Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A ) Schematic illustration of the Jak2 V617F knock-in MPN transplant mouse model used, treatment regimen, and analyses performed. ( B ) Hemoglobin (Hb) levels, ( C ) hematocrit (HCT) percentage and ( D ) red blood cell (RBC) counts in peripheral blood of recipient mice are shown for each treatment group four weeks post treatment initiation. ( E ) Percentage of Ter119 med CD71 high proerythroblasts (R1), ( F ) Ter119 high CD71 high basophilic erythroblasts (R2) and ( G ) Ter119 high CD71 med late basophilic and polychromatophilic erythroblasts (R3) and Ter119 high CD71 low orthochromatophilic erythroblasts (R4) in the BM of recipient mice four weeks post treatment initiation. ( H ) Spleen weight and percentage of ( I ) R1, ( J ) R2 and ( K ) R3+R4 cells in the spleen of recipient mice four weeks post treatment initiation. ( B-K ) Box plots show distribution of data from 10 mice per group. Statistical analyses were performed using Mann-Whitney test. Statistically significant p -values are reported. See also supplemental figures 6 and 7 . ( L-M ) Cellular viability of JAK2 V617F -positive ( L ) HEL and ( M ) SET-2 cells treated with either DMSO-vehicle control (Ctrl) or with increasing doses of the ULK1 inhibitor SBP-7455 for five days, as indicated, was measured using WST-1 cell viability reagent. Data are expressed as percent cell viability relative to Ctrl-treated cells and represent means ± SEM of ( L ) four and ( M ) three independent experiments. IC50 values are shown. ( N ) Clonogenic capability of HEL cells treated with either vehicle-control (DMSO) or with increasing doses of the ULK1 inhibitor SBP-7455, as indicated. Data are expressed as percent colony formation relative to DMSO-treated cells (control) and represent means ± SEM of three independent experiments. Statistical analysis was performed using Welch’s ANOVA test that accounts for unequal variances followed by Dunnett’s T3 pairwise multiple comparisons test. Statistically significant p -values are reported. Test for linear trend based on the one-way ANOVA model showed a significant decrease in colony formation with increased dose of ULK1 inhibitor ( p <0.0001). See also supplemental figure 8 .

Article Snippet: HEL cells (#TIB-180; ATCC) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS and antibiotics.

Techniques: Inhibition, Activity Assay, In Vivo, Knock-In, MANN-WHITNEY, Control

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A-B ) Immunoblotting analysis of the indicated proteins in lysates from non-targeting (NT) control, Atg7 KO and Ulk1 KO Jak2 V617F -positive Ba/F3-EpoR + GFP-LC3-RFP-expressing cells. ( C ) Percentage of autophagy measured as percentage of GFP negative cells assessed by flow cytometry analyses of NT control, Atg7 KO and Ulk1 KO Jak2 V617F -positive Ba/F3-EpoR + GFP-LC3-RFP-expressing cells cultured under normal nutrient conditions (RPMI 1640 media supplemented with 10% FBS and 10 ng/mL of IL-3, no starvation) or under starvation conditions (RPMI 1640 media without serum and IL-3) for 48 hours. Scatter dot plot shows means ± SD. Statistical analysis was performed using Welch’s ANOVA test that accounts for unequal variances followed by Dunnett’s T3 pairwise multiple comparisons test. Statistically significant p -values are reported. ( D-F ) Immunoblotting analysis of the indicated proteins in lysates from ( D ) Cas9-control compared to ULK1 KO HEL cells and from ( E ) HEL cells treated with either DMSO-vehicle control (C), 5μM SBP-7455 (S), 100nM bafilomycin A1 (B) or with the combination of SBP-7455 and bafilomycin A1 (S+B) for 4 hours under either nutrient-starvation conditions (HBSS) or normal complete nutrient-conditions (RPMI). Immunoblots shown are representative of three independent experiments. ( F ) SET-2 cells treated with either DMSO-vehicle control (C), 5μM SBP-7455 (S), 100nM bafilomycin A1 (B) or with the combination of SBP-7455 and bafilomycin A1 (S+B) for 4 hours under either nutrient-starvation conditions (HBSS) or normal complete nutrient-conditions (RPMI). Immunoblots shown are representative of three independent experiments.

Article Snippet: HEL cells (#TIB-180; ATCC) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS and antibiotics.

Techniques: Inhibition, Western Blot, Control, Expressing, Flow Cytometry, Cell Culture

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A-B ) HEL cells were treated with either vehicle (DMSO, Ctrl), 5μM SBI-0206965 (SBI) or 5μM ULK101 for 6 hours followed by RNA-seq and analysis of transcript expression. ( A ) Heatmap of differentially expressed genes between treatment groups (adj. p val. < 0.01). ( B ) MA plot of genes significantly changed by drug-targeted inhibition of ULK1 in HEL cells versus control (adj. p val. < 0.01). In purple are represented up-regulated genes and in green are represented down-regulated genes. ( C and D ) Enriched ontology clusters for genes that are ( C ) increased or ( D ) decreased after drug-targeted inhibition of ULK1 in HEL cells. ( A-D ) Data are from 4 biological replicates per condition. See also supplemental figures 9 – 11 .

Article Snippet: HEL cells (#TIB-180; ATCC) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS and antibiotics.

Techniques: Inhibition, Activity Assay, Gene Expression, RNA Sequencing, Expressing, Control

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A-E ) Scatter dot plots of PIM1 , ID2 , HES1 , CTR9 and PDCD2 expression in healthy individuals (normal, n = 11) and patients with ET (n = 47), PV (n = 28) and MF (n = 18). Data extracted from GSE5464672. Shown are means ± SEM of Log2 mRNA expression. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparisons adjustment comparing each MPN group vs. Normal group. Statistically significant p -values are reported. ( F-J ) mRNA expression of the indicated genes was assessed by qRT-PCR analysis for HEL cells treated for 6 hours with either vehicle (DMSO, Control) or SBP-7455 (SBP), as indicated. Data are means ± SEM from three independent experiments. Fold changes relative to control (dashed line) were calculated for each experimental replicate (SBP/DSMO), and fold changes were compared to FC=1 using a one-sample t-test, p values are reported. See also supplemental figure 12 .

