Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Representative images show DAPI (blue), EdU (green), pATM (magenta), SFPQ (cyan), and merged (right) staining in siNTC-treated DIvA U2OS cells under break and no break conditions. Breaks were induced with 4-hydroxytamoxifen for 24 hours, and images were captured using a 10X magnification. Cells (∼20,000 per well) were imaged across three wells per condition (16 fields per well; 48 images total) and quantified in Cell Profiler for nuclear intensity, foci count, and cell-cycle stage based on EdU/DAPI. Data are representative of n=48 images. (B) Quantification of SFPQ–pATM and pATM–γH2AX co-localization in G2-phase cells. Violin plots show correlation coefficients of SFPQ and pATM (left) and pATM and γH2AX (right) in G2 cells with or without DNA breaks. (C) ChIP-seq data representing SFPQ-bound chromatin at 122 defined AsiSI sites under uncut (noDSB) and cut (+4OHT, 4 hours) conditions (left). ChIP-seq data representing SFPQ bound to RNU sites (right). Immunoprecipitation was performed using SFPQ polyclonal antibody. Normalized ChIP-seq signal was plotted for ±1.5 kb around AsiSI sites. SFPQ occupancy profiles are shown for two independent replicates with DSB induction (dark blue and light blue) and for the noDSB control (yellow).

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Staining, ChIP-sequencing, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) (Top) SFPQ mean intensity: Violin plots (with embedded boxplots) show the single-cell distribution of nuclear SFPQ mean fluorescence intensity in DIvA U2OS cells under no break (untreated) and break (4-hydroxytamoxifen, 4-OHT) conditions. Each dot is one nucleus; boxplots denote median and interquartile range. Cell-cycle phase (G1, S, G2) was assigned per cell using EdU incorporation (green) and DAPI DNA content (blue). (Bottom) SFPQ foci per cell: Violin plots (with embedded boxplots) show the number of SFPQ nuclear foci per cell under the same conditions and cell-cycle stratification. Quantification: Cells were left untreated or treated with 4-OHT to induce AsiSI-mediated DSBs, then stained for SFPQ (cyan), EdU, and DAPI. Images were analyzed in Cell Profiler to segment nuclei, call SFPQ foci, compute per-nucleus mean intensity and foci counts, and assigned cell-cycle stage from EdU/DAPI features. (B) Non–pre-extracted immunofluorescence staining of pATM and SFPQ in DIvA U2OS cells with or without DSB induction. Cells were left untreated or treated with 4-hydroxytamoxifen (4-OHT) to induce AsiSI-mediated DSBs and stained for DNA (DAPI, blue), EdU incorporation (green), phosphorylated ATM (pATM, magenta), and SFPQ (cyan). Images were acquired without cytoskeletal (CSK) pre-extraction to visualize total nuclear staining patterns. Merged images show nuclear co-localization of pATM and SFPQ signals in the presence and absence of DNA damage.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Fluorescence, Staining, Immunofluorescence, Extraction

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) mRNA-seq log₂ fold changes of RAD51 paralogs and pooled transcripts in the indicated Gene Ontology (GO) categories in DIvA U2OS cells treated with siSFPQ compared to siNTC control for 72 hours in the absence of DSBs. Data represent the mean of three biological replicates. Individual p-values were adjusted for multiple comparisons. Aggregate p-values were combined by Fisher’s method. (B) Differential transcript utilization analysis for RAD51 paralogs. mRNA-seq data from siNTC versus siSFPQ DIvA U2OS cells were analyzed for transcript isoform usage. Bars represent the likelihood ratio statistic for each gene, with blue bars indicating genes showing significant shifts in transcript utilization (RAD51B, RAD51C) upon SFPQ depletion. Grey bars indicate genes without significant changes. (C) Western blot analysis of SFPQ and RAD51 protein levels of the three biological replicates used for mRNA-seq following siNTC or siSFPQ treatment. Total protein staining is shown as a loading control. (D) Representative images show DAPI (blue), EdU (green), RAD51 (magenta), SFPQ (cyan), and merged (right) staining in siNTC-treated DIvA U2OS cells under break and no break conditions, pre-extracted with CSK. Breaks were induced with 4-OHT for 4 hours. Cells (∼20,000 per well) were imaged across four wells per condition (16 fields per well; 64 images total) and quantified in Cell Profiler for nuclear intensity, foci count, and cell-cycle stage based on EdU/DAPI. (E) Quantification of SFPQ and RAD51 foci per cell in DIvA U2OS cells following siRNA treatment and DNA damage induction. Violin plots show the distribution of foci counts across conditions with or without 4-hydroxytamoxifen (4-OHT) treatment and following transfection with non-targeting control (NTC), RAD51-targeting, or SFPQ-targeting siRNAs. Data are representative of n=64 images. (F) Violin plots showing correlation coefficients of SFPQ and RAD51 in G2 cells with or without DNA breaks. Quantification of SFPQ-RAD51 foci co-localization in G2-phase cells was performed using Cell Profiler analysis of single-cell fluorescence signals.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Control, Western Blot, Staining, Transfection, Fluorescence

