Identification of the key gene CSNK1D and correlation analysis. (a) Intersection of differential genes with circadian rhythm‐related genes and melatonin‐related genes. (b) Scatter plot of correlation analysis between CSNK1D and HLA‐DRA . (c) Scatter plot of correlation analysis between CSNK1D and CCL2 . (d) Scatter plot of correlation analysis between CSNK1D and ANGPTL4 .

Article Snippet: Various antibodies, such as CSNK1D (Proteintech, catalogue number 14388‐1‐AP), Matrix metalloproteinase‐9 (MMP9) and β‐actin (Cell Signaling Technology, catalogue numbers 3852 and 4967), NFATc1 and c‐Fos (Santa Cruz Biotechnology, catalogue numbers sc‐7294 and sc‐166940), Runt‐related transcription factor 2 (RUNX2) and osteopomtin (OPN, Abcam, catalogue numbers ab236639 and ab283656) were sourced from the respective companies.

Techniques:

Journal: Experimental Physiology

Article Title: Unveiling the role of melatonin‐related gene CSNK1D in osteoclastogenesis and its implications for osteoporosis treatment

doi: 10.1113/EP092189

Figure Lengend Snippet: Construction of protein−protein interaction (PPI) network, functional enrichment and potential transcription factor analysis of CSNK1D . (a) PPI networks between CSNK1D and other circadian rhythm‐related proteins. (b) GO/KEGG enrichment analysis of CSNK1D . (c) PPI network analysis of CSNK1D and bone metabolic protein. (d) WikiPathways enrichment analysis of CSNK1D . (e) Potential transcription factor SP2 binding sites of CSNK1D .

Article Snippet: Various antibodies, such as CSNK1D (Proteintech, catalogue number 14388‐1‐AP), Matrix metalloproteinase‐9 (MMP9) and β‐actin (Cell Signaling Technology, catalogue numbers 3852 and 4967), NFATc1 and c‐Fos (Santa Cruz Biotechnology, catalogue numbers sc‐7294 and sc‐166940), Runt‐related transcription factor 2 (RUNX2) and osteopomtin (OPN, Abcam, catalogue numbers ab236639 and ab283656) were sourced from the respective companies.

Techniques: Functional Assay, Binding Assay

Journal: Experimental Physiology

Article Title: Unveiling the role of melatonin‐related gene CSNK1D in osteoclastogenesis and its implications for osteoporosis treatment

doi: 10.1113/EP092189

Figure Lengend Snippet: High expression of CSNK1D promotes osteoclast differentiation. (a) Relative expression of marker genes ( MMP9 , NFATc1 and c‐Fos ) and CSNK1D at the protein level after stimulation of RAW264.7 cells with or without RANKL for 4 days. (b) Relative expression of marker genes ( MMP9 , NFATc1 and c‐Fos ) and CSNK1D at the protein level after treatment of RANKL‐induced RAW264.7 cells with si‐NC and si‐CSNK1D, respectively, for 4 days. (c) Quantification of MMP9, NFATc1, c‐Fos and CSNK1D relative to β‐actin. (d) Quantification of MMP9, NFATc1 and CSNK1D relative to β‐actin. (e) TRAP staining and F‐actin fluorescence staining were performed on representative images of RAW264.7 cells induced for 4 days without RANKL, with RANKL or with RANKL + si‐CSNK1D. (f) Quantification of TRAP + multinucleated osteoclasts (≥3 nuclei) per well. (g,h) Quantitative analysis of the number of osteoclasts and average osteoclast area. n = 3 per group, Data represent the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 based on one‐way ANOVA. si‐NC, Negative control small interfering RNA; si‐CSNK1D, Small interfering RNA targeting the CSNK1D gene.

Article Snippet: Various antibodies, such as CSNK1D (Proteintech, catalogue number 14388‐1‐AP), Matrix metalloproteinase‐9 (MMP9) and β‐actin (Cell Signaling Technology, catalogue numbers 3852 and 4967), NFATc1 and c‐Fos (Santa Cruz Biotechnology, catalogue numbers sc‐7294 and sc‐166940), Runt‐related transcription factor 2 (RUNX2) and osteopomtin (OPN, Abcam, catalogue numbers ab236639 and ab283656) were sourced from the respective companies.

Techniques: Expressing, Marker, Staining, Fluorescence, Negative Control, Small Interfering RNA

Journal: Experimental Physiology

Article Title: Unveiling the role of melatonin‐related gene CSNK1D in osteoclastogenesis and its implications for osteoporosis treatment

doi: 10.1113/EP092189

Figure Lengend Snippet: Increased expression of CSNK1D in ovariectomized rat femur. (a) Mico‐CT of femur in sham operation (NC) group and ovariectomy (OP) group. (b) Comparison of bone volume fraction (BV/TV), trabecular thickness (Tb.Th) and the number of trabeculae (Tb.N) in each group. (c) Immunofluorescence staining of femur in NC group and OP group. (d) Semi‐quantitative analysis of CSNK1D fluorescence intensity in femur sections from OP group and NC group. n = 6 per group; data represent the mean ± SD. ** P < 0.01 based on one‐way ANOVA.

Article Snippet: Various antibodies, such as CSNK1D (Proteintech, catalogue number 14388‐1‐AP), Matrix metalloproteinase‐9 (MMP9) and β‐actin (Cell Signaling Technology, catalogue numbers 3852 and 4967), NFATc1 and c‐Fos (Santa Cruz Biotechnology, catalogue numbers sc‐7294 and sc‐166940), Runt‐related transcription factor 2 (RUNX2) and osteopomtin (OPN, Abcam, catalogue numbers ab236639 and ab283656) were sourced from the respective companies.

Techniques: Expressing, Comparison, Immunofluorescence, Staining, Fluorescence

Journal: Experimental Physiology

Article Title: Unveiling the role of melatonin‐related gene CSNK1D in osteoclastogenesis and its implications for osteoporosis treatment

doi: 10.1113/EP092189

Figure Lengend Snippet: Effects of CSNK1 D on osteoblast differentiation in vitro and in vivo. (a) Western blot bands showing the expression of RUNX2, OPN and CSNK1D at day 0 and day 7 of osteoblast induction. GAPDH was used as a loading control ( n = 3 per group). (b) Quantitative analysis of RUNX2, OPN and CSNK1D expression based on band intensity ( n = 3 per group). (c) Immunofluorescence staining of femur in sham operation (NC) group and ovariectomy (OP) group ( n = 6 per group). (d) Semi‐quantitative analysis of CSNK1D and OPN fluorescence intensity in femur sections from the OP group and the NC group ( n = 6 per group). Data represent the mean ± SD. * P < 0.05 and ** P < 0.01 based on one‐way ANOVA.

Article Snippet: Various antibodies, such as CSNK1D (Proteintech, catalogue number 14388‐1‐AP), Matrix metalloproteinase‐9 (MMP9) and β‐actin (Cell Signaling Technology, catalogue numbers 3852 and 4967), NFATc1 and c‐Fos (Santa Cruz Biotechnology, catalogue numbers sc‐7294 and sc‐166940), Runt‐related transcription factor 2 (RUNX2) and osteopomtin (OPN, Abcam, catalogue numbers ab236639 and ab283656) were sourced from the respective companies.

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Control, Immunofluorescence, Staining, Fluorescence