Journal: bioRxiv

Article Title: Lysosome-Dependent Sphingolipid Regulation as a potential therapeutic Target for Cohen Syndrome

doi: 10.1101/2025.09.04.674037

Figure Lengend Snippet: A) Schematic 2D representation of screening output, plotting 2 parameters (Golgi perimeter average, Features count/cell) for WT (magenta) and untreated VPS13B KO (black) replicates and compound-treated VPS13B KO cells (green), each dot representing a different compound. Analysis based on 1000-1500 cells/point (N=2). B) Representation of the data as the distance to WT based on 4 parameters (Golgi fragment average area, Golgi fragment average perimeter, shape factor, Golgi fragments/cell) plotted for WT and untreated VPA13B KO cells and for individual compound-treated VPS13B KO cells (CPD). (N=2). C) Representative screening images stained for GM130 (green) and Hoechst 33342 (magenta) showing WT and VPS13B KO HeLa cells and 4 different compound-treated cells: dilazep (DLZ); azelastine (AZL); raloxifene (ral); budesonide (BUD), 10 µM. D) Distribution of top 50 screening hits by primary therapeutic protein target (upper panel) and 2nd level ATC classification group (lower panel). Data obtained from PubChem ( pubchem.ncbi.nlm.nih.gov ). E) Screening library compound plotted according to predicted basic pKa and AlogP value. Top 50 hits are shown in magenta. Data obtained from ChEMBL (ebi.ac.uk/chembl). F) Expected and observed hit frequency of CAD compounds in the library. *p<0.0001, Wilson-Brown Binomial test.

Article Snippet: Compounds used in further follow-up experiments were purchased separately: azelastine hydrochloride (MedChemExpress, cat. HY-B0462 for HeLa cells experiments; Sigma-Aldrich, cat. A7611 for brain organoid experiments); brefeldin A (Merck, cat. B6542) cyclobenzaprine hydrochloride (Merck, cat. T8516); dilazep dihydrochloride (MedChemExpress, cat. HY-100957); fumonisin B1 ((Merck, cat. F1147); HPA-12 (MedChemExpress, cat. HY-132182); lovastatin (MedChemExpress, cat. HY-N0504); nocodazole (Apex Bio, cat. A8487); raloxifene (MedChemExpress, cat. HY-13738 for HeLa cells experiments; Sigma-Aldrich, cat. PHR1852 for brain organoid experiments); sertraline hydrochloride (Merck, cat. S6319); trifluoperazine dihydrochloride (Merck, cat. C4542); U18666A (Merck, cat. U3633).

Techniques: Staining

Journal: bioRxiv

Article Title: Lysosome-Dependent Sphingolipid Regulation as a potential therapeutic Target for Cohen Syndrome

doi: 10.1101/2025.09.04.674037

Figure Lengend Snippet: A) Representative images of VPS13B KO HeLa cells treated with: azelastine (AZL); dilazep (DLZ); trifluoperazine (TFPZ); raloxifene (RLX); U18666A (U18). 10 µM, 24 h. cells were fixed and stained with an anti-LBPA antibody (upper panel) of with Filipin (lower panel). B) Integrated intensity/cell quantification of LBPA and Filipin staining at different compound doses (1.1, 3.3, 10 µM) for 24h. Relative to untreated (a.u). Analysis based on 1000-1500 cells/point (N=2). C) Representative images of LipidTox accumulation in cells treated with azelastine (AZL); dilazep (DLZ); trifluoperazine (TFPZ). 10 µM, 24 h (upper panel). Integrated intensity/cell quantification of LipidTox at different compound doses (2.5, 5, 10 µM) for 24h. Relative to untreated (a.u). Analysis based on 1000-1500 cells/point (N=2). D) Representative images of WT or VPS13B KO transfected with non-targeting (mock) or targeting NPC1 or SMPD1 for 72 hours. Cells were stained for GM130 (green) and Hoechst 33342 (magenta). E) Analysis of Golgi average area (value relative to WT cells) in NPC1 and SMPD1 KD. Analysis based on 1000-1500 cells/point. (*p<0.001, **p<0.0001. paired t-test, 2-tails; N=3)

