Journal: bioRxiv

Article Title: Lysosomes cell autonomously regulate myeloid cell states and immune responses

doi: 10.1101/2024.11.11.623074

Figure Lengend Snippet: Pharmacological perturbation of lysosomal properties drives an acute functional and transcriptional remodeling of BMDMs. (a) Schematic displaying pharmacological approaches to perturb various lysosomal properties [BafA1, v-ATPase inhibitor; U18666A, NPC1 inhibitor; E64/leupeptin/pepstatin (E/L/P) cocktail, protease inhibitors; VPS34IN1, VPS34 inhibitor; monosodium urate (MSU), lysosome membrane permeabilizing agent]. (b) Dose response of WT BMDM live nuclei count in response to increasing doses of each lysosomal drug after 22 hours of treatment. Selected doses for downstream assays are displayed in the legend and above each curve (n=1 biological replicate/dose/drug). (c) Quantification of relative live nuclei count following a 22 hour treatment with various lysosomal drugs at selected doses (% of WT; n=5 biological replicates/genotype). (d) Quantification of EdU + nuclei count following a 22 hour treatment with various lysosomal drugs at selected doses to measure proliferation (% of WT; n=4 biological replicates/genotype). (e,f) Quantification of pSIVA + cells over time (e) and after 16 hours (f) of treatment with various lysosomal drugs at selected doses to measure transient phosphatidylserine exposure and apoptosis (fraction of total cells; n=5 biological replicates/genotype). (g) Representative images and quantification of zymosan-488 phagocytosis in WT BMDMs following a 22 hour pre-treatment with selected lysosomal drugs (n=5 biological replicates/genotype). (h) Schematic displaying experimental workflow and quantification to measure BMDM transwell migration towards chemoattractant C5a following a 22 hour pre-treatment with selected lysosomal drugs (n=5 biological replicates/genotype). (i) Heatmap displaying relative cytokine levels in conditioned media of BMDMs treated with lysosomal drugs for 22 hours (n=5 biological replicates/genotype). (j) Multidimensional scaling plot displaying overall transcriptional (dis)similarity between BMDMs treated with various lysosomal drugs for 22 hours. (k) Number of significant up- and downregulated differentially expressed genes (DEGs) over time in each condition (sampled at 1, 7, 22 hours). (l) Venn diagram displaying overlap in DEGs between conditions after 22 hours of treatment. (m) Bar graph displaying high degree of concordance in directionality of common DEGs between all treatment conditions (795/861 changed in same direction across all 5 treatments). (n,o) Gene ontology (GO: molecular function) analysis of (n) up- and (o) downregulated common genes. (p) Average expression of shared up- (top) and downregulated (bottom) shared DEGs in each microglia subcluster from aged Grn KO microglia scRNA-seq. (q) Heatmap displaying relative expression of constitutive Grn KO BMDM DEGs in BMDMs treated with lysosomal drugs for 22 hours (n=5 biological replicates/genotype). Data presented in (c-h) are mean±s.e.m. One-way ANOVA tests with multiple comparisons were performed to compare across treatment conditions in (c,d,f,g). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: The following primary antibodies were used: sheep anti-PGRN (R&D Systems; Cat# 2557; 0.5μg/mL), rabbit anti-GPNMB (Cell Signaling Technology; Cat# 90205; 1:500), rabbit anti-FABP5 (Cell Signaling Technology; Cat# 39926; 1:500), rabbit anti-CatB (Cell Signaling Technology; Cat# 31718; 1:500), rabbit anti-LPL (Biotechne; Cat# AF7197; 1μg/mL), mouse anti-Vinculin (Sigma-Aldrich; Cat# V9264; 1:1000), mouse anti-β-Actin (Sigma-Aldrich; Cat# A2228; 1:10000), mouse anti-LC3 (Cell Signaling Technology; Cat# 83506; 1:500), mouse anti-p62 (Biotechne; Cat# MAB8028; 2μg/mL), mouse anti-AMPKα (Cell Signaling Technology; Cat# 2793; 1:500), rabbit anti-phospho-AMPKα (T172) (Cell Signaling Technology; Cat# 2535; 1:500), mouse anti- GAPDH (Abcam; Cat# ab8245; 1:2000), rabbit anti-ATP6V1A (Cell Signaling Technology; Cat# 39517; 1:500), rabbit anti-NPC1 (Abcam; Cat# ab134113; 1:500), rabbit anti-VPS34 (Cell Signaling Technology; Cat# 4263; 1:500), rabbit anti-CatD (Cell Signaling Technology; Cat# 88239; 1:500), sheep anti-TREM2 (Biotechne; Cat# AF1729; 0.1μg/mL), rabbit anti-ATG7 (Cell Signaling Technology; Cat# 8558; 1:500), rabbit anti-ATG14 (Cell Signaling Technology; Cat# 96752; 1:500), and rabbit anti-UVRAG (Abcam; Cat# ab313627; 1:500).