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Differential expression analysis of mRNA-seq data comparing DSB versus no-DSB conditions in siNTC-treated DIvA U2OS cells (n=3 biological replicates). Mean log₂ fold change for the same targets is shown as . No significant expression differences were detected for these targets upon DSB induction in control cells. (B) ChIP-seq data showing SFPQ abundance at sites upstream and downstream of RAD51-paralog genes both without (noDSB) or with (+DSB) 4 hours of DSB induction. Data displayed is the average signal across all 6 RAD51 paralogs. (C) mRNA-seq log₂ fold changes of transcript expression of the indicated gene or GO category in DIvA U2OS cells treated with siSFPQ compared to siNTC control for 72 hours in the absence of DSBs. Data represent the mean of three biological replicates. Individual p-values were adjusted for multiple comparisons. Aggregate p-values were combined by Fisher’s method. (D) Western blot of DIvA U2OS cells treated with siSFPQ with or without p53 inhibition by PFT-α (30 µM) for 24 hours. Lysates were blotted for SFPQ, HSP70, MDM2 and RAD51. (E) (Left) Western blot of p53-null K562 cells treated with siSFPQ. Total protein staining is shown as a loading control. (Right) Quantification of SFPQ and RAD51 normalized band intensities relative to total protein is graphed.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Quantitative Proteomics, Expressing, Control, ChIP-sequencing, Western Blot, Inhibition, Staining

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Cycloheximide (CHX) ± carfilzomib (Carf) protein stability assay in DIvA U2OS cells. Cells were transfected with either non-targeting control (siNTC) or SFPQ-targeting (siSFPQ) siRNAs for 72 h, then treated with CHX alone or CHX + Carf to inhibit protein synthesis and proteasomal degradation, respectively. Lysates were collected at 0-, 2-, and 4-hours post-drug treatment from three independent biological replicates. (B) RAD51 abundance from normalized to total protein and then to 0 hr. condition. Data points represent individual replicates; lines indicate the mean. (C) RIP-seq analysis of SFPQ binding across RAD51 family paralogs in melanoma cells. Read coverage tracks show SFPQ-associated RNA fragments aligned to the genomic loci of RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. Peaks indicate regions of enriched SFPQ binding, with annotations of exon–intron structure shown below each track. Model for SFPQ-mediated stabilization of RAD51 mRNA and its impact on homologous recombination (HR). In the presence of SFPQ, the protein binds to RAD51 mRNA, promoting transcript stabilization. Stable RAD51 mRNA ensures sufficient RAD51 protein production, enabling efficient RAD51 filament formation on DNA and supporting robust HR (left). Upon SFPQ loss, RAD51 family mRNAs are destabilized, leading to reduced RAD51 protein abundance. This reduction impairs HR efficiency (right).

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Stability Assay, Transfection, Control, Binding Assay, Homologous Recombination, Quantitative Proteomics

Journal: Scientific Reports

Article Title: Novel millimeter-wave-based method for in situ cell isolation and other applications

doi: 10.1038/s41598-018-32950-w

Figure Lengend Snippet: Nuclear “leakage” after 50 °C exposure. U2OS cells exposed to 50 °C for 2–3 seconds lost their nuclear envelope selectivity. Borders between MMW treated and untreated areas are shown as dashed lines. ( a ) In cells expressing NLS-RFP, heating induced the exit of NLS-RFP from the nuclei. 20x objective, the scale bar is 100 µm. ( b ) In cells expressing GFP-tubulin and BFP-lamin B1, heating induced the entry of GFP-tubulin into the nuclei while BFP-lamin distribution remained unchanged. 40x objective, the scale bar is 50 µm. Experiments were repeated at least 5 times, with similar results obtained each time; representative data from a single experiment are shown.

Article Snippet: Triple-tagged U2OS cell line (BFP-LMNB1/GFP-TUBA1B/RFP-ACTB, CLL1218) was obtained from Sigma-Aldrich.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Novel millimeter-wave-based method for in situ cell isolation and other applications

doi: 10.1038/s41598-018-32950-w

Figure Lengend Snippet: Cell viability assayed after 50 °C exposure. A spot in a confluent culture of U2OS cells expressing NLS-RFP was heated to 50 °C by local application of MMW radiation for 2–3 seconds. ( a ) Borderline (dashed) between MMW treated (right) and untreated (left) cells. 1 µg/ml Calcein-AM and 0.5 µg/ml DAPI were added after heating and images were acquired 30 minutes later. Cells with leaky nuclei and cells with intact nuclear selectivity were separated at the borderline as seen from NLS-RFP distribution. All cells were capable of cutting off the AM ester groups and retaining freed Calcein as well as preventing DAPI from entering (a few DAPI stained cells were most probably dead before MMW application). 40x objective, the scale bar is 50 µm. ( b ) The area within the dashed circle was MMW treated and the cells were incubated under environmentally controlled conditions for about 17 hours (see Supplementary Movie ). Then an apoptotic marker YO-PRO-1 (1 µM) was added and the cells were imaged 30 minutes later. Cells in the heated area were rounded and detached, some of them have floated outside the heated area. Some cells surrounding the heated area have migrated inside it and did not lose their nuclear selectivity. Some of the detached cells became YO-PRO-1 positive and also NLS-RFP negative as there are only a few yellow cells on the overlay. 20x objective, the scale bar is 100 µm. Experiments were repeated at least 3 times, with similar results obtained each time; representative data from a single experiment are shown.

Article Snippet: Triple-tagged U2OS cell line (BFP-LMNB1/GFP-TUBA1B/RFP-ACTB, CLL1218) was obtained from Sigma-Aldrich.

Techniques: Expressing, Staining, Incubation, Marker

Journal: Scientific Reports

Article Title: Novel millimeter-wave-based method for in situ cell isolation and other applications

doi: 10.1038/s41598-018-32950-w

Figure Lengend Snippet: Cell viability assayed after 60 °C exposure. A spot in a confluent culture of U2OS cells expressing NLS-RFP was heated to 60 °C by local application of MMW radiation for 2–3 seconds. 1 µg/ml Calcein-AM and 0.5 µg/ml DAPI were added after heating and the cells were imaged 30 minutes later. ( a ) Areas where the temperature (T) reached 60 °C and gradually dropped from 60 °C to 50 °C are shown by the dashed circles for all three viability markers. The sizes of 50 °C and 60 °C dashed lines were determined by melting two LCA films formed in close proximity to each other. The LCAs were 1-Hexadecanol (1-HXD, MP = 50 °C) and 1-Octadecanol (1-OD, MP = 59 °C) - see Table . NLS-RFP channel serves as another (indirect) indicator of the temperature: RFP starts to leak out of the nuclei at 50 °C and out of the cell at 60 °C, 4x objective. ( b ) Zoomed-in image of the heated center spot in A overlaying all three markers, 10x objective. ( c ) Further zoomed-in image of the transition area at the edge of the heated spot (dashed square) where the temperature gradually dropped from 60 °C to 50 °C. The image is an overlay of Calcein and DAPI channels, 20x objective. Ibidi chambered coverslip with 500 µm grid, the scale bar is 100 µm. Experiments were repeated at least 3 times, with similar results obtained each time; representative data from a single experiment are shown.

Article Snippet: Triple-tagged U2OS cell line (BFP-LMNB1/GFP-TUBA1B/RFP-ACTB, CLL1218) was obtained from Sigma-Aldrich.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Novel millimeter-wave-based method for in situ cell isolation and other applications

doi: 10.1038/s41598-018-32950-w

Figure Lengend Snippet: Heat-induced Trask phosphorylation. U2OS cells expressing NLS-RFP were heated to 50 °C for 2–3 seconds using MMWs. This led to cell rounding and partial detachment from the substrate within 30 minutes. Then an area within the rounded cell culture (to the left of the dashed line) was “thermofixed” by heating to 70 °C for 2–3 seconds in order to denature the proteins and permeabilize the membrane. Finally the cells were blocked, p-Trask (pY743) primary and secondary antibodies were applied, and 0.5 µg/ml DAPI was added. The images show the edge of the “thermofixed” area. The right image is an overlay of all three fluorescence channels. 40x objective, Ibidi chambered coverslip with 500 µm grid, the dots of the grid line were made at every 20 µm. Experiments were repeated twice with similar results obtained.

Article Snippet: Triple-tagged U2OS cell line (BFP-LMNB1/GFP-TUBA1B/RFP-ACTB, CLL1218) was obtained from Sigma-Aldrich.

Techniques: Expressing, Cell Culture, Fluorescence

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Immunoblot analysis of G3BP1 and G3BP2 in U2OS WT and ΔΔG3BP1/2 cells. ( B ) Quantification by high-content microscopy (HCM) of polyA RNA (Cy3-oligo[dT]) by FISH (i) and LAMP2 (ii) in U2OS WT and ΔΔG3BP1/2 cells. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries (primary objects); red masks, computer-identified polyA RNA or LAMP2 puncta respectively (target objects). ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells. Cells were treated with 2 mM LLOMe in the presence or absence of 10 μg/ml cycloheximide (CHX) for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( D ) Cell death analysis of supernatants of U2OS cells by a LDH release assay. Cells were treated with 2 mM LLOMe in the presence or absence of 10 μg/ml CHX for 30 min. ( E ) Quantification by HCM of G3BP1 puncta in human monocytic THP-1 cells. Cells were treated with 1 mM LLOMe in the presence or absence of 200 nM ISRIB for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( F ) Immunoblot analysis of ATF4 in THP-1 cells treated with 1 mM LLOMe in the presence or absence of 200 nM ISRIB for 30 min. ( G ) Cell death analysis of supernatants of THP-1 cells by a LDH release assay. Cells were treated with 1 mM LLOMe in the presence or absence of 200 nM ISRIB for 30 min. ( H ) Quantification of cell death by HCM using a propidium iodide (PI) uptake assay in U2OS G3BP1&2 double knockout (ΔΔG3BP1/2) cells overexpressing either FLAG or FLAG-G3BP1 & FLAG-G3BP2. Cells were treated with 2 mM LLOMe for 30 min, and then stained with propidium iodide (PI) (dead cells) and Hoechst-33342 (total cells). White masks, algorithm-defined cell boundaries; red masks, computer-identified PI+ nuclei. ( I ) Immunoblot analysis of the protein level of G3BP1 and G3BP2 in hMDM transfected with scrambled siRNA as control (SCR) or G3BP1 and G3BP2 siRNA for double knockdown (DKD). CTR, control; NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Western Blot, Microscopy, Lactate Dehydrogenase Assay, Double Knockout, Staining, Transfection, Control, Knockdown

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by high-content microscopy (HCM) of cell death by a propidium iodide (PI) uptake assay in U2OS wild type (WT) and G3BP1&2 double knockout (ΔΔG3BP1/2) cells. Cells were treated with 2 mM LLOMe for 30 min, and then stained with propidium iodide (PI) (dead cells) and Hoechst-33342 (total cells). White masks, algorithm-defined cell boundaries (primary objects); red masks, computer-identified PI + nuclei (target objects). ( B ) Cell death analysis of supernatants of U2OS WT and ΔΔG3BP1/2 cells by a LDH release assay. Cells were treated with 2 mM LLOMe for 30 min. ( C ) Quantification by HCM of cell death by a PI uptake assay in human peripheral blood monocyte-derived macrophages (hMDM). Cells were treated with 2 mM LLOMe in the presence or absence of 10 μg/ml cycloheximide (CHX) for 30 min, and then stained with PI (dead cells) and Hoechst-33342 (total cells). ( D ) Confocal microscopy analysis of G3BP1 (Alexa Fluor 488) in hMDM treated with 2 mM LLOMe with or without CHX for 30 min. Scale bar, 10 μm. ( E ) Quantification using AMNIS of cell death by Live/Dead TM stain kit in hMDM. Cells were treated with 2 mM LLOMe with or without CHX for 30 min, and then stained using Live/Dead TM stain kit (ThermoFisher). ( F ) Quantification by HCM of cell death by a PI uptake assay and SG formation by eIF4G in hMDM transfected with scrambled siRNA as control (SCR) or G3BP1 and G3BP2 siRNA for double knockdown (DKD). Cells were treated with 2 mM LLOMe for 30 min, and then stained with PI (dead cells), Hoechst-33342 (total cells) or eIF4G. (i) HCM images: white masks, algorithm-defined cell boundaries; green masks, computer-identified eIF4G puncta; red masks, computer-identified PI+ nuclei (target objects); (ii and iii) corresponding HCM quantification. Scale bar, 10 μm. ( G ) Cell death analysis of supernatants of hMDM transfected with either scrambled siRNA as control (SCR) or G3BP1 and G3BP2 siRNA for double knockdown (DKD) using a LDH release assay. Cells were treated with 2 mM LLOMe for 30 min. ( H ) Quantification by HCM of SG formation by G3BP1 in hMDM treated with 20 µM FAZ3532 or 20 µM FAZ3780 for 20 min, followed by exposure to 2 mM LLOMe for 30 min. Control cells were treated with DMSO. Green masks, computer-identified G3BP1 puncta. ( I ) Quantification by HCM of cell death by a PI uptake assay in hMDM treated with 20 µM FAZ3532 or 20 µM FAZ3780 for 20 min, followed by exposure to 2 mM LLOMe for 30 min. Control cells were treated with DMSO. Red masks, computer-identified PI+ nuclei. ( J ) Cell death analysis of supernatants of hMDM treated with 20 µM FAZ3532 or 20 µM FAZ3780 for 20 min, followed by exposure to 2 mM LLOMe for 30 min using a LDH release assay. Control cells were treated with DMSO. ( K ) Schematic summary of the findings in Fig. 1 and . CTR, control; NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), * p < 0.05, ** p < 0.01, ANOVA. See also Fig. . .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Microscopy, Double Knockout, Staining, Lactate Dehydrogenase Assay, Derivative Assay, Confocal Microscopy, Transfection, Control, Knockdown

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylation of eIF2α (S51), 4EBP1 (Ser65), S6K (Thr389), ULK1 (Ser757) and TFEB (Ser142) in U2OS cells treated with the indicated dose of LLOMe for 30 min. ( B ) Quantification by HCM of overlaps between mTOR and LAMP2 or G3BP1 puncta in U2OS cells. Cells were treated with EBSS, 2 mM LLOMe or 100 µM NaAsO2 for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified overlap between mTOR and LAMP2; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) and S6K1 (T389) in U2OS cells treated as in ( B ). ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) and cell death analysis by a LDH release assay in HEK293T cells expressing APEX2-eIF2α. Cells were treated with 1 mM LLOMe for the indicated durations. ( E ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells. Cells were treated with 2 mM LLOMe for the indicated durations. ( F ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with either scrambled siRNA as control (SCR) or MARK2 siRNA for knockdown (MARK2 KD ). Cells were treated with 2 mM LLOMe for 30 min. ( G ) Quantification by HCM of dsRNA puncta in U2OS cells. Cells were treated with 2 mM LLOMe or 100 ng/mL Poly (I:C) for 30 min. Green masks, computer-identified dsRNA puncta. ( H ) Immunoblot analysis of phosphorylation of PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or RNASET2 siRNA for knockdown (RNASET2 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( I ) Immunoblot analysis of phosphorylation of PKR (T446) in PKR KO U2OS G3BP1-GFP cells, overexpressing GFP, GFP-PKR and GFP-PKR K60A&K150A . Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( J ) Immunoblot analysis of phosphorylation of PKR (T446) in U2OS PACT knockdown cells (PACT KD ) overexpressing FLAG or FLAG-PACT. Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Figs. and .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Western Blot, Lactate Dehydrogenase Assay, Expressing, Transfection, Control, Knockdown

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or eIF2α siRNA for knockdown (eIF2α KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of mTORC1 activity by phosphorylation of 4EBP1 (Ser65), S6K (Thr389), ULK1 (Ser757), and TFEB (Ser142) in U2OS cells transfected with either scrambled siRNA as control (SCR) or eIF2α siRNA for knockdown (eIF2α KD ). Cells were treated with 2 mM LLOMe for 30 min. Quantification is based on three independent experiments. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells overexpressing wild-type RagB (RagB WT ) or constitutively active RagB mutant (RagB Q99L ) treated with 2 mM LLOMe for 30 min. Quantification is based on three independent experiments. ( D ) Quantification by HCM of G3BP1 puncta in U2OS cells overexpressing wild-type RagB (RagB WT ) or constitutively active RagB mutant (RagB Q99L ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( E ) Quantification by HCM of G3BP1 puncta in eIF2α knockdown (eIF2α KD ) U2OS cells transfected with FLAG, FLAG- eIF2α WT or FLAG- eIF2α S51A . Cells were treated with 2 Mm LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( F ) Schematic summary of the findings in Figs. 2 and . NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Transfection, Control, Knockdown, Western Blot, Activity Assay, Mutagenesis

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantitative liquid chromatography-tandem mass spectrometry (LC/MS/MS) using the data-independent acquisition (DIA) technique to identify eIF2α binding partners that were proximity-biotinylated by APEX2-eIF2α during lysosomal damage (1 mM LLOMe for 1 h). Scatter (volcano) plot shows log2 fold change (LLOMe/CTR; spectral counts) and –log10 p value for the proteins identified and quantified in three independent experiments. Green dots indicate increase in proximity to eIF2α (log2 fold change ≥ 1), and red dots indicate decrease in proximity to eIF2α (log2 fold change ≤ −1) during LLOMe treatment. Orange dots indicate values below the statistical significance cut-off ( P ≥ 0.05). Bubble size represents a normalized value for the total amount of spectral counts for the protein indicated. PACT, PKR and ALIX proteins are highlighted as purple circles (see Dataset EV ). ( B ) Quantification by HCM of G3BP1-GFP puncta in wild type (WT) or PKR knockout (PKR KO ) U2OS G3BP1-GFP cells. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in WT or PKR KO U2OS G3BP1-GFP cells, as well as in cells overexpressing FLAG-PKR in PKR KO U2OS G3BP1-GFP cells. Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( D ) Co-IP analysis of interactions between eIF2α and PKR/PACT during lysosomal damage. HEK293T cells expressing FLAG (control) or FLAG-eIF2α were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or PACT siRNA for knockdown (PACT KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta; (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in SCR or PACT KD cells; 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( F ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells treated with 1 mM LLOMe in the presence or absence of 210 nM imidazolo-oxindole C16 for 1 h. TMEM192-2xFLAG, control. The level of PKR, eIF2α and PACT in LysoIP was quantified based on three independent experiments shown in Fig. . ( G ) Schematic summary of the findings in Figs. 3 and . NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Knock-Out, Western Blot, Co-Immunoprecipitation Assay, Expressing, Control, Immunoprecipitation, Transfection, Knockdown, Purification

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Summary of the literature on the detected peptide count of PKR, PACT and eIF2α in the proteomic analysis of lysosomes based on LysoIP LC/MS/MS analysis. ( B ) Quantification of Fig. ; the level of PKR, eIF2α and PACT in LysoIP was quantified based on three independent experiments. ( C ) Confocal microscopy imaging of GFP-PKR and LAMP2 in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( D ) Confocal microscopy imaging of GFP-PACT and LAMP2 in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( E ) Confocal microscopy imaging of GFP-eIF2α and LAMP2 in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. * p < 0.05, ** p < 0.01, ANOVA. See also Fig. .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Imaging

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Transfection, Control, Knockdown, Western Blot

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of LAMP2 in U2OS cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). White masks, algorithm-defined cell boundaries; green masks, computer-identified LAMP2 puncta. ( B ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ), subjected to 2 mM LLOMe treatment for 30 min. ( D ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ), or pre-treated with 15 µM BAPTA-AM for 1 h. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALG2 knockdown cells (ALG2 KD ) overexpressing FLAG or FLAG-ALG2. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Transfection, Control, Knockdown, Western Blot

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) AlphaFold 2 predicted the interaction between PKR and ALIX, with the C-terminal PRD domain removed. ( B ) AlphaFold 2 predicted the interaction between PACT and ALIX, with the C-terminal PRD domain removed. ( C ) GST pulldown assay of in vitro translated His-tagged PKR with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( D ) GST pulldown assay of in vitro translated His-tagged PACT with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( E ) Confocal microscopy imaging of GFP-PKR/PACT and ALIX in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( F ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), PKR siRNA for knockdown (PKR KD ), or PACT siRNA for knockdown (PACT KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 1 mM LLOMe for 1 h. NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ANOVA. See also Fig. .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: GST Pulldown Assay, In Vitro, Confocal Microscopy, Imaging, Transfection, Control, Knockdown, Purification

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or galectin-3 (Gal3) siRNA for knockdown (Gal3 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with scrambled siRNA as control (SCR), or galectin-3 (Gal3) siRNA for knockdown (Gal3 KD ), subjected to 2 mM LLOMe treatment for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Co-IP analysis of interactions among FLAG-Gal3, ALIX, PKR and PACT in HEK293T cells during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis for ALIX, PKR, and PACT based on three independent experiments. ( D ) Co-IP analysis of interactions between FLAG-PKR and PACT in HEK293T cells transfected with scrambled siRNA as control (SCR), or Gal3 siRNA for knockdown (Gal3 KD ) during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( E ) Co-IP analysis of interactions between Myc-PACT and PKR in HEK293T cells transfected with FLAG, or FLAG-Gal3 during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( F ) Co-IP analysis of interactions among FLAG-ALIX, PKR and PACT in HEK293T cells transfected with GFP, GFP-Gal3 or GFP-Gal3 R186S during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( G ) Schematic summary of the findings in Fig. 6. NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). ** p < 0.01, ANOVA. .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Transfection, Control, Knockdown, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells infected with wild-type human adenovirus C2 (HAdV-C2 WT ) or C2 TS1 mutant (HAdV-C2 TS1 ) at MOI = 10 for 1 h. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells infected with wild type human adenovirus C2 (HAdV-C2 WT ) or C2 TS1 mutant (HAdV-C2 TS1 ) at MOI = 10 for 1 h. ( C ) Quantification by HCM of cell death by a propidium iodide (PI) uptake assay in U2OS wild type (WT) and G3BP1&2 double knockout (ΔΔG3BP1/2) cells during adenovirus infection. Cells were infected with wild-type human adenovirus C2 (HAdV-C2 WT ) at MOI = 10 for 1 h, and then stained with propidium iodide PI (dead cells) and Hoechst-33342 (total cells). White masks, algorithm-defined cell boundaries; red masks, computer-identified PI + nuclei. ( D ) Cell death analysis of supernatants of U2OS WT and ΔΔG3BP1/2 cells by a LDH release assay during SARS-Cov-2 ORF3a expression. Cells were transfected with the GFP-SARS-Cov-2 ORF3a construct overnight. ( E ) Cell death analysis of supernatants of human peripheral blood monocyte-derived macrophages (hMDM) by a LDH release assay during hemozoin exposure. Cells were treated with 10 µg/ml hemozoin for 4 h in the presence or absence of 1 μg/ml cycloheximide (CHX). ( F ) Quantification using AMNIS of cell death by Live/Dead TM stain kit in hMDM during silica treatment. Cells were treated with 200 µg/mL silica for 4 h in the presence or absence of 1 μg/ml cycloheximide (CHX), and then stained using Live/Dead TM stain kit (ThermoFisher). ( G ) Quantification using AMNIS of cell death by Live/Dead TM stain kit in hMDM during the treatment of tau oligomer. Cells were treated with 10 µg/mL tau oligomer for 4 h in the presence or absence of 1 μg/ml cycloheximide (CHX), and then stained using Live/Dead TM stain kit (ThermoFisher). CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). * p < 0.05, ** p < 0.01, ANOVA. See also Appendix Fig. S . .

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Infection, Mutagenesis, Western Blot, Double Knockout, Staining, Lactate Dehydrogenase Assay, Expressing, Transfection, Construct, Derivative Assay, Control

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: U2OS G3BP1-GFP cell line was generated using Flp-In system (ThermoFisher), details below.

Techniques: Recombinant, Sequencing, Mutagenesis, Control, CRISPR, Staining, Magnetic Beads, Transfection, Lysis, Cytotoxicity Assay, Protease Inhibitor, Software