Article Snippet: Compounds used in further follow-up experiments were purchased separately: azelastine hydrochloride (MedChemExpress, cat. HY-B0462 for HeLa cells experiments; Sigma-Aldrich, cat. A7611 for brain organoid experiments); brefeldin A (Merck, cat. B6542) cyclobenzaprine hydrochloride (Merck, cat. T8516); dilazep dihydrochloride (MedChemExpress, cat. HY-100957); fumonisin B1 ((Merck, cat. F1147); HPA-12 (MedChemExpress, cat. HY-132182); lovastatin (MedChemExpress, cat. HY-N0504); nocodazole (Apex Bio, cat. A8487); raloxifene (MedChemExpress, cat. HY-13738 for HeLa cells experiments; Sigma-Aldrich, cat. PHR1852 for brain organoid experiments); sertraline hydrochloride (Merck, cat. S6319); trifluoperazine dihydrochloride (Merck, cat. C4542); U18666A (Merck, cat. U3633).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: Lysosome-Dependent Sphingolipid Regulation as a potential therapeutic Target for Cohen Syndrome

doi: 10.1101/2025.09.04.674037

Figure Lengend Snippet: A) Abundance of indicated lipid classes in VPS13B KO cells (clone 2D9) treated with the indicated compounds (10 µM, 24 hours), relative to untreated cells. DLZ (dilazep); AZL (azelastine); RLX (raloxifene); TFPZ (trifluoperazine); CBZP (cyclobenzaprine); SER (sertraline). (*q<0.05, **q<0.01. multiple paired t-tests with Benjamini-Hochberg correction; N=4) B) Volcano plot illustrating relatives changes in individual lipid species in compound treated VPS13B KO cells (clone 2D9) ( upper panel , AZL; lower panel , TFPZ) relative to WT. Cells at 10 µM for 24 hours. sphingomyelin (green); ceramides (magenta) are highlighted. (AZL (N=12); TFPZ (N=6). C) Relative amounts of SM 36:1 (calculated as a fraction of total SM) in VPS12B KO cells treated with the indicated compounds for 6 or 24 hours (10 µM). (*p<0.05, **p<0.01. one-way ANOVA with Šidák correction; N=4) D) Relative amounts of total SM, total CER, SM 36:1 (calculated as a fraction of total SM) and CER 36:1 (calculated as a fraction of total CER) in light (LM) or Heavy (HM) membranes prepared from VPS12B KO cells treated (TFPZ) with not (UT) with TFPZ 10 µM for 24 hours. (*p<0.05, **p<0.01. paired t-test, 2-tails; N=2). E) Relative amounts of SM 36:1 and CER 36:1 (calculated as a fraction of the respective lipid class) in cells treated (AZL) or not (UT) with 10µM AZL for 6 hours in combination with 20 µM fumonisin B1 (FB1) or 5 µM HPA-12. (*p<0.05, **p<0.01. paired t-test, 2-tails; N=3).

Article Snippet: Compounds used in further follow-up experiments were purchased separately: azelastine hydrochloride (MedChemExpress, cat. HY-B0462 for HeLa cells experiments; Sigma-Aldrich, cat. A7611 for brain organoid experiments); brefeldin A (Merck, cat. B6542) cyclobenzaprine hydrochloride (Merck, cat. T8516); dilazep dihydrochloride (MedChemExpress, cat. HY-100957); fumonisin B1 ((Merck, cat. F1147); HPA-12 (MedChemExpress, cat. HY-132182); lovastatin (MedChemExpress, cat. HY-N0504); nocodazole (Apex Bio, cat. A8487); raloxifene (MedChemExpress, cat. HY-13738 for HeLa cells experiments; Sigma-Aldrich, cat. PHR1852 for brain organoid experiments); sertraline hydrochloride (Merck, cat. S6319); trifluoperazine dihydrochloride (Merck, cat. C4542); U18666A (Merck, cat. U3633).