Techniques: Functional Assay, Membrane, Migration, Expressing

Journal: Cell Death & Disease

Article Title: TMEM232 is required for the formation of sperm flagellum and male fertility in mice

doi: 10.1038/s41419-024-07200-9

Figure Lengend Snippet: A Western blotting analysis for the comparison of ARMC3 protein levels between Tmem232 −/− ( n = 2) and Tmem232 +/+ ( n = 2) mouse testes and sperms at 2-months-old. α-Tubulin served as a loading control. The corresponding optical density readings for each image are shown. The representative image of biological duplicates is shown. B Western blotting analysis revealed the protein levels of four subunits (VPS15, VPS34, ATG14, and Beclin1) of PIK3C3-C1 complex in the testes of 2-month-old Tmem232 +/+ ( n = 2) and Tmem232 −/− ( n = 2) male mice. α-Tubulin served as a loading control. The representative image of biological duplicates is shown. C Co-immunoprecipitation assay results revealed the interaction between the TMEM232 and ATG14 protein in cells. Anti-Flag beads were used for immunoprecipitation. Anti-Flag and anti-GFP antibodies were used for western blotting analysis. The results shown are representative of three independent experiments. The representative image of biological duplicates is shown. D TMEM232-GFP was partly colocalized with LC3 in HeLa cells. Subcellular localization of target proteins (red) was probed with an anti-GOLGI97 antibody (upper panel, marker of Golgi apparatus), an anti-LAMP1 antibody (middle panel, marker of lysosome), and an anti-LC3 antibody (lower panel, marker of autophagosome). Cell nuclei were counterstained with DAPI. Scale bars, 10 μm. E The absence of TMEM232 in mice resulted in the accumulation of P62 and LC3. Tmem232 +/+ ( n = 2) and Tmem232 −/− ( n = 2) mouse testes were separated and prepared for western blotting analysis. α-Tubulin served as a loading control. The corresponding optical density readings for each image are shown. The representative image of biological duplicates is shown. F Immunofluorescence staining of the P62 protein performed on frozen sections of Tmem232 +/+ ( n = 2) and Tmem232 −/− ( n = 2) mouse testes. Blue, DAPI; green, P62. Scale bars, 20 μm. G Western blotting analysis of LC3B protein levels in HeLa cells after Flag-TMEM232 overexpression treatment with or without bafilomycin A1 (BafA1, 50 nM) treatment. HeLa cells were pretreated with Baf-A1 for 1 h and transfected with p-TMEM232×3FLAG-Myc-CMV-24 plasmid for 24 h. α-Tubulin served as a loading control. The corresponding optical density readings for each image are shown below. The results shown are representative of three independent experiments. The representative image of biological duplicates is shown. H A proposed model for the assumption that TMEM232 is involved in autophagy to regulate sperm formation in mice.