Article Snippet: HEL cells (#TIB-180; ATCC) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS and antibiotics.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Control

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A-E ) qRT-PCR analysis of the indicated genes mRNA expression in murine c-kit + bone marrow Jak2 V617F/+ VavCre + cells from 3 mice treated for 6 hours with either vehicle (DMSO, Control) or 5μM SBP-7455 (SBP), as indicated. Data are means ± SD across three different mice. Fold changes were calculated relative to gene expression from one randomly selected vehicle control-treated mouse sample. Statistical analyses were performed using two-tailed unpaired t test with Welch’s correction. Statistically significant p -values are reported. ( F-J ) qRT-PCR analysis of the indicated genes mRNA expression in Cas9-control and ULK1 KO HEL cells, as indicated. Data are means ± SD across three independent experiments. Fold changes were calculated relative to gene expression from one randomly selected Cas9 control sample. Statistical analyses were performed using two-tailed unpaired t test with Welch’s correction. Statistically significant p -values are reported. See also supplemental figures 13 – 15 .

Article Snippet: HEL cells (#TIB-180; ATCC) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS and antibiotics.

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Control, Gene Expression, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Adipocyte Low Density Lipoprotein Receptor-related Protein Gene Expression and Function Is Regulated by Peroxisome Proliferator-activated Receptor γ

doi: 10.1074/jbc.m212989200

Figure Lengend Snippet: FIG. 2. Increased cellular degradation of 125I-2M* by cells treated with PPAR ligands. A, differentiated primary human adi- pocytes were treated with various concentrations of rosiglitazone in the absence of insulin for 24 h prior to the experiment. The cells were then incubated at 37 °C with HBSS, 25 mM HEPES, pH 7.45, 10 mg/ml BSA containing 1 mg/ml 125I-2M* with or without RAP (30 g/ml). At the beginning of the incubation period and at each time point, the medium was precipitated with ice-cold tricarboxylic acid, and the tricarboxylic acid-soluble material was used as a measure of the degraded protein. The data points represent the means from triplicate experiments, and the error bars represent the standard error of the mean. *, p 0.009; **, p 0.007; ***, p 0.002. B and C, SW872 cells (treated with vehicle, 500 nM rosiglitazone, and 160 M arachidonic acid) were washed at 37 °C to remove any residual fetal calf serum. The cells were incubated at 37 °C for the indicated times in HBSS, 25 mM HEPES, pH 7.45, 10 mg/ml BSA containing 1 mg/ml 125I-2M*. At the beginning of the incubation period and at each time point, the medium was precipitated with ice-cold tricarboxylic acid, and the tricarboxylic acid-soluble ma- terial was used as a measure of the degraded protein. Trichloroacetic acid-soluble material from untreated cells (vehicle) was compared with the trichloroacetic acid-soluble material from cells treated with 500 nM rosiglitazone (B) or 160 M arachidonic acid (C). The data points rep- resent means from triplicate experiments, and the error bars represent the standard error of the mean. *, p 0.006; **, p 0.03. DMSO, dimethyl sulfoxide.

Article Snippet: Cell Culture—The human liposarcoma cell line SW872 (American Type Culture Collection, Manassas, VA) was previously characterized and has been shown to be a good cell model for adipocyte gene expression (42–44).

Techniques: Incubation

Journal: Journal of Biological Chemistry

Article Title: Adipocyte Low Density Lipoprotein Receptor-related Protein Gene Expression and Function Is Regulated by Peroxisome Proliferator-activated Receptor γ

doi: 10.1074/jbc.m212989200

Figure Lengend Snippet: FIG. 3. LRP protein levels are increased in SW872 cells upon exposure to PPAR ligands. A, cells were incubated with 1 g/ml fluorescently labeled activated 2M (2M*) (Cy3 monofunctional reac- tive dye) on ice for 45 min in 3:1 Dulbecco’s modified Eagle’s medium/ Ham’s F-12 medium supplement with 2 mg/ml BSA buffered with 10 mM HEPES after an equilibration period of 1 h. The cells were washed with ice-cold PBS three times prior to being fixed with 4% paraformal- dehyde for 10 min at 0 °C. The cells were rinsed with PBS and kept in 2 ml of PBS at room temperature for fluorescence microscopy. The photographs are representative of cells treated with the ligands indi- cated. A 30-fold excess of unlabeled 2M* was used to compete for binding with the fluorescently labeled 2M*. The photographs are normalized so that an increase in intensity on the cell surface and the cell circumference between photographs represents an increase in the fluorescence (i.e. total binding). B, Western blot of cells treated with Me2SO (vehicle control), 160 M arachidonic acid, or 500 nM rosiglita- zone for 24 h. 5 g of total protein was loaded in each lane. The blot was developed using chemiluminescent techniques, and the bands were visualized using the ChemiDoc apparatus. The band at 515 kDa corre- sponds to the -subunit of LRP. C, quantification of Western blot. The 515-kDa band corresponding to LRP was quantified using Quantity One software. All of the samples were normalized to the vehicle-treated cells (control). The Results are shown as the means of triplicate exper- iments, and the error bars represent the standard error of the mean. *, the two-tailed p value is 0.002; **, the two-tailed p value is 0.003.

Article Snippet: Cell Culture—The human liposarcoma cell line SW872 (American Type Culture Collection, Manassas, VA) was previously characterized and has been shown to be a good cell model for adipocyte gene expression (42–44).

Techniques: Incubation, Labeling, Modification, Fluorescence, Microscopy, Binding Assay, Western Blot, Control, Software, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Adipocyte Low Density Lipoprotein Receptor-related Protein Gene Expression and Function Is Regulated by Peroxisome Proliferator-activated Receptor γ

doi: 10.1074/jbc.m212989200

Figure Lengend Snippet: FIG. 4. Endogenous LRP mRNA levels in SW872 cells are mod- ulated by PPAR ligands at the transcriptional level. The effect of PPAR ligands on levels of LRP mRNA was determined by incubating SW872 cells for 24 h in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (3:1) supplemented with complete serum in the presence or absence of the PPAR ligands. A, oleic acid. B, arachidonic acid. C, rosiglitazone. Also a potent inhibitor of RNA polymerase II, -amanitin, was added to SW872 cells in the presence or absence of 500 nM rosigli- tazone to inhibit transcription (C). Reverse transcription was performed on total RNA isolated from these cells, and multiplex PCR was per- formed using LRP and 18 S gene-specific primers (relative quantitative RT-PCR). PCR products were visualized by EtBr staining on the Chemi- Doc, and the band intensities were determined using Quantity One software. The intensity of the LRP product was divided by the intensity of the 18 S product to obtain a value termed relative intensity. These results were normalized to the control and shown as fold increases. The bars in each graph represent the means of triplicate experiments, and the error bars represent the standard error of the mean. One-tailed p values from paired t test are shown above the bars to indicate signifi- cant differences.

Article Snippet: Cell Culture—The human liposarcoma cell line SW872 (American Type Culture Collection, Manassas, VA) was previously characterized and has been shown to be a good cell model for adipocyte gene expression (42–44).

Techniques: Modification, Reverse Transcription, Isolation, Multiplex Assay, Quantitative RT-PCR, Staining, Software, Control, One-tailed Test

Journal: Journal of Biological Chemistry

Article Title: Adipocyte Low Density Lipoprotein Receptor-related Protein Gene Expression and Function Is Regulated by Peroxisome Proliferator-activated Receptor γ

doi: 10.1074/jbc.m212989200

Figure Lengend Snippet: FIG. 6. PPAR-RXR heterodimers selectively bind PPRE in the LRP promoter. EMSA were performed on oligomers correspond- ing the PPREs of LRP, hACOX, and LRPmut. A, the PPREs of the human LRP and ACOX genes are shown compared with the consensus PPRE along with the sequence of the mutant LRP PPRE. The mis- matches for each PPRE are underlined. B, oligomers were incubated on ice for 20 min with various components. Unprogrammed reticulocyte lysate (TNT reaction containing empty pSPORT1 vector) was used as a control (lanes 1, 7, and 13). TNT in vitro transcribed/translated PPAR and RXR were incubated with radiolabeled PPRE oligomers (lanes 2, 8, and 14). To control for specificity of binding competition experiments were performed by adding increasing amounts of unlabeled oligomer to the binding reactions. LRP binding reactions were competed using cold hACOX oligomers at 5, 10, 25, and 50 excess (lanes 3–6), and the hACOX binding reactions were competed using cold LRP oligomers at 5, 10, 25, and 50 excess (lanes 9–12). C, nuclear extracts from primary human adipocytes were incubated with radiolabeled oligomer (lanes 1, 6, and 11) on ice for 20 min. Specificity of binding was again tested using unlabeled oligomers in competition experiments as de- scribed above (lanes 2–5 and 7–10). D, gel supershift analysis was used to determine whether PPAR was present in shift seen with nuclear extracts. Free probe (first lane) was used as a control reaction. The nuclear extracts (18 g) were incubated with labeled oligomer and run in the second lane. NuShift anti-PPAR antibody (4 l) was incubated with 18 g of nuclear extracts from SW872 cells prior to being added to the EMSA reaction mix containing labeled oligomers (third lane). A nonspecific antibody (mouse monoclonal anti--actin) was used as a negative control for the supershift (fourth lane). The arrows indicate the shift and the supershift.

Article Snippet: Cell Culture—The human liposarcoma cell line SW872 (American Type Culture Collection, Manassas, VA) was previously characterized and has been shown to be a good cell model for adipocyte gene expression (42–44).

Techniques: Sequencing, Mutagenesis, Incubation, Plasmid Preparation, Control, In Vitro, Binding Assay, Labeling, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Adipocyte Low Density Lipoprotein Receptor-related Protein Gene Expression and Function Is Regulated by Peroxisome Proliferator-activated Receptor γ

doi: 10.1074/jbc.m212989200

Figure Lengend Snippet: FIG. 7. LRP promoter confers regulation of luciferase activity by rosiglitazone. A, the promoter region of human LRP (0 to 1200) was cloned into pGL3-Basic. This construct was then transiently co- transfected into SW872 cells along with the Renilla luciferase reporter vector pRL-CMV using the calcium-phosphate precipitation method. Luciferase activities for each of firefly and Renilla luciferase were determined using the dual luciferase assay system. The intensity of firefly luciferase is shown as a function of Renilla luciferase (relative activity). The relative intensity is normalized to the control (vehicle treatment) values. The means of triplicate experiments are graphed, and the error bars represent the standard error of the mean. The one-tailed p values from paired t tests are shown for significant differ- ences. B, pGL3-LRP was subjected to site-directed mutagenesis, in which the half-site of the DR-1 to which PPAR binds, was mutated so that 5 of 6 nucleotides no longer matched the consensus. PPREs of pGL3-LRP and pGL3-LRPmutant PPRE are shown with the consensus sequence for comparison. Point mutations are underlined, and the mismatches present in endogenous PPRE are in italics. C, pGL3- LRPmutant PPRE, was transiently co-transfected with pRL-CMV into SW872 cells, and their respective luciferase activities were determined using the dual luciferase assay system. The relative intensity was calculated, and the means of triplicate experiments are shown. The values of relative intensity are normalized to the control (vehicle-treat- ed) cells. The error bars represent the standard error of the mean. Rosiglitazone has a significant effect on pGL3-LRP; however, it is not significant for pGL3-LRPmutant PPRE. The one-tailed p values from t tests were 0.02 and 0.2, respectively.

Article Snippet: Cell Culture—The human liposarcoma cell line SW872 (American Type Culture Collection, Manassas, VA) was previously characterized and has been shown to be a good cell model for adipocyte gene expression (42–44).

Techniques: Luciferase, Activity Assay, Clone Assay, Construct, Transfection, Plasmid Preparation, Control, One-tailed Test, Mutagenesis, Sequencing, Comparison

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 1. ELK3 depletion increases NK cell cytotoxicity regardless of E-cadherin expression. (a) Immunofluorescence images of caspase 3/7 activity (green) in MDA- MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells. The E:T ratio is presented as 1:1, 1:5, and 1:10. Scale bar, 100 μm. (b) Rate of target cell death in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5 after 4 h of coculture with CFSE-labeled NK92MI cells. The E:T ratio is 1:10. Error bars represent standard deviation; **p < 0.01 (one-way ANOVA). (c) Immunofluorescence images of perforin and granzymeB in MDA-MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells. The E:T ratio is 1:10. Scale bar, 50 μm. (d) The quantified graft of granzymeB and perforin protein. Error bars represent standard deviation; ***p < 0.001 (unpaired two-tailed Student’s t-test). (e) The percentage of CD107a-positive and IFNγ-positive NK92MI cells in MDA-MB231 cells expressing shCon and shELK3#1 after 4 h of coculture with NK92MI cells. Error bars represent standard deviation; *p < 0.05 (unpaired two-tailed Student’s t-test). (f-g) Quantitative analysis of indicated ligands in MDA-MB231 cells expressing shCon and shELK3#1. Error bars represent standard deviation; *p < 0.05, NS is non-significant (unpaired two-tailed Student’s t-test). (h-i) Quantitative analysis of indicated receptors in MDA-MB231 cells expressing shCon and shELK3#1. Error bars represent standard deviation; *p < 0.05, NS is non-significant (unpaired two-tailed Student’s t-test).

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunofluorescence, Activity Assay, Labeling, Standard Deviation, Two Tailed Test

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 2. ELK3 depletion increases CXCL16 expression in TNBC cells. (a) Experimental scheme for assessing NK cell cytotoxicity using conditioned medium (CM) from MDA-MB231 organoids expressing shCon and shELK3#1. CM from MDA-MB231 organoids was applied to MDA-MB231 organoids expressing shELK3#1 for NK92MI cell coculture; conversely, CM from MDA-MB231 organoids expressing shELK3#1 was applied to MDA-MB231 organoids expressing shCon for NK92MI cell coculture. N. M. represents normal media. Immunofluorescence images of caspase 3/7 activity (green) in MDA-MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells under the indicated conditions. The E:T ratio is 1:10. Scale bar, 100 μm. (b) Rate of target cell death in MDA-MB231 cells expressing shCon and shELK3#1 after 4 h of coculture with CFSE-labeled NK92MI cells under the indicated conditions. The E:T ratio is 1:10. Error bars represent standard deviation; *p < 0.05 (unpaired two-tailed Student’s t-test). NS is non-significant. (c) Quantification of secreted CXCL16 protein in MDA-MB231 cells transfected with siCon and siELK3. Error bars represent standard deviation; ***p < 0.001 (unpaired two-tailed Student’s t-test). (d) Quantitative analysis of CXCL16 mRNA levels in MDA- MB231 cells expressing shCon and shELK3#1. Error bars represent standard deviation; **p < 0.01 (unpaired two-tailed Student’s t-test). (e) Immunoblot analysis of the CXCL16 protein in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5. GAPDH was used as the loading control. (f) Heatmap analysis of immune- related genes in MDA-MB231 cells expressing shCon and shELK3#1. (g) Correlation analysis of ELK3 and CXCL16 in various breast cancer cells. Orange line indicates TNBC

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunofluorescence, Activity Assay, Labeling, Standard Deviation, Two Tailed Test, Transfection, Western Blot, Control

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 3. The chemotactic activity of CXCL16 recruits NK cells to target TNBC cells. (a) Immunofluorescence images of caspase 3/7 activity (green) in MDA-MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells under the indicated conditions. The E:T ratio is 1:10. Scale bar, 100 μm. (b) Rate of target cell death in MDA-MB231 cells expressing shCon after 4 h of coculture with CFSE-labeled NK92MI cells under the indicated conditions. The final concentration of CXCL16 protein was 200 ng/ml. The E:T ratio is 1:10. Error bars represent standard deviation; *p < 0.05, ***p < 0.001 (one-way ANOVA). (c) Immunofluorescence images of migrated NK92MI cells in response to recombinant CXCL16 protein. Scale bar, 200 μm. (d) The graph indicates the number of migrated NK92MI cells in CXCL16-mediated chemotaxis. Error bars represent standard deviation; **p < 0.01 (one-way ANOVA). (e) Migrated NK92MI cells in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5. Scale bar, 100 μm, 200 μm. (f) The graph indicates the number of migrated NK cells in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5. Error bars represent standard deviation; **p < 0.01, ***p < 0.001 (one-way ANOVA). (g-i) Migrated NK92MI cells in Hs578T cells expressing shCon and shELK3#1. The graph indicates the number of migrated NK92MI cells in Hs578T cells expressing shCon and shELK3#1. Scale bar, 100 μm, 200 μm. Error bars represent standard deviation; ***p < 0.001, ****p < 0.0001 (unpaired two-tailed Student’s t-test).

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Immunofluorescence, Expressing, Labeling, Concentration Assay, Standard Deviation, Recombinant, Chemotaxis Assay, Two Tailed Test

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 4. The ELK3-CXCL16 axis determines the response of NK cells in TNBC cells. (a) Immunofluorescence images of caspase 3/7 activity (green) in MDA-MB231 shELK3#1 organoids transfected with siCon, siCXCL16#1, and siCXCL16#2 after 4 h of coculture with FarRed-labeled NK92MI cells. The E:T ratio is 1:10. Scale bar, 50 μm. (b) Rate of target cell death in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2 after 4 h of coculture with CFSE-labeled NK92MI cells. Error bars represent standard deviation; *p < 0.05, **p < 0.01 (one-way ANOVA). (c) Migrated NK92MI cells in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Scale bar, 200 μm. (d) The graph indicates the number of migrated NK92MI cells in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; *p < 0.05 (one-way ANOVA). (e) Migrated primary NK cells in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; **** p < 0.0001 (one-way ANOVA). (f) Rate of target cell death in MDA- MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2 after 4 h of coculture with CFSE-labeled primary cells. Error bars represent standard deviation; *p < 0.05, **p < 0.01 (one-way ANOVA). (g) Migrated NK92MI cells in Hs578T shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; ****p < 0.0001 (one-way ANOVA). (h) Migrated primary NK cells in Hs578T shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; ****p < 0.0001 (one-way ANOVA). (i) Rate of target cell death in Hs578t and Hs578t shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2 after 4 h co-cultured with CFSE-labeled NK92MI cells. Error bars represent the standard deviation; *p < 0.05 (one-way ANOVA).

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Immunofluorescence, Activity Assay, Transfection, Labeling, Standard Deviation, Cell Culture

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 5. CXCL16 promotes NK cell chemotaxis and cytotoxicity in vivo. (a) Quantitative analysis of CXCL16 mRNA levels in MDA-MB231-GFP-luciferase cells expressing control and CXCL16 (231-control and 231-CXCL16). Error bars represent standard deviation; ***p < 0.001 (unpaired two-tailed Student’s t-test). (b) Immunoblot analysis of CXCL16 in 231-control and 231-CXCL16 cells. GAPDH was used as the loading control. (c) Migrated NK92MI cells in 231-control and 231-CXCL16 cells. Scale bar, 100 μm. The graph indicates the number of migrated NK92MI cells in 231-control and 231-CXCL16 cells. Error bars represent standard deviation; *p < 0.05 (unpaired two- tailed Student’s t-test). (d) Experimental scheme for NK cell recruitment in the experimental metastasis model. NK92MI cells were intravenously injected three times after 231-control and 231-CXCL16 cell injection. (e) The percentage of GFP-positive cells in the lungs from mice harboring 231-control and 231-CXCL16 cells after administration of NK92MI cells. GFP indicates tumor cells in the lung. FarRed indicates NK92MI cells in the lung. Error bars represent standard deviation; *p < 0.05 (unpaired two-tailed Student’s t-test). NS is non-significant. (f) Immunofluorescence images of NK92MI cells in the lungs from mice harboring 231-control and 231- CXCL16 cells after NK92MI cell administration. Scale bar, 100 μm, 200 μm. (g) Experimental scheme for NK cell recruitment and NK cell cytotoxicity in the orthotopic mouse model. NK92MI cells were intravenously injected six times after visualization of primary tumors. (h) Primary tumor growth in mice harboring 231-control and 231-

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Chemotaxis Assay, In Vivo, Luciferase, Expressing, Control, Standard Deviation, Two Tailed Test, Western Blot, Injection, Immunofluorescence

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 6. Negative correlation between ELK3 and CXCL16 in human breast cancer. (a) Expression analysis of ELK3 and CXCL16 in 421 human breast cancer patient samples (TCGA database). (b-c) Kaplan–Meier survival plots showing the overall survival of breast cancer patients. (d) Heatmap analysis of ELK3, CXCL16, and NK cell- relatedgenes37 in 421 human breast cancer patient samples. The white box indicates the patient samples with high expression of the ELK3 gene and low expression of the CXCL16 gene. The yellow box indicates patient samples with low expression of the ELK3 gene and high expression of the CXCL16 gene. (e) Correlation analysis of CXCL16 and NK cell-related genes in human breast cancer patient samples. (f) The biological role of ELK3-CXCL16 axis on NK cell cytotoxicity. ELK3 depletion increased secreted CXCL16 protein to recruit NK cells and resulted in improved NK cell cytotoxicity in TNBC cells.

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: Food Science & Nutrition

Article Title: Dihydromyricetin regulates KEAP1‐Nrf2 pathways to enhance the survival of ischemic flap

doi: 10.1002/fsn3.4049

Figure Lengend Snippet: Dihydromyricetin (DHM) promotes angiogenesis in ischemic flaps. (a) Immunofluorescence (IF) staining of CD31 and EMCN in ischemic flaps on POD7. Scale bars: 50 μm. Quantification of CD31/EMCN‐positive blood vessel density between the two groups ( n = 5). (b) Expression of Cadherin 5, MMP9, and VEGF proteins in ischemic flaps for both groups on POD7. Measurement of the expression of proteins associated with angiogenesis in both groups ( n = 5). (c) Comparative analysis of relative Vegf mRNA expression in ischemic flaps for both groups on POD7 ( n = 5). (d) Comparative analysis of relative Vegfr mRNA expression in ischemic flaps for both groups on POD7 ( n = 5). Error bars are SEM. The band density was normalized and the loading control was β‐actin. Significance: * p < .05, substantially distinct as stated; two‐tailed, unpaired t ‐test.

Article Snippet: Antibodies against KEAP1 (cat# 10503‐2‐AP), NRF2 (cat# 16396‐1‐AP), MMP9 (cat# 10375‐2‐AP), HO‐1 (cat# 10701‐1‐AP), SOD1 (cat# 10269‐1‐AP), Bax (cat# 50599‐2‐Ig), Bcl‐2 (COX2, cat# 26593‐1‐AP), and histone‐H3 (H3; cat# 17168‐1‐AP) were supplied by the Proteintech Group (Chicago, IL, USA).

Techniques: Immunofluorescence, Staining, Expressing, Control, Two Tailed Test

Journal: Oncotarget

Article Title: Inflammation and stem markers association to PIM1/PIM2 kinase-induced tumors in breast and uterus

doi: 10.18632/oncotarget.19438

Figure Lengend Snippet: ( A ) The figure shows a heat map comparing immunology-related genes with PIM1 and PIM2 (See ) in breast tumors (Halfwerk dataset, R2). Heat map shows a clear correlation of PIM1 and PIM2 with the expression of antigen presentation genes. ( B ) The figure shows a heat map comparing immunology-related genes with PIM1 and PIM2 (See ) in endometrial tumors (EXPO dataset, R2). Heat map shows a clear correlation of PIM1 and PIM2 with the expression of antigen presentation genes. ( C ) The figure shows a heat map comparing immunology-related genes with PIM1 and PIM2 (See ) in ovarian tumors (Wong dataset, R2). Heat map shows a clear correlation of PIM1 and PIM2 with the expression of antigen presentation genes. ( D ) A Venn diagram showing the number of immune system-dependent genes that correlated with PIM1 and PIM2 overexpression in Breast, endometrial and ovary tumors. ( E ) A list of system-dependent genes in Breast, endometrial and ovary tumors that correlated with both PIM1 and PIM2 overexpression (common) and that was unique to each tumor type. ( F , G ) Pro-inflammatory microenvironment PIM-dependent characterization by qPCR. The figure shows a number of immune system-dependent genes that were correlated with PIM overexpression in breast, endometrium and ovarian datasets: CD4, CD8a, CD74, Ifng, Tap1 and Tap2; and mouse Major Histocompatibility Complex markers (H2 complex). We decided to focus on the ones which has a mouse ortholog: HLA-DMA (H2-DMa), HLA-DRA (H2-A), HLA-DPB1 (CD74), and also H2-K (classical MHC class I), H2-Q (non-classical MHC class I), H2-E (classical MHC class II). They were measured by qRT-PCR of mammary gland of WT mice with non-proliferative status (MG-N) and carcinoma grades (MG-T) in both transgenic models (F), and the same way in uterus (UT-N, normal; and UT-T, carcinoma) (G). ( H ) The overexpression levels of the human transgenes PIM1 or PIM2 in the models causes an increase in the expression of NFAT and NFAT effectors (OSM and IL6), as well as the phosphorylation of mTOR. The p -value was obtained using a one-tailed student's t -test. (* p < 0.05), (** p < 0.01), and (*** p < 0.001).

Article Snippet: We used the following TaqMan Gene Expression Assays probes (ThermoFisher) after RT-PCR: PIM1 (Hs01065498_m1), PIM2 (Hs00179139_m1), Pim1 (Mm00435712_m1), Pim2 (Mm00454579_m1), Meox1 (Mm00440285_m1), Sox15 (Mm00839542_g1), Pou5f1 (Mm03053917_g1), Nanog (Mm02019550_s1), Sox2 (Mm03053810_s1), Klf4 (Mm00516104_m1), Klf10 (Mm00449812_m1), Serpine1 (Mm00435858_m1), Thbd (Mm00437014_s1), CD4 (Mm00442754_m1), CD8a (Mm01182107_g1), CD74 (Mm00658576_m1), Ifng (Mm01168134_m1), Tap1 (Mm00443188_m1), Tap2 (Mm01277033_m1).

Techniques: Expressing, Immunopeptidomics, Over Expression, Quantitative RT-PCR, Transgenic Assay, Phospho-proteomics, One-tailed Test

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Doxycycline inducible system for controlled expression of Muc1 or Muc1-GFP possessing 42 tandem repeats (TRs) in MCF10A epithelial cells. b , Isolation of stable cell clones with titratable expression of Muc1-GFP (1E7 clone) or Muc1 (B2 clone). c , Fluorescence and brightfield images of 1E7 cells induced at the indicated concentration of doxycycline. Scale bar, 10 μm. d , Flow cytometry analysis of 1E7 cells showing titratable Muc1-GFP levels across the indicated concentrations of doxycycline inducer. e , NK-92 cell-mediated cytotoxicity against 1E7 cells treated with control buffer or 100 nM StcE mucinase for 1 hour prior to co-culture; Muc1-GFP induction is at the indicated doxycycline concentration. Results are the mean ± s.d. of n=3 independent experimental replicates. f , NK-92 cell-mediated cytotoxicity against an MCF10A cell line expressing a Muc1-GFP construct with the TRs deleted (Muc1 0TR) under the control of a tetracycline inducible promoter at the indicated doxycycline induction level. Results are the mean ± s.d of n=3 technical replicates. g , Primary human NK cell-mediated cytotoxicity against 1E7 cells and 1E7 GNE KO cells at the indicated doxycycline concentration. NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of n=3 biological replicates corresponding to three distinct human blood cell donors. h, Primary equine PBMC-mediated cytotoxicity against 1E7 cells at the indicated concentration of doxycycline; NK cell to target cell ratio is 20:1. Results are the mean ± s.d. of n=3 biological replicates corresponding to unique equine blood donors. i , NK-92 cell-mediated cytotoxicity against ZR-75-1 breast cancer cells sorted into sub-populations with low, intermediate, and high endogenous Muc1 expression. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 experimental replicates. j , NK-92 cell-mediated cytotoxicity against ZR-75-1 treated with control buffer or 100 nM StcE prior to co-culture. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 independent experimental replicates. Statistical analysis by two-tailed t tests. In panels e , g , and h , statistical analysis by one-way ANOVA with Tukey’s post-hoc tests.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Expressing, Isolation, Stable Transfection, Clone Assay, Fluorescence, Concentration Assay, Flow Cytometry, Control, Co-Culture Assay, Construct, Two Tailed Test

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Schematic illustration of Ring-SAIM; interference of direct and reflected laser light off a silicon mirror creates standing waves of excitation light that depend on the laser incidence angle, θ, and are used to probe the glycocalyx thickness, which is measured as the gap between the substrate and fluorescently labelled membrane. b, Optical configuration for Ring-SAIM showing azimuthally scanned excitation light at varying θ; AOTF, Acousto-optic tunable filter. c , Representative widefield image and glycocalyx thickness map of live 1E7 cells expressing Muc1-GFP at the indicated doxycycline induction level. Scale bars, 10 μm. d , Glycocalyx thickness in ZR-75-1 cells before and after treatment with 100 nM StcE. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 6 cells from a representative experiment (n=3 independent experiments). Statistical analysis by two-tailed t test. e, Biosynthetic pathway for mucin-type O-glycans. f, Relative O-glycan distributions measured by the Cellular O-glycome Reporter Amplification (CORA) analysis in 1E7 wild-type, 1E7 GCNT1 overexpressing (OE), 1E7 C1GALT1 knockout (KO), and 1E7 GNE KO cells. g Glycocalyx thickness in the 1E7 cell panel at the indicated doxycycline concentration; some cellular treatments include 10 μM recombinant human galectin-1, 5 μM recombinant human galectin-3, or 10 μM TD139. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 13 cells from a representative experiment (n=3 independent experiments). Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. h , NK-92 cell-mediated cytotoxicity against the wild-type and engineered 1E7 cells at the indicated doxycycline induction level. NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of at least n=3 independent experimental replicates. Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. i , NK-92 cell-mediated cytotoxicity is inversely proportional to the measured glycocalyx thickness with Pearson correlation coefficient, r = −0.91; plotted data is from ( g ) and ( h ); dashed line indicates a linear fit to the data.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Membrane, Expressing, Two Tailed Test, Glycoproteomics, Amplification, Knock-Out, Concentration Assay, Recombinant

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Representative fluorescence images of endogenous GzmB-Gamillus in NK-92 cells following conjugation with Muc1-expressing B2 cells at minimal and maximal doxycycline induction of Muc1 expression; B2 cells also express cytosolic mScarlet-I as a red fluorescent marker. Scale bar, 10 μm. b , Polarity index for Gamillus-labelled cytolytic granules in NK-92 cell conjugates with B2 cells at the indicated doxycycline induction of Muc1 (0 and 1,000 ng/ml). Results are the mean ± s.d. of at least 38 cells over n=3 independent experimental replicates. Statistical analysis by two-tailed t test. c , Schematic showing structure of chimeric antigen receptor (CAR) targeting HER2 and CD19. d , Western blot analysis of HER2 in wild-type and HER2 overexpressing 1E7 cells (+ HER2 OE). e , NK-92 and HER2 CAR NK-92 cell-mediated cytotoxicity against 1E7 HER2 OE cells at the indicated concentration of doxycycline. f , HER2 CAR-T cell-mediated cytotoxicity against 1E7 HER2 OE cells at the indicated concentration of doxycycline. g , HER2 CAR T cell-mediated cytotoxicity against 1E7 HER2 OE cells at the indicated doxycycline induction level and target:effector ratio. h , Flow cytometry analysis of CD19 surface levels on 1E7 wild-type and CD19 overexpressing 1E7 cells (+ CD19 OE) cells at the indicated concentrations of doxycycline. i , NK-92 and CD19 CAR NK-92 cell-mediated cytotoxicity against 1E7 or CD19 OE 1E7 cells at the indicated concentration of doxycycline. n=3 independent experimental replicates. j , CD19 CAR-T cell-mediated cytotoxicity against 1E7 and 1E7 CD19 OE cells at the indicated concentration of doxycycline. k , CD19 CAR T cell-mediated cytotoxicity against 1E7 and 1E7 CD19 OE cells at the indicated doxycycline induction level and target:effector ratio. Unless otherwise indicated, effector cell to target cell ratio is 5:1. All results in e - g and i-k are shown as mean ± s.d. of n=3 independent experimental replicates. In panels e - f and i - j , statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. HER2 CAR NK-92/T and CD19 CAR-T cells are second-generation CARs, while CD19 CAR NK-92 is a third-generation CAR.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Activation Assay, Fluorescence, Conjugation Assay, Expressing, Marker, Two Tailed Test, Western Blot, Concentration Assay, Flow Cytometry

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , StcE NK-92 cells prepared through tethering of StcE mucinase to the NK-92 cell surface by the native StcE X409 lectin domain. b , Surface levels of StcE and X409-deleted StcE (StcE ΔX409) on NK-92 cells following mucinase incubation at the indicated concentrations. c , ZR-75-1 cell killing by StcE NK-92 cells prepared with the indicated StcE concentration. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 technical replicates. d , Killing of 1E7 cells by NK-92, StcE NK-92, NK-92 cells tethered with catalytically dead StcE E447D, and NK-92 cells treated with StcE ΔX409. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 independent experimental replicates. e , Brightfield and Muc1-GFP fluorescence images of 1E7 cells conjugated with NK-92 or StcE NK-92 cells; lower images show zoomed-in regions of the NK-1E7 cell interface. Scale bar, 10 μm. f , Mean Muc1-GFP intensity (dark lines) and standard deviations (lighter bounding areas) along line profiles across the contact interface between 1E7 cell and NK-92 cells (n=6) or StcE NK-92 cells (n=10); the intensity is normalized to the mean intensity outside of the contact interface; red dotted arrows in e show representative profile lines. g , Glycocalyx thickness before and after treatment with 100 nM StcE. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 11 cells from a representative experiment (n=3 independent experiments). Statistical analysis by two-tailed t test. h, Cytotoxicity mediated by control and engineered NK-92 cells. Results are the mean ± s.d. of n=3 target cancer cell lines with individual data points indicating average cytotoxicity against a specific cell line (SKOV3, SKBR3, and 1E7 HER2 OE). NK cell to target cell ratio is 5:1 for 1E7 HER2 OE and 10:1 for SKOV3 and SKBR3. In d and h , statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. For all experiments, Muc1-GFP expression in 1E7 and 1E7 HER2 OE cells was with 1,000 ng/ml doxycycline.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Incubation, Concentration Assay, Fluorescence, Two Tailed Test, Control, Expressing

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a . Cartoon showing the design of Zip-NK-92 cells for modular surface display of glycocalyx-editing (GE) enzymes using leucine zippers. b , Flow cytometry analysis of sfGFP and ALFA-Tag on wild-type NK-92 and Zip-NK-92 cells expressing the membrane-anchored RR zipper. c , Binding isotherms for StcE-EE zipper fusion protein (StcE-EE) incubated with Zip-NK-92 cells (red) and wild-type StcE incubated with NK-92 cells (black). Representative normalized intensity plot from n=3 independent experimental replicates. d , Cytotoxicity mediated by Zip-NK-92 cells coupled with StcE-EE at the indicated concentration against Muc1-expressing B2 cells at the indicated doxycycline induction level. NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of n=3 technical replicates for each doxycycline level. e, f, Cytotoxicity mediated by Zip-NK-92 cells coupled with the indicated concentration of StcE-EE ( e ) and Sialidase-EE ( f ) against target cancer cell lines. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=4 or 5 target cancer cell lines (Capan-2, SKBR3, KPL-1, T47D, ZR-75-1). Statistical analysis by two-tailed t test.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Flow Cytometry, Expressing, Membrane, Binding Assay, Incubation, Concentration Assay, Two Tailed Test

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a,b , Representative confocal and brightfield images of two clonally expanded MCF10A cell lines expressing Muc1-GFP with 42 tandem repeats (TRs) under the control of a tetracycline inducible promoter at the indicated doxycycline induction level. Scale bar, 10 μm. c , Representative confocal and brightfield images of Muc1 expresssing B2 clonal cells labelled with anti-Human Muc1 Janelia Fluor 549; the doxycycline induction level is indicated. Scale bar, 10 μm. d , NK-92 cell-mediated cytotoxicity against the polyclonal Muc1-GFP and three Muc1-GFP expressing clonal cell lines (1E7, 2E4, and 2G9) expressing MCF10A line from which the clonal lines were isolated, and a clonal MCF10A cell line expressing the Muc1 construct without the GFP reporter (B2 clone); the doxycycline induction levels are indicated. Results are the mean ± s.d of n=3 experimental replicates. NK cell to target cell ratio is 5:1. e . Mean fluorescence intensity of cell-surface Muc1-GFP in 1E7 cells treated with the indicated concentration of StcE mucinase; cell-surface Muc1-GFP probed with Alexa Fluor 647 conjugated GFP nanobody. Results are shown for induction of Muc1-GFP at 100 ng/ml and 1000 ng/ml doxycycline. Results are the mean ± s.d. of at least 14 cells from one representative of 3 independent experiments. Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Expressing, Control, Isolation, Construct, Fluorescence, Concentration Assay

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a, b, Surface expression levels of CD3 (a) and CD56 (b) in NK-92 cells and primary NK cells analyzed by flow cytometry, validating isolation of CD56+/CD3− NK cells. c, d, Surface levels of Siglec-7 (c) and Siglec-9 (d) on NK-92 and primary human NK cells evaluated by flow cytometry, demonstrating minimal to no detectable levels of the receptors on NK-92. e , Western blot analysis confirming UDP-N-acetylglucosamine 2-epimerase ( GNE ) knockout (KO) in 1E7 cells to ablate sialylation. Flow cytometry data for primary NK cells are representative results for one of n=3 human blood donors.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Expressing, Flow Cytometry, Isolation, Western Blot, Knock-Out

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: Using CRISPR/Cas9 and an appropriate homology directed repair template, the gene sequence for a flexible GGSGGSGGS linker and the pH-tolerant green fluorescent protein, Gamillus, was inserted immediately before the endogenous GZMB stop codon in NK-92 cells. a , Representative brightfield and fluorescence images of NK-92 GzmB-Gamillus cells stained with Lysotracker Deep Red. Scale bar, 10μm. b , Pearson’s coefficients of correlation between GzmB-Gamillus and Lysotracker. Results are the mean ± s.d. of 20 cells. c , d , NK-92 GzmB-Gamillus mediated cytotoxicity against 1E7 with Muc1-GFP expression induced at the indicated doxycycline concentration. Results are the mean ± s.d. of n=3 independent experimental replicates. NK cell to target cell ratio is 10:1. Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. e , Western blot showing the relative expression level of Granzyme B (GzmB) in the NK-92 cells and NK-92 GzmB-Gamillus knock-in (KI) cells. Note that the acidlabile linker between GzmB and Gamillus is expected to undergo cleavage in the low pH of lysosomes and cytolytic granules.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Biomarker Discovery, Labeling, CRISPR, Sequencing, Fluorescence, Staining, Expressing, Concentration Assay, Western Blot, Knock-In

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a, Representative confocal images of Muc1-GFP on 1E7 cells treated with control buffer or 100 nM StcE, StcE ΔX409, StcE ΔX409 fused to the EE leucine zipper (StcE-EE); Alexa Fluor 647 conjugated GFP nanobody is used to probe cell-surface Muc1-GFP constructs. Scale bar, 10 μm. b , Viability of NK-92 and Zip-NK-92 after treatment with indicated concentration of StcE-EE. Propidium iodide (20 μg/ml) was used to detect dead cell population by flow cytometry. c , Cytotoxicity mediated by Zip-NK-92 cells coupled with the indicated concentration of Sialidase-EE zipper fusion protein (Sialidase-EE) against B2 cells at the indicated doxycycline induction level; NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of n=3 technical replicates for each doxycycline concentration.

Article Snippet: NK-92 cells (ATCC; CRL-2407) were cultured in ɑ-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1x penicillin/streptomycin at 37°C in 5% CO 2.

Techniques: Control, Construct, Concentration Assay, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: A small‑molecule HDAC/PDE modulator activates human adipocyte UCP1 and resolves inflammatory signaling

doi: 10.1186/s12967-026-07970-0

Figure Lengend Snippet: Proinflammatory cytokine enrichment in obesity and white adipocytes. a multiplexed Elisa of pro-inflammatory markers in plasma of obese subjects and overweight subjects compared to lean controls. b multiplexed Elisa of pro-inflammatory markers done on supernatant from white adipocytes SW872 cells at day 0 and day 7 of differentiation and brown adipocytes PAZ6 cells at Day 0 and Day 14 of differentiation. c IPA analysis of differentially expressed inflammatory-related genes in undifferentiated versus differentiation SW872 and PAZ6 cells. The genes are placed relative to their cellular localization. The color of each gene reflects it differential relative expression in differentiated versus undifferentiated PAZ6 cells where green is downregulated and red is upregulated. The bar plot adjacent to each gene display its differential expression in both PAZ6 (left) and SW872 (right) cell lines respectively in differentiated versus undifferentiated state. Direct interactions are depicted by solid gray lines and indirect interactions by dashed gray lines. d THP-1 migration towards conditioned media derived from pre-differentiated and differentiated SW872 and PAZ6 adipocytes. e FACS analysis of CD36 levels in blood-derived-macrophages treated with conditioned media from pre-differentiated and differentiated SW872 and PAZ6 adipocytes ( N = 3). SW, SW872; CTRL, control; OW, overweight; OB, obese, pre, pre-differentiated; diff, differentiated. All data are presented as mean ± S.D. p values were calculated by unpaired two-tailed Student’s t test. ms, marginally significant (0.05 < P < 0.08); *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Human SW872 white preadipocytes (ATCC® HTB-92TM, RRID: CVCL_1730) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 medium (Thermo Fisher Scientific, 11330032), supplemented with 8% fetal bovine serum (FBS) (Thermo Fisher Scientific, 16000044), 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122), and 15 mM HEPES (Thermo Fisher Scientific, 15630080).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing, Quantitative Proteomics, Migration, Derivative Assay, Control, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: A small‑molecule HDAC/PDE modulator activates human adipocyte UCP1 and resolves inflammatory signaling

doi: 10.1186/s12967-026-07970-0

Figure Lengend Snippet: A high-throughput small compound-UCP1 inducers screening platform. a diagram of the establishment of screening platform showing two human UCP1 promoter regions (4 K and 6K) inserted above the luciferase reporter, respectively. b Validation of screening platform through luciferase activity induction in SW872 (upper panel) and PAZ6 (lower panel) treated with known UCP1 inducers, rosiglitazone (ROS) and genistein (GEN). UNT, untransfected; CTRL, control. c screening of an MCR compound library in SW872 preadipocytes with UCP1 luciferase reporter ( N = 3). Luciferase signal Fold change was calculated relative to DMSO-treated cells after 24 hours of compound treatment. d secondary screen of the candidates in SW872 preadipocytes. Luciferase signal Fold change in SW872 preadipocytes after 24 hours of treatment with the hits from the primary screen relative to the control ( N = 3). e luciferase signal Fold change in PAZ6 cells treated with candidate compounds identified in the SW872 screen ( N = 3). f cell viability (measured by CellTiter-Glo luminescence) of SW872 preadipocytes treated with the indicated compounds (at 10uM) or vehicle for 24 hours ( N = 3). g and h relative mRNA expression level of UCP1(g) and ADRB3 (h) in SW872 differentiated white adipocytes upon treatment with 10 μM of candidate compounds and vehicle ( N = 3). The results of the replicates and independent experiment analyses were presented as mean ± S.D. Statistical analyses have been performed using Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Human SW872 white preadipocytes (ATCC® HTB-92TM, RRID: CVCL_1730) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 medium (Thermo Fisher Scientific, 11330032), supplemented with 8% fetal bovine serum (FBS) (Thermo Fisher Scientific, 16000044), 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122), and 15 mM HEPES (Thermo Fisher Scientific, 15630080).

Techniques: High Throughput Screening Assay, Luciferase, Biomarker Discovery, Activity Assay, Control, Drug discovery, Expressing

Journal: Journal of Translational Medicine

Article Title: A small‑molecule HDAC/PDE modulator activates human adipocyte UCP1 and resolves inflammatory signaling

doi: 10.1186/s12967-026-07970-0

Figure Lengend Snippet