Techniques:

Journal: bioRxiv

Article Title: Lysosome-Dependent Sphingolipid Regulation as a potential therapeutic Target for Cohen Syndrome

doi: 10.1101/2025.09.04.674037

Figure Lengend Snippet: Abundance of indicated lipid classes in VPS13B KO cells (clone 2D9) treated with the indicated compounds (10 µM, 24 hours), relative to untreated cells. DLZ (dilazep); AZL (azelastine); RLX (raloxifene); TFPZ (trifluoperazine); CBZP (cyclobenzaprine); SER (sertraline). (*q<0.05, **q<0.01. multiple paired t-tests with Benjamini-Hochberg correction; N=4).

Article Snippet: Compounds used in further follow-up experiments were purchased separately: azelastine hydrochloride (MedChemExpress, cat. HY-B0462 for HeLa cells experiments; Sigma-Aldrich, cat. A7611 for brain organoid experiments); brefeldin A (Merck, cat. B6542) cyclobenzaprine hydrochloride (Merck, cat. T8516); dilazep dihydrochloride (MedChemExpress, cat. HY-100957); fumonisin B1 ((Merck, cat. F1147); HPA-12 (MedChemExpress, cat. HY-132182); lovastatin (MedChemExpress, cat. HY-N0504); nocodazole (Apex Bio, cat. A8487); raloxifene (MedChemExpress, cat. HY-13738 for HeLa cells experiments; Sigma-Aldrich, cat. PHR1852 for brain organoid experiments); sertraline hydrochloride (Merck, cat. S6319); trifluoperazine dihydrochloride (Merck, cat. C4542); U18666A (Merck, cat. U3633).

Techniques:

Journal: bioRxiv

Article Title: Lysosome-Dependent Sphingolipid Regulation as a potential therapeutic Target for Cohen Syndrome

doi: 10.1101/2025.09.04.674037

Figure Lengend Snippet: A) Ratios of ceramide (CER) and sphingomyelin (SM) lipid species in treated (Tr) versus untreated (UT) cells. Values for individual lipid species were normalized to their respective lipid class prior to ratio calculation. Cells were treated for 24 hours at 10 µM with the following compounds: DLZ (dilazep), AZL (azelastine), RLX (raloxifene), TFPZ (trifluoperazine), CBZP (cyclobenzaprine), and SER (sertraline). Color coding indicates statistical significance: magenta for ratios >1 and green for ratios <1, both with p < 0.05 (multiple paired ratio t-tests, N = 4). Non-significant changes (ratios not different from 1) are shown in grey. B) Relative amounts of CER 36:1 (calculated as a fraction of total SM) in VPS12B KO cells treated with the indicated compounds for 6 or 24 hours. (*p<0.05, **p<0.0001. one-way ANOVA with Šidák correction; N=4).

Article Snippet: Compounds used in further follow-up experiments were purchased separately: azelastine hydrochloride (MedChemExpress, cat. HY-B0462 for HeLa cells experiments; Sigma-Aldrich, cat. A7611 for brain organoid experiments); brefeldin A (Merck, cat. B6542) cyclobenzaprine hydrochloride (Merck, cat. T8516); dilazep dihydrochloride (MedChemExpress, cat. HY-100957); fumonisin B1 ((Merck, cat. F1147); HPA-12 (MedChemExpress, cat. HY-132182); lovastatin (MedChemExpress, cat. HY-N0504); nocodazole (Apex Bio, cat. A8487); raloxifene (MedChemExpress, cat. HY-13738 for HeLa cells experiments; Sigma-Aldrich, cat. PHR1852 for brain organoid experiments); sertraline hydrochloride (Merck, cat. S6319); trifluoperazine dihydrochloride (Merck, cat. C4542); U18666A (Merck, cat. U3633).

Techniques:

Journal: bioRxiv

Article Title: Lysosome-Dependent Sphingolipid Regulation as a potential therapeutic Target for Cohen Syndrome