Article Snippet: The following primary antibodies were used: rabbit anti-Lamin B1 (Cat # 12987-1-AP; Proteintech, Chicago, IL, USA, 1:5000), mouse anti-α-Tubulin (Cat # 66031-1-lg; Proteintech, Chicago, IL, USA, 1:10000), rabbit anti-SEPTIN2 (Cat # 11397-1-AP; Proteintech, USA, 1:2000), rabbit anti-SEPTIN4 (Cat # 12476-1-AP; Proteintech, Chicago, IL, USA, 1:1000), rabbit anti-SEPTIN6 (Cat # 12805-1-AP; Proteintech, Chicago, IL, USA, 1:1000), rabbit anti-SEPTIN7 (Cat # 13818-1-AP; Proteintech, Chicago, IL, USA, 1:3000), rabbit anti-SEPTIN12 (Cat # PA5-98593; Invitrogen, Waltham, MA, USA, 1:1000), rabbit anti-SEPTIN14 (Cat # 24590-1-AP; Proteintech, Chicago, IL, USA, 1:2000), rabbit anti-TSSK4 (Cat # A7861; ABclonal Biotechnology, Wuhan, China, 1:2000), rabbit anti-Ac-α-Tubulin (Cat # 5335; CST, Danvers, Massachusetts, USA, 1:1000), rabbit anti-CatSper2 (Cat # orb354243; Biorbyt, Cambridge, UK, 1:2000), rabbit anti-ODF1 (Cat # 24736-1-AP; Proteintech, Chicago, IL, USA, 1:1000), rabbit anti-ODF2 (Cat # 12058-1-AP; Proteintech, Chicago, IL, USA, 1:2000), rabbit anti-PDHA2 (Cat # 17134-1-AP; Proteintech, Chicago, IL, USA, 1:2000), rabbit anti-COX6B2 (Cat # 11437-1-AP; Proteintech, Chicago, IL, USA, 1:2000), rabbit anti-VDAC3 (Cat # 14451-1-AP; Proteintech, Chicago, IL, USA, 1:2000), rabbit anti-GFP (Cat # 50430-2-AP; Proteintech, Chicago, IL, USA, 1:3000), mouse anti-FLAG (Cat # 66008-3-Ig, Proteintech, Chicago, IL, USA, 1:2000), anti-GST tag (91G1) (Cat # 2625; CST, Danvers, Massachusetts, USA, 1:1000), rabbit anti-TMEM232 (generated by ABclonal Biotechnology, Wuhan, China, 1:1000), anti-ATAT1 (Cat # 28828-1-1-AP; ProteinTech Group, Chicago, IL, USA), anti-RPL3 (Cat # 11005-1-AP; ProteinTech Group, Chicago, IL, USA), anti-RPL6 (Cat # A15094; ABclonal Biotechnology, Wuhan, China, 1:200), anti-RPS6 (Cat # A11874; ABclonal Biotechnology, Wuhan, China, 1:200), anti-RPS8 (Cat # A21124; ABclonal Biotechnology, Wuhan, China, 1:200), anti-LC3 (Cat # BM4827; Boster, Wuhan, China), anti-ARMC3 (Cat # 28418-1-AP), ATG14 (Cat # 19491-1-AP), anti-VPS15 (Cat # 17894-1-AP), anti-VPS34 (Cat # 12452-1-AP), anti-Beclin1 (Cat # 17894-1-AP), and anti-P62 (Cat # 29503-1-AP) (ProteinTech Group, Chicago, IL, USA).

Techniques: Western Blot, Comparison, Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Marker, Immunofluorescence, Staining, Over Expression, Transfection, Plasmid Preparation

Journal: eLife

Article Title: Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance

doi: 10.7554/eLife.96178

Figure Lengend Snippet: ( A ) Percentage of suspended MCF-10A cells showing GUVac formation after treatment with the indicated drugs for 18 hr. Dimethyl sulfoxide (DMSO) (n = 1122), latrunculin B (LatB) (n = 1386), LatB + VPS34-IN1 (n = 989), LatB + LY294002 (n = 876), LatB + GDC-0941 (n = 787), and LatB + TGX-221 (n = 801). ( B ) Percentage of cells showing GUVac formation for MCF-10A cells transfected with the indicated siRNAs and suspended in the presence of LatB for 24 hr. siRNAs: control (n = 456), p110α (n = 718), p110β (n = 709), PI3K-C2α (n = 421), and VPS34 (n = 295). ( C ) Percentage of cells showing GUVac formation for nontargeting control (NTC), PI3K-C2α KO, or VPS34 KO MCF-10A cells suspended in the presence of DMSO or LatB for 24 hr. NTC/DMSO (n = 746), NTC/LatB (n = 730), PI3K-C2α KO/LatB (n = 824), and VPS34 KO/LatB (n = 939). ( D ) Representative transmission electron microscopy (TEM) images of suspended WT, PI3K-C2α KO, or VPS34 KO MCF-10A cells treated with LatB for the indicated times. Scale bars, 2 µm. ( E ) Representative super-resolution structured illumination microscopy (SIM) fluorescence images of MCF-10A cells expressing membrane-targeted EGFP that had been suspended in the presence of the indicated drugs for the indicated times. The nucleus is denoted by the letter ‘N’ and outlined with a dashed line. Arrows indicate vacuoles. Scale bars, 5 μm. ( F ) Maximum diameter of vacuoles in each cell for experiments as in ( E ). LatB: 3 hr (n = 29), 6 hr (n = 39), and 12 hr (n = 36). LatB + VPS34-IN1: 3 hr (n = 47), 6 hr (n = 43), and 12 hr (n = 39). ( G ) Representative super-resolution SIM fluorescence images of MCF-10A cells expressing membrane-targeted EGFP and either mCherry-2xFYVE or mCherry–PH-TAPP1 that had been suspended in the presence of DMSO or LatB for 6 hr. All quantitative data are means ± SD. The n values represent the total number of cells examined in three independent experiments (A–C, F). *p<0.05, **p<0.01, ****p<0.0001; ns, not significant by one-way ANOVA with Tukey’s multiple comparisons ( A–C ) or two-tailed unpaired t -test ( F ). Figure 4—source data 1. Quantification data corresponding to . Figure 4—source data 2. Quantification data corresponding to . Figure 4—source data 3. Quantification data corresponding to .

Article Snippet: Antibodies to BCL2 (15,071T), Bim (2933T), phospho-Akt (Ser473, 4060S; Thr308, 13038S), cleaved caspase-3 (9664S), Na,K-ATPase 1 (3010S), Vinculin (13901S), and VPS34 (4263S) were obtained from Cell Signaling Technology; those to CHC (ab21679), LAMP1 (ab25630), LC3B (ab192890), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245), and GFP (ab13970) were from Abcam; those to dynamin 2 (sc-166669) and PI3K-C2α (sc-365290) were from Santa Cruz Biotechnology; those to EEA1 (610457) were from BD Biosciences; those to Transferrin receptor (13-6800) were from Invitrogen; and those to β-actin (A5316) were from Sigma-Aldrich.

Techniques: Transfection, Control, Transmission Assay, Electron Microscopy, Microscopy, Fluorescence, Expressing, Membrane, Two Tailed Test

Journal: eLife

Article Title: Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance

doi: 10.7554/eLife.96178

Figure Lengend Snippet: ( A ) Quantitative RT-PCR analysis of relative p110α mRNA abundance in MCF-10A cells transfected with control or p110α siRNAs. Data are means ± SD from three independent experiments. ***p<0.001 (two-tailed unpaired t -test). ( B ) Quantitative RT-PCR analysis of relative p110β mRNA abundance in MCF-10A cells transfected with control or p110β siRNAs. Data are means ± SD from three independent experiments. ****p<0.0001 (two-tailed unpaired t -test). ( C ) Immunoblot analysis of PI3K-C2α and VPS34 in MCF-10A cells transfected with the indicated siRNAs. β-actin was examined as a loading control. ( D ) Immunoblot analysis of PI3K-C2α in nontargeting control (NTC) and PI3K-C2α KO MCF-10A cell clones. ( E ) Immunoblot analysis of VPS34 in VPS34 KO MCF-10A clones and siRNA KD cells. Figure 4—figure supplement 1—source data 1. Uncropped blot images with sample labeling used in . Figure 4—figure supplement 1—source data 2. Original blot images used in . Figure 4—figure supplement 1—source data 3. Uncropped blot images with sample labeling used in . Figure 4—figure supplement 1—source data 4. Original blot images used in . Figure 4—figure supplement 1—source data 5. Uncropped blot images with sample labeling used in . Figure 4—figure supplement 1—source data 6. Original blot images used in .

Article Snippet: Antibodies to BCL2 (15,071T), Bim (2933T), phospho-Akt (Ser473, 4060S; Thr308, 13038S), cleaved caspase-3 (9664S), Na,K-ATPase 1 (3010S), Vinculin (13901S), and VPS34 (4263S) were obtained from Cell Signaling Technology; those to CHC (ab21679), LAMP1 (ab25630), LC3B (ab192890), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245), and GFP (ab13970) were from Abcam; those to dynamin 2 (sc-166669) and PI3K-C2α (sc-365290) were from Santa Cruz Biotechnology; those to EEA1 (610457) were from BD Biosciences; those to Transferrin receptor (13-6800) were from Invitrogen; and those to β-actin (A5316) were from Sigma-Aldrich.

Techniques: Quantitative RT-PCR, Transfection, Control, Two Tailed Test, Western Blot, Clone Assay, Labeling

Journal: eLife

Article Title: Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance

doi: 10.7554/eLife.96178

Figure Lengend Snippet: ( A – C ) MCF-10A cells were suspended in the presence of latrunculin B (LatB) or EDTA for the indicated times and then immunostained for cleaved caspase-3. Nuclei were stained with Hoechst 33342. Representative differential interference contrast (DIC) and fluorescence images at 24 hr (scale bar, 20 μm) ( A ), the percentage of cells positive for GUVac formation at 24 hr ( B ), and the time course for the percentage of cells positive for cleaved caspase-3 ( C ) are shown. Dimethyl sulfoxide (DMSO): 0 hr (n = 1030), 6 hr (n = 1742), 12 hr (n = 1423), and 24 hr (n = 1273). LatB: 6 hr (n = 1479), 12 hr (n = 1508), and 24 hr (n = 1443). EDTA: 6 hr (n = 1976), 12 hr (n = 1578), and 24 hr (n = 1296). (D–F) MCF-10A cells were suspended with EDTA and in the presence of DMSO or LatB for the indicated times and then stained with trypan blue. Representative bright-field images (scale bar, 20 μm) ( D ) as well as the percentage of cells positive for GUVac formation ( E ) and the percentage of trypan blue-positive cells ( F ) are shown. DMSO: 24 hr (n = 1003), 48 hr (n = 797), and 72 hr (n = 943). LatB: 24 hr (n = 1473), 48 hr (n = 2100), and 72 hr (n = 1350). ( G ) Percentage of trypan blue-positive cells for suspended MCF-10A cells that either were simultaneously treated with both LatB and the indicated concentrations of sFasL for 24 hr (a condition under which cell death signaling precedes GUVac formation) or were treated with LatB for 24 hr and then incubated in the presence of sFasL for 24 hr (a condition under which GUVac formation precedes cell death signaling). GUVac(–): sFasL at 0 ng/ml (n = 1193), 100 ng/ml (n = 1441), or 500 ng/ml (n = 994). GUVac(+): sFasL at 0 ng/ml (n = 1240), 100 ng/ml (n = 1418), or 500 ng/ml (n = 1529). ( H, I ) Nontargeting control (NTC), PI3K-C2α KO, and VPS34 KO MCF-10A cells were suspended with EDTA in the presence of DMSO or LatB for 48 hr, after which the percentage of cells showing GUVac formation ( H ) and the percentage of trypan blue-positive cells ( I ) were determined. NTC: DMSO (n = 927), LatB (n = 1369). PI3K-C2α KO: DMSO (n = 627), LatB (n = 883). VPS34 KO: DMSO (n = 1026), and LatB (n = 885). ( J ) Time-lapse bright-field images of cells with GUVacs after matrix reattachment. MCF-10A cells were suspended in the presence of LatB for 24 hr, washed to eliminate LatB, and then cultured in an adhesive confocal dish for 14 hr before imaging for the indicated times (hour:minute). The yellow dotted line represents the largest vacuole in each cell, while the white dotted line indicates the spread cell area. Scale bar, 10 μm. All quantitative data are means ± SD. The n values represent the total number of cells examined in three ( B , C , G – I ) or four ( E, F ) independent experiments. **p<0.01, ****p<0.0001; ns, not significant (two-tailed unpaired t -test). V, vacuole. Figure 5—source data 1. Quantification data corresponding to . Figure 5—source data 2. Quantification data corresponding to . Figure 5—source data 3. Quantification data corresponding to . Figure 5—source data 4. Quantification data corresponding to .

Article Snippet: Antibodies to BCL2 (15,071T), Bim (2933T), phospho-Akt (Ser473, 4060S; Thr308, 13038S), cleaved caspase-3 (9664S), Na,K-ATPase 1 (3010S), Vinculin (13901S), and VPS34 (4263S) were obtained from Cell Signaling Technology; those to CHC (ab21679), LAMP1 (ab25630), LC3B (ab192890), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245), and GFP (ab13970) were from Abcam; those to dynamin 2 (sc-166669) and PI3K-C2α (sc-365290) were from Santa Cruz Biotechnology; those to EEA1 (610457) were from BD Biosciences; those to Transferrin receptor (13-6800) were from Invitrogen; and those to β-actin (A5316) were from Sigma-Aldrich.

Techniques: Staining, Fluorescence, Incubation, Control, Cell Culture, Adhesive, Imaging, Two Tailed Test

Journal: eLife

Article Title: Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance

doi: 10.7554/eLife.96178

Figure Lengend Snippet: In matrix-attached cells, cortical actin and extracellular matrix (ECM) attachment suppresses plasma membrane (PM) fluctuation and maintains membrane tension. However, in matrix-detached cells with disrupted cortical actin, the PM exhibits micron-scale membrane fluctuations. This promotes the recruitment of septin to the micrometer-sized inwardly curved plasma membrane via its amphipathic helix (AH) domain and the subsequent recruitment further accelerates the PM invagination. As a result of this macropinocytosis-like process, followed by dynamin-mediated pinching off, vacuoles accumulate within the cell and gradually coalesce, a process facilitated by the synthesis of PI(3)P, involving the activity of VPS34 and PI3K-C2α. Eventually, these processes lead to the formation of GUVac. Cells that possess GUVac demonstrate resistance to anoikis. The schematic was generated using Biorender.

Article Snippet: Antibodies to BCL2 (15,071T), Bim (2933T), phospho-Akt (Ser473, 4060S; Thr308, 13038S), cleaved caspase-3 (9664S), Na,K-ATPase 1 (3010S), Vinculin (13901S), and VPS34 (4263S) were obtained from Cell Signaling Technology; those to CHC (ab21679), LAMP1 (ab25630), LC3B (ab192890), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245), and GFP (ab13970) were from Abcam; those to dynamin 2 (sc-166669) and PI3K-C2α (sc-365290) were from Santa Cruz Biotechnology; those to EEA1 (610457) were from BD Biosciences; those to Transferrin receptor (13-6800) were from Invitrogen; and those to β-actin (A5316) were from Sigma-Aldrich.

Techniques: Membrane, Activity Assay, Generated

Journal: Molecular Cancer

Article Title: Overcoming multi-drug resistance in SCLC: a synergistic approach with venetoclax and hydroxychloroquine targeting the lncRNA LYPLAL1-DT/BCL2/BECN1 pathway

doi: 10.1186/s12943-024-02145-1

Figure Lengend Snippet: LYPLAL1-DT facilitates the assembly of the core players of autophagy, BECN1/PIK3C3 complex. (A-B) The expression level of BECN1 of H446 or H196 cells with cDDP, VP-16 or PTX treatment at different times and concentrations. (C) The expression level of BECN1 in cDDP-, VP-16- and PTX-resistant H446 or H196 cells. (D) The sections of tumor tissues from chemosensitive patients ( n = 6) and chemoresistant patients ( n = 6) patients were stained with BECN1. (E) IF/FISH images showing the colocalization of LYPLAL1-DT and BECN1, PIK3C3 in H446/cDDP cells with cDDP treatment by fluorescence confocal microscopy. (F) IF/FISH images showing the colocalization of LYPLAL1-DT and BECN1, PIK3C3 in H446/cDDP cells transfected with scramble or LYPLAL1-DT shRNA with cDDP treatment by fluorescence confocal microscopy. (G) Western blot showed the expression level of BECN1 in LYPLAL1-DT-overexpressing H446 cells or LYPLAL1-DT-knockdown H446/cDDP cells. (H) H446/cDDP cells were transfected with scramble or shLYPLAL1-DT, and the coupling between BECN1 and PIK3C3 was shown by immunoprecipitation. ( I ) RNA pull-down assay confirmed the interaction of LYPLAL1-DT and BECN1/PIK3C3 complex (**, P < 0.01; ***, P < 0.001)

Article Snippet: Commercially available antibodies are as follows: Anti-β-actin (HUABIO, Cat#R1207-1), anti-p62/SQSTM1 (Proteintech, Cat#18420-1-AP), anti-LC3B (HUABIO, ET1701-65), anti-casepase3 (Abcam, ab184787), anti-BCL2 (Abways, Cat#CY6717), anti-Beclin1/BECN1(Proteintech, Cat#11306-1-AP), anti-PIK3C3/VPS34 (Proteintech, Cat#12452-1-AP), anti-ATG14 (ABclonal, Cat#A7526), anti-Ago2 (CST, Cat#2897S), ANTI-FLAG (Proteintech, Cat#80010-1-RR).

Techniques: Expressing, Staining, Fluorescence, Confocal Microscopy, Transfection, shRNA, Western Blot, Knockdown, Immunoprecipitation, Pull Down Assay

Journal: Journal of Medicinal Chemistry

Article Title: New Mitochondria-Targeted Fisetin Derivative Compromises Mitophagy and Limits Survival of Drug-Induced Senescent Breast Cancer Cells

doi: 10.1021/acs.jmedchem.4c01664

Figure Lengend Snippet: Mito-fisetin (mF3)-mediated changes in mitochondrial parameters (A,B) and mitophagic pathway (C) in tamoxifen-induced noncancerous and breast cancer cells. Senescent cells were treated with fisetin (F) and mito-fisetin (mF3) (1 or 5 μM) for 24 h (A,C) or 5 μM F or mF3 for 6 h (B). (A) Depolarization of the MMP (Δψ m ) was assayed using a dedicated fluorogenic probe and flow cytometry. The effects in nonsenescent proliferating cells are also shown. (B) Mitochondrial function was assayed as real-time measurements of mitochondrial oxidative phosphorylation (OXPHOS) as selected OCR parameters (pmol/min or %), namely, basal respiration, ATP production, proton leak (stimulation with oligomycin), maximal respiration (stimulation with the uncoupler FCCP), and spare respiratory capacity (stimulation with rotenone and antimycin A). Uncoupling efficiency [%] and nonmitochondrial oxygen consumption [pmol/min] are also presented. A summarizing scheme showing mF3-mediated effects on mitochondrial function is also provided. (A,B) Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to the corresponding untreated control (CTR, proliferating cells or CTR SEN, senescent cells) (ANOVA and Dunnett’s a posteriori test), ### p < 0.001, # p < 0.05 compared to fisetin treatment (F) (ANOVA and Tukey’s a posteriori test). (C) Cytoplasmic levels of selected markers of mitophagy and autophagy (PINK1, Parkin, ubiquitin, LC3B, ULK1, VPS34, beclin 1, BNIP3, and Rab9) were studied using dedicated antibodies, an immunostaining protocol, and imaging cytometry. Protein levels are presented as relative fluorescent units (RFU). Box and whisker plots are shown, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to the corresponding untreated proliferating control (CTR) (ANOVA and Dunnett’s a posteriori test), ∧∧∧ p < 0.001, ∧∧ p < 0.01, ∧ p < 0.05 compared to the corresponding untreated senescent control (CTR SEN) (ANOVA and Dunnett’s a posteriori test), ### p < 0.001, ## p < 0.01, # p < 0.05 compared to fisetin treatment (F) (ANOVA and Tukey’s a posteriori test).

Article Snippet: The following primary and secondary antibodies were used, namely, anti-PINK1 (PA5-86941, 1:200), anti-Parkin (PA5–13399, 1:100), anti-Ubi1 (13–1600, 1:100), anti-LC3B (PA5–32254, 1:500), anti-ULK1 (20986–1-AP, 1:100), anti-BECN1 (TA502527, 1:100), anti-VPS34 (38-2100, 1:100), anti-BNIP3 (710728, 1:250), anti-Rab9 (MA3-067, 1:200), anti-SOD1 (PA1–30195, 1:200), anti-SOD2 (MA1-106, 1:200), anticaspase 3 (PA5-77887, 1:100), anticaspase 9 (PA5-17913, 1:100), antiphospho-AMPK alpha-1,2 (Thr183, Thr172) (PA5-17831, 1:200), anti-HSP90 (MA-110373, 1:200), anti-IL-8 (M801, 1:500), antimouse IgG conjugated to Texas Red-X (T-6390, 1:1000), antirabbit IgG conjugated to PE-cyanine5.5 (L43018, 1:1000), and antimouse IgG conjugated to FITC (F2761, 1:1000) (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Flow Cytometry, Control, Immunostaining, Imaging, Cytometry, Whisker Assay