Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Correlation between log2-fold change (L2FC) in RAD51 foci number and the Rule Set 2 on-target score. All guides targeting RAD51 regulators including RAD51 paralogs (RAD51D, RAD51C, XRCC3), BRCA1, and BRCA2 in the library are shown. Splice and nonsense variants with high Rule Set 2 score shows more significant L2FC. Pearson correlation (r) equals −0.30. (B) Quantification of RAD51 foci in irradiated S/G2-phase cells with guides targeting RAD51 regulators that have low Rule Set 2 score. Cells are grouped by sgRNA category. No. or moderate significant separation from the cells with control guides is observed. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. (Outliers are not shown in the CDFs.) (C) as in B) for guides with high on-target score, showing significant reduction in RAD51 foci in cells with nonsense and splice guides, compared to the control guides. (D) as in A) for L2FC in BRCA1 foci for BRCA1-targeting guides, showing a strong negative correlation with the Rule Set 2 score. Pearson correlation (r) equals −0.75. (E) same as B) for BRCA1 foci and guides targeting BRCA1 that have low Rule Set 2 score, showing no significant separation from the control guides. (F) same as E) for guides with high Rule Set 2 score, showing significant reduction in BRCA1 foci in cells with missense, nonsense, and splice guides. (G) Volcano plot showing no guides targeting AAVS1 and non-targeting control (NTC) guides shows statistically significant changes in RAD51 foci under irradiation. Significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.55, all AAVS1-targeting non-targeting control (NTC) guides are shown. Benjamini-Hochberg corrected two-sided KS test. (H) Same as G) showing guides that result in significant changes in RAD51 foci. (I) Gene enrichment analysis of guides targeting RAD51D among guides causing significant changes in RAD51 foci in the irradiated condition. Fisher exact test. Enriched genes are defined as p.adj <0.05. (J) same as G) for BRCA1 foci. (K) same as H) for guides that result in significant changes in BRCA1 foci (L) same as I) for BRCA1 foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Irradiation, Control

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Wasserstein distance of cells with DDR gene-targeting guides (Perturb) or control guides (Controls) to control cells for RAD51 foci in irradiated cells. Hits of RAD51 foci identified in the pooled screening are marked. (B) same as A) for BRCA1 foci. (C) Volcano plots showing variants yielding significant changes in the proportion of cells with high p21 expression in untreated (left) or irradiated (right) conditions. Significance is defined by p.adj < 0.05, Beta-Binomial test. The proportion of control cells with high p21 is marked with the vertical line (UNT: 0.376; IR: 0.576). sgRNA category and ClinVar category for significant guides are displayed by colors and shapes, respectively. (D) Correlation of RAD51 foci log2-fold change (L2FC) in guides delivered in the pooled library (pooled) and transduced individually (individual). The Pearson correlation (r) is 0.90. (E) same as D) for BRCA1 foci. The Pearson correlation (r) is 0.95. (F) MCF7-BE3 cells were lentivirally transduced with the DDR364 library, selected in puromycin-containing medium for 2 days, and cultured 2 days without puromycin. Cells were either untreated (UNT) or treated with DNA damaging agents Camptothecin (CPT), Olaparib (OLAP), Cisplatin (CISP) or Etoposide (ETOP) for 24 hours, then fixed before optical phenotyping and barcode detection. (G) Correlation between L2FC in RAD51 foci in CISP-treated cells and the Rule Set 2 on-target score, showing a negative correlation. All guides targeting RAD51 regulators including RAD51D, RAD51C, XRCC3, BRCA1, and BRCA2 are shown. The Pearson correlation (r) equals −0.29. (H) same as G) for OLAP-treated cells. The Pearson correlation (r) equals −0.30. (I) Volcano plot showing variants yielding significant changes in RAD51 foci in CISP-treated cells. Statistical significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.5, all AAVS1-targeting and non-targeting control (NTC) guides are shown. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. Same figure legend as in C). (J) same as I) for OLAP-treated cells. (K) Gene enrichment analysis in variants that result in significant changes in RAD51 foci. Fisher exact test. Enrichment is defined as p.adj <0.05. (L) Same as I) for variants yielding significant changes in large γH2AX foci in CISP-treated cells. (M) same as L) for OLAP-treated cells. (N) same as K) for large γH2AX foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Control, Irradiation, Expressing, Transduction, Cell Culture

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Immunoblots on guides targeting BRCA1 showing the reduction of full-length BRCA1 protein in the splice variant BRCA1.234, compared to the missense variants BRCA1.416 and.BRCA1.476, and the AAVS1 variant AAVS1.86. Cells transduced with Firefly siRNA (siFirefly) and BRCA1 siRNA (siBRCA1) were included to show the specificity of BRCA1 detection. Cells were treated with or without irradiation and the induction of DNA damage is shown with the phospho-KAP1 (pKAP1) staining. Tubulin is used as the loading control. (B) As in A) for BRCA2 variants showing the reduction of full-length BRCA2 protein in the nonsense variant BRCA2.207, compared to the missense variant BRCA2.438 and the AAVS1 variant AAVS1.86.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Western Blot, Variant Assay, Transduction, Irradiation, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress oxidative stress–induced parthanatos. A , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: MTS Assay, Control, Concentration Assay, Labeling, Western Blot, Software, Fluorescence, FACS

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress the ALIS formation. A , HT1080 and p62 KO HT1080 cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. B , HT1080 cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. C , HT1080 cells were treated with CTX (1 mg/ml) and/or NAC (2 mM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. D , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. Scale bar represents 10 μm. G , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. I , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were treated with the indicated reagents for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. CTX (1 mg/ml). I3MT-3 (10 μM). PAG (5 mM). Scale bar represents 10 μm. K , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, I3MT-3 0 μM PAG 0 mM cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; PAG, DL-propargylglycine; NAC, N-acetylcysteine; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence, Staining, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS disaggregate the ALIS by inducing HSP70. A , HT1080 cells were treated with the indicated reagents for 24 h and then incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. CTX (1 mg/ml). PAG (5 mM). I3MT-3 (20 μM). Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM, I3MT-3 0 μM cells). B , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h and then incubated with 10 μM DCFH-DA. Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), N.S. p > 0.05 ( versus CTX 1 mg/ml Na 2 S 4 0 μM cells). C , HT1080 cells were treated with the indicated reagents for 24 h, and then the detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. Bafilomycin A1 (5 nM). CTX (1 mg/ml). Na 2 S 4 (100 μM). D , HT1080 cells were treated with CTX (1 mg/ml) for 20 h and then treated with MG132 (10 μM) and/or Na 2 S 4 (100 μM) for 4 h. The detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the indicated concentration of Na 2 S 4 for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. G , HT1080 cells were treated with CTX (1 mg/ml) and Na 2 S 4 (100 μM) for 18 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then whole cell lysate were subjected to immunoblotting with the indicated antibodies. I , HT1080 and PARP-1 KO cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. L , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05, ∗∗ p < 0.01, ( versus siRNA Ctr, CTX 1 mg/ml cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; HSP, heat shock protein; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; ROS, reactive oxygen species; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Incubation, Fluorescence, Control, Western Blot, Concentration Assay, Transfection, Negative Control, Immunofluorescence, Staining, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS activate HSF1 by promoting its dissociation from HSP90. A , HT1080 cells were treated with the Na 2 S 4 (100 μM) for indicated period, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗∗ p < 0.001, ( versus control cells). B , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. C , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. D , HT1080 and PARP-1 KO cells were treated with the Na 2 S 4 (100 μM) for 6 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus control cells), ## p < 0.01 ( versus Na 2 S 4 100 μM, KRIBB11 0 μM cells). G , HT1080 cells were transfected with FLAG-HSP90 and/or Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with indicated concentration of Na 2 S 4 for 4 h, then immunoprecipitated protein G-Sepharose beads with the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. I , HSP90 was immunoprecipitated using anti-HSP90 antibody with protein G beads. Beads were washed four times with PBS and then treated with Na 2 S 4 (100, 1000 μM) for 1 h. After reaction, beads were washed four times with PBS and subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with FLAG-HSP90 (WT/C412A/C564A/C521A) and Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with FLAG-Empty or FLAG-HSP90 (WT/C521A) plasmid for 24 h and treated with Na 2 S 4 (100 μM) for indicated periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. All data are representative of at least three independent experiments. HSF, heat shock factor; HSP, heat shock protein; PARP-1, poly (ADP-ribose) polymerase-1; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Concentration Assay

Journal: BMC Cell Biology

Article Title: μ-Slide Chemotaxis: A new chamber for long-term chemotaxis studies

doi: 10.1186/1471-2121-12-21

Figure Lengend Snippet: Microscopic image of the observation area and representative cell trajectory plots . A: Representative HT-1080 microscopic image of the chamber observation area (2 mm × 1 mm) at time 24 h with an overlay of the tracked cell trajectories. Data from a positive chemotaxis experiment using 10% FCS as attractant (+/-) is shown. Figure 4B shows the corresponding trajectory plot. Figures 4B-D: Representative HT-1080 cell trajectory plots in the time period from 0 to 24 hours. (B) Chemotaxis experiment using 10% FCS as attractant. Cells move in the positive direction of the║-axis, which is by definition the direction of the gradient (+/-). (C) In the negative control, both chambers are filled with FCS-free medium (-/-). Directed cell migration is not visible. (D) In the positive control group, both chambers are filled with FCS 10% (+/+). As in the negative control group, no directed migration can be measured. Cell motility is enhanced. Figures 4E-G: Representative HUVEC trajectory plots in the time period from 0 to 24 hours. (E) Similar to HT-1080, HUVEC also move in the direction of the FCS gradient (+/-). Negative (F, -/-) and positive (G, +/+) controls show no directed migration and a smaller displacement of centre of mass.

Article Snippet: Human fibrosarcoma epithelial cell line HT-1080 (obtained from Toni Lindl, Munich, Germany, ATCC No.: CCL-121) was grown in Dulbecco's modified Eagle's Medium (DMEM; PromoCell, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37°C and 5% CO2.

Techniques: Chemotaxis Assay, Negative Control, Migration, Positive Control

Journal: BMC Cell Biology

Article Title: μ-Slide Chemotaxis: A new chamber for long-term chemotaxis studies

doi: 10.1186/1471-2121-12-21

Figure Lengend Snippet: Analyzed migration parameters . A-D: Analyzed migration parameters of one set of HT-1080 experiments. Each symbol represents a data point (or its - or ┴-component) of a single measurement. (A) Forward migration indices in parallel (FMI ║ ) and perpendicular (FMI ┴ ) directions are compared to the values of negative (-/-) and positive controls (+/+). The horizontal line indicates zero. All FMI except FMI ║ of the chemotaxis experiment (+/-) are close to 0, indicating arbitrary movements. The FMI ║ (+/-) varies between 0.2 and 0.3, representing directed migration. (B) The displacement of the centre of mass (COM) is shown from the same experimental set as A. Like in the case of the FMI, all COM are close to 0, but only the displacement in parallel direction (COM ║ ) of the chemotaxis experiment differs strongly from 0. (C) Average cell velocities of HT-1080. In case of the positive control, the velocity is enhanced roughly by a factor of two compared to the chemotaxis experiment (+/-) and the negative control (-/-). (D) Corresponding p-values of the Rayleigh test in logarithmic scale. The dashed line indicates the 0.05 threshold. Only in the (+/-) case is the p-value smaller than 0.05, showing an inhomogeneous cell distribution. Figures 5E-H: Analyzed data of HUVEC cells. Circles (HUVEC 1: self prepared) and squares (HUVEC 2: Lonza) represent parameters of single experiments of two different cell sources. (E) Only the FMI ║ of the (+/-) case is clearly larger than 0. No difference between the two different cell sources is visible. (F) COM ║ of the (+/-) case of HUVEC 2 is larger than all other COM. (G) Average cell velocities differ dependent on the cell sources. (H) The p-values of the (+/-) data are below 0.05 with one exception. There is no significant difference between the two cell sources.

Article Snippet: Human fibrosarcoma epithelial cell line HT-1080 (obtained from Toni Lindl, Munich, Germany, ATCC No.: CCL-121) was grown in Dulbecco's modified Eagle's Medium (DMEM; PromoCell, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37°C and 5% CO2.

Techniques: Migration, Chemotaxis Assay, Positive Control, Negative Control

Journal: BMC Cell Biology

Article Title: μ-Slide Chemotaxis: A new chamber for long-term chemotaxis studies

doi: 10.1186/1471-2121-12-21

Figure Lengend Snippet: Time development of ┴ and ║ components of COM and FMI in one representative HT-1080 chemotaxis group (+/-) (data from Figure 4B) . The FMI ║ increases volatile during five hours at the beginning of the experiment and remains nearly constant afterwards. COM ║ increases during the whole experimental time with a constant slope. Both, COM ┴ and FMI ┴ level off around zero indicating no directed migration perpendicular to the direction of the gradient.

Article Snippet: Human fibrosarcoma epithelial cell line HT-1080 (obtained from Toni Lindl, Munich, Germany, ATCC No.: CCL-121) was grown in Dulbecco's modified Eagle's Medium (DMEM; PromoCell, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37°C and 5% CO2.

Techniques: Chemotaxis Assay, Migration

Journal: BMC Cell Biology

Article Title: μ-Slide Chemotaxis: A new chamber for long-term chemotaxis studies

doi: 10.1186/1471-2121-12-21

Figure Lengend Snippet: Single HT-1080 cell, migrating towards a FCS gradient pointing from the right to the left side (higher concentration is on the left side) . The image was acquired with an epi fluorescence mode using a 60× oil immersion objective (Nikon). Scale bar is 20 μm.

Article Snippet: Human fibrosarcoma epithelial cell line HT-1080 (obtained from Toni Lindl, Munich, Germany, ATCC No.: CCL-121) was grown in Dulbecco's modified Eagle's Medium (DMEM; PromoCell, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37°C and 5% CO2.

Techniques: Concentration Assay, Fluorescence

Journal: Communications Biology

Article Title: piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

doi: 10.1038/s42003-021-02844-1

Figure Lengend Snippet: a The IC 50 values of DOX in HT1080 cells determined by MTT assay. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01 vs 0 μM, Dunnett’s multiple comparisons test. b The IC 50 values of DOX in HT1080/DOX cells determined by MTT assay. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, *** P < 0.001, **** P < 0.0001 vs 0 μM, Dunnett’s multiple comparisons test. c The viability of HT1080/DOX cells in 0.4 µM DOX is ~30% higher than HT1080 cells. Bars, mean ± SEM; n = 3 independent experiments; * P < 0.05, t -test. d The viability of HT1080 cells in 2.0 µM DOX is ~40% lower than HT1080/DOX cells. Bars, mean ± SEM; n = 3 independent experiments; ** P < 0.01, t -test. e Relative expression levels of piR-39980 in parental HT1080 and HT1080/DOX cell lines were determined via qRT-PCR. The expression level of piR-39980 was lower in HT1080/DOX cells compared with HT1080 cells, p < 0.01. Bars, mean ± SEM; n = 3 independent experiments; ** P < 0.01, t -test. f The effect of piR-39980 on viability of HT1080 cells was determined by MTT assay upon transfection with 20 nM piR-39980 mimic/NC_Mimic and treatment with 0.4 µM DOX. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, *** P < 0.001, Sidak’s multiple comparisons test. g The effect of piR-39980 on viability of HT1080 cells was determined by MTT assay upon transfection with 20 nM piR-39980 inhibitor/NC_inhibitor and treated with 0.4 µM DOX. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, Sidak’s multiple comparisons test.

Article Snippet: HT1080 cell line was cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Nashik, India; AL007A), and HEK293 cell line was cultured with Minimum Essential Medium Eagle (MEM, Himedia; AL047A).

Techniques: MTT Assay, Expressing, Quantitative RT-PCR, Transfection

Journal: Communications Biology

Article Title: piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

doi: 10.1038/s42003-021-02844-1

Figure Lengend Snippet: a Transfection efficiency showing relative expression of piR-39980 increased by ~250-fold in HT1080/DOX cells transfected with 20 nM piR-39980 mimics compared to controls. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, Tukey’s multiple comparisons test. b Effect of piR-39980 on HT1080/DOX cell viability determined by MTT assay. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, Tukey’s multiple comparisons test. c The colony-forming ability of HT1080/DOX cells transfected with piR-39980 mimic. Bars, mean ± SEM; n = 3 independent experiments; ns non-ignificant, * P < 0.05, ** P < 0.01, Tukey’s multiple comparisons test. d Effect of piR-39980 on HT1080/DOX cell morphology upon 0.4 µM DOX treatment. Scale bar: 100 μm.

Article Snippet: HT1080 cell line was cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Nashik, India; AL007A), and HEK293 cell line was cultured with Minimum Essential Medium Eagle (MEM, Himedia; AL047A).

Techniques: Transfection, Expressing, MTT Assay

Journal: Communications Biology

Article Title: piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

doi: 10.1038/s42003-021-02844-1

Figure Lengend Snippet: a Intracellular DOX-accumulation in HT1080/DOX cells in different conditions. Scale bar: 50 μm. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, Tukey’s multiple comparisons test. b AO/EB dual staining assay showing an increase in apoptotic cell death. Blue, white, and pink arrows indicate live cells, early apoptotic cells, and late apoptotic cells, respectively. Scale bar: 75 μm. c Flow cytometric PE Annexin-V apoptosis assay showing an increase in apoptotic cell death upon transfection with piR-39980 mimic and DOX. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, *** P < 0.001, Tukey’s multiple comparisons test.

Article Snippet: HT1080 cell line was cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Nashik, India; AL007A), and HEK293 cell line was cultured with Minimum Essential Medium Eagle (MEM, Himedia; AL047A).

Techniques: Staining, Apoptosis Assay, Transfection

Journal: Communications Biology

Article Title: piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

doi: 10.1038/s42003-021-02844-1

Figure Lengend Snippet: a Relative expression of RRM2 detected in parental HT1080 and resistant HT1080/DOX cell lines by qRT-PCR. Bars, mean ± SEM; n = 3 independent experiments; * P < 0.05, t -test. b The expression of RRM2 in HT1080/DOX cells upon transfected with 20 nM piR-39980 mimic/inhibitor as detected by qRT-PCR. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, *** P < 0.001, Tukey’s multiple comparisons test. c The expression of RRM2 in HT1080 cells upon transfected with 20 nM piR-39980 inhibitor/mimic as detected by qRT-PCR. Bars, mean ± SEM; n = 5 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, Tukey’s multiple comparisons test. d Relative expression of CYP1A2 detected in parental HT1080 and resistant HT1080/DOX cell lines by qRT-PCR. Bars, mean ± SEM; n = 3 independent experiments; * P < 0.05, t -test. e The expression of CYP1A2 in HT1080/DOX cells upon transfected with 20 nM piR-39980 mimic/inhibitor compared to control, detected by qRT-PCR. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, Tukey’s multiple comparisons test. f The expression of CYP1A2 in HT1080 cells transfected with 20 nM piR-39980 mimic/inhibitors detected by qRT-PCR. Bars, mean ± SEM; n = 5 independent experiments; ns nonsignificant, ** P < 0.01, *** P < 0.001, Tukey’s multiple comparisons test. g Luciferase reporter constructs containing wild-type/mutant piR-39980 target site in RRM2 3′-UTR were co-transfected with mimic into the HEK293 cells. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, *** P < 0.001, Sidak’s multiple comparisons test. h Luciferase reporter constructs containing wild-type/mutant piR-39980 target site in CYP1A2 3′-UTR were co-transfected with mimic into the HEK293 cells. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, *** P < 0.001, Sidak’s multiple comparisons test.

Article Snippet: HT1080 cell line was cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Nashik, India; AL007A), and HEK293 cell line was cultured with Minimum Essential Medium Eagle (MEM, Himedia; AL047A).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Luciferase, Construct, Mutagenesis

Journal: Communications Biology

Article Title: piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

doi: 10.1038/s42003-021-02844-1

Figure Lengend Snippet: a Relative expression of RRM2 was detected by qRT-PCR in HT1080/DOX cells transfected with pcDNA3.1_RRM2 compared with pcDNA3.1. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Tukey’s multiple comparisons test. b MTT assay showing role of RRM2/piR-39980 axis in HT1080 cell viability. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Tukey’s multiple comparisons test. c PE Annexin-V flow cytometry assay showing role of RRM2/piR-39980 axis on apoptosis of HT1080 cells and quantification of apoptotic cells upon transfection with pcDNA3.1_RRM2 and piR-39980. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Tukey’s multiple comparisons test.

Article Snippet: HT1080 cell line was cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Nashik, India; AL007A), and HEK293 cell line was cultured with Minimum Essential Medium Eagle (MEM, Himedia; AL047A).

Techniques: Expressing, Quantitative RT-PCR, Transfection, MTT Assay, Flow Cytometry

Journal: Communications Biology

Article Title: piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

doi: 10.1038/s42003-021-02844-1

Figure Lengend Snippet: a Comet assay showing the impact of RRM2/piR-39980 axis on DNA damage of HT1080 cells. Scale bar: 10 μm. b Comet tail length and c comet tail moment measured by ImageJ. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Tukey’s multiple comparisons test. d γH2AX accumulation assay detected by fluorescence imaging using anti-γH2AX antibody and quantification of γH2AX- foci upon transfection with pcDNA3.1_RRM2 and piR-39980. Green fluorescence, DyLight 488 conjugated secondary antibody showing accumulation of γH2AX. Blue fluorescence of DAPI showing nucleus. Scale bar: 20 μm. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, Tukey’s multiple comparisons test.

Article Snippet: HT1080 cell line was cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Nashik, India; AL007A), and HEK293 cell line was cultured with Minimum Essential Medium Eagle (MEM, Himedia; AL047A).

Techniques: Single Cell Gel Electrophoresis, Fluorescence, Imaging, Transfection

Journal: Communications Biology

Article Title: piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

doi: 10.1038/s42003-021-02844-1

Figure Lengend Snippet: a Relative expression of CYP1A2 was detected by qRT-PCR in HT1080/DOX cells transfected with pcDNA3.1_CYP1A2 and empty pcDNA3.1 vector. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001, Tukey’s multiple comparisons test. b Role of CYP1A2/piR-39980 axis in HT1080 cell viability determined by MTT assay. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, ** P < 0.01, Tukey’s multiple comparisons test. c Role of CYP1A2/piR-39980 axis in intracellular DOX accumulation determined by fluorescence microscopy. Scale bar: 50 μm. d The intensity of intracellular DOX was quantified by ImageJ using the images of Fig. 10c. Bars, mean ± SEM; n = 3 independent experiments; * P < 0.05, ** P < 0.01, Tukey’s multiple comparisons test. e PE Annexin-V flow cytometry assay showing the role of CYP1A2/piR-39980 axis in HT1080 cell death. f Quantification of apoptotic cells upon transfection with pcDNA3.1_CYP1A2 and piR-39980. Bars, mean ± SEM; n = 3 independent experiments; ns nonsignificant, * P < 0.05, Tukey’s multiple comparisons test.

Article Snippet: HT1080 cell line was cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Nashik, India; AL007A), and HEK293 cell line was cultured with Minimum Essential Medium Eagle (MEM, Himedia; AL047A).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, MTT Assay, Fluorescence, Microscopy, Flow Cytometry

Journal: Oncotarget

Article Title: Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2

doi: 10.18632/oncotarget.10062

Figure Lengend Snippet: Digestion activity is more prominent in cells treated by C16 ( A. , white arrow in gelatin-FITC panel). Red lines (A, overlay panel) indicate points of XY image projected to generate orthogonal planes XZ and YZ. Colocalization of actin protrusions with digested areas (invadopodia) is evident in overlay panel (A, white arrow), and in orthogonal projections XZ and YZ (A, black arrows in orthogonal projections magnifications). Figure B is a three-dimensional reconstruction from the boxed area in A, overlay panel. C16-treated sample exhibits actin projections invading the gelatin-FITC substrate ( B. , white arrows). HT1080 cells treated by C16SX control peptide exhibit few areas of degraded substrate C. Measurements of digested areas per cell demonstrate that invadopodia activity induced by C16 is 11-fold increased compared to C16SX D. Results represent mean ± standard error of 20 cells, in three different experiments. Scale bars: 10 μm.

Article Snippet: HT1080 cell line, from human fibrosarcoma [ ], was cultured in minimum Eagle's medium (MEM, Vitrocell) with 10% FBS.

Techniques: Activity Assay, Control

Journal: Oncotarget

Article Title: Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2

doi: 10.18632/oncotarget.10062

Figure Lengend Snippet: Time-lapse image sequence of cells transfected with cortactin-GFP and cultured on Alexa-568 fluorescent gelatin reveal that C16 enhances invadopodia activity in both CAL27 A. and HT1080 C. cells over time. Evident digestion area in gelatin matrix is observed 12 hours after treatment with the peptide in both cells (A and C, white arrows). Measurements of digested areas per cell (expressed as fold increase of initial digested area) demonstrate that invadopodia activity of C16-treated CAL27 cells is evident 6 hours after C16 treatment B. , while in HT1080 cells, this activity increases 2 hours after peptide treatment D. Asterisks in B and D indicate significant data compared to controls (P<0.05). Results represent mean ± standard error of 10 cells. Scale bars: 10 μm.

Article Snippet: HT1080 cell line, from human fibrosarcoma [ ], was cultured in minimum Eagle's medium (MEM, Vitrocell) with 10% FBS.

Techniques: Sequencing, Transfection, Cell Culture, Activity Assay

Journal: Oncotarget

Article Title: Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2

doi: 10.18632/oncotarget.10062

Figure Lengend Snippet: In 3D reconstructions of fluorescence assays with rhodamine-conjugated peptides ( A and D. , C16 and C16SX panels), cell staining with phalloidin (blue channel) identifies actin filaments. C16 (red channel) is found decorating the cell edge (A and D, C16), while C16SX apparently do not adhere to cell surface (A and D, C16SX). Active β1 integrin expression is increased in both CAL27 and HT1080 C16-treated cells compared to C16SX, as demonstrated by flow cytometry ( B and E. ). Tumor cells were transfected with a plasmid containing β1 integrin miRNA (Block-iT, Invitrogen) and subjected to fluorescent substrate degradation assays ( C and F. ). Measurements of digested areas per cell show a decrease in invadopodia activity over time in β1 integrin-silenced cells treated with C16, compared to negative control (pcDNA 6.2-GW/EmGFPmiR-neg control – Block-iT). Results in B and E represent mean ± standard error of three experiments. Results in C and F represent mean ± standard error of 10 cells.

Article Snippet: HT1080 cell line, from human fibrosarcoma [ ], was cultured in minimum Eagle's medium (MEM, Vitrocell) with 10% FBS.

Techniques: Fluorescence, Staining, Expressing, Flow Cytometry, Transfection, Plasmid Preparation, Blocking Assay, Activity Assay, Negative Control, Control

Journal: Oncotarget

Article Title: Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2

doi: 10.18632/oncotarget.10062

Figure Lengend Snippet: Immunoblot and densitometry show that C16 stimulates Src phosphorylation in HT1080 cells A. Increased ERK phosphorylation is consistently observed in immunoblot, but not significant in densitometry B. A decrease in C16-mediated invadopodia formation is observed in HT1080 cells with ERK 1/2 signaling pathway inhibited by U0126 C. This reduction in invadopodia activity is also observed over time, in cortactin-GFP-transfected HT1080 cells treated with U0126 and incubated with C16 D. No differences are detected in ERK-inhibited cells incubated with C16SX and positive non-peptide control (C). Immunoblot confirms U0126 inhibition efficiency (C, box). Asterisks in C, D indicates significant data compared to methanol (vehicle) controls (P<0.05). Results in A and B represent mean ± standard error of three different experiments. Results in C represent mean ± standard error of 20 cells, in three different experiments. Results in D represent mean ± standard error of 10 cells.

Article Snippet: HT1080 cell line, from human fibrosarcoma [ ], was cultured in minimum Eagle's medium (MEM, Vitrocell) with 10% FBS.

Techniques: Western Blot, Phospho-proteomics, Activity Assay, Transfection, Incubation, Control, Inhibition