doi: 10.1101/2025.09.04.674037

Figure Lengend Snippet: A, B) Representative images of control and VPS13B KO neurons treated with 500 nM azelastine (AZL) and raloxifene (RAL). Cells stained with Hoechst (blue) and Tuj1 (red). Scale bar: 100 μm. C, D) Quantification of mean neurite length in control and VPS13B KO neurons following AZL or RAL treatment. Neurite length values were normalized to DMSO- treated control neurons. Each data point represents the mean neurite length of approximately 9–16 individual neurons. Data are presented as mean ± SD. (Welch ANOVA test with Dunnett T3 multiple comparisons correction; ***p<0.001; **p<0.01; *p<0.05; ns: non-significant). E, F ) Representative brightfield images of control (top) and VPS13B KO neural organoids (bottom) at the 2-month stage, treated with 1 µM of AZL and RAL for 35 days. Scale bar: 1 mm. G, H ) Quantification of organoid area following AZL and RAL treatment. Organoid sizes were normalized to untreated control organoids. (Welch ANOVA test with Dunnett T3 multiple comparisons correction; ***p<0.001; **p<0.01; *p<0.05; ns: non-significant; n= ∼16 organoids).

Article Snippet: Compounds used in further follow-up experiments were purchased separately: azelastine hydrochloride (MedChemExpress, cat. HY-B0462 for HeLa cells experiments; Sigma-Aldrich, cat. A7611 for brain organoid experiments); brefeldin A (Merck, cat. B6542) cyclobenzaprine hydrochloride (Merck, cat. T8516); dilazep dihydrochloride (MedChemExpress, cat. HY-100957); fumonisin B1 ((Merck, cat. F1147); HPA-12 (MedChemExpress, cat. HY-132182); lovastatin (MedChemExpress, cat. HY-N0504); nocodazole (Apex Bio, cat. A8487); raloxifene (MedChemExpress, cat. HY-13738 for HeLa cells experiments; Sigma-Aldrich, cat. PHR1852 for brain organoid experiments); sertraline hydrochloride (Merck, cat. S6319); trifluoperazine dihydrochloride (Merck, cat. C4542); U18666A (Merck, cat. U3633).

Techniques: Control, Staining

Journal: Breast Cancer Research : BCR

Article Title: Medications to reduce breast cancer risk: a network meta-analysis of randomized controlled trials

doi: 10.1186/s13058-025-02059-w

Figure Lengend Snippet: Comparative efficacy of risk-reducing medications for overall breast cancer incidence. Treatment vs. placebo: Thirteen risk-reducing medications that have been compared to other medications more than once are compared with placebo by network meta-analysis; Treatment vs. Treatment: Six risk-reducing medications that have significant efficacy (SU, TZDs, 3rd-gen SERMs, ARINs, raloxifene, and tamoxifen) are compared with each other by network meta-analysis. Effects are measured by risk ratios (RRs, ratio of breast cancer incidence in the two arms). Predictive intervals (PrIs) represent the expected range of effects in a future study, considering both the current effect estimate and between-study heterogeneity. Abbreviations: ARINs, Aromatase inhibitors; CI, Confidence interval; PrI, Predictive interval; RR, Risk ratio; SU, Sulfonylurea; TZDs, Thiazolidinediones; 3rd-gen SERMs, Third-generation selective estrogen receptor modulators

Article Snippet: Vogel 2010 [ ] , NSABP STAR P-2 , Raloxifene, 9754 , Tamoxifen, 9736 , Raloxifene 60 mg/d and tamoxifen 20 mg/d , 81.0.

Techniques: Medications

Journal: Breast Cancer Research : BCR

Article Title: Medications to reduce breast cancer risk: a network meta-analysis of randomized controlled trials

doi: 10.1186/s13058-025-02059-w

Figure Lengend Snippet: Network and comparative efficacy of risk-reducing medications for invasive breast cancer incidence. Top: Network of risk-reducing medications evaluated for invasive breast cancer incidence. Bottom: Three risk-reducing medications (ARINs, tamoxifen, and raloxifene) are compared with placebo by network meta-analysis. All three medications are effective compared with placebo. Abbreviations: ARINs, Aromatase inhibitors; CEE, Conjugated equine estrogen; CI, Confidence interval; PrI, Predictive interval; RR, Risk ratio; 3rd-gen SERMs, Third-generation selective estrogen receptor modulators

Article Snippet: Vogel 2010 [ ] , NSABP STAR P-2 , Raloxifene, 9754 , Tamoxifen, 9736 , Raloxifene 60 mg/d and tamoxifen 20 mg/d , 81.0.

Techniques: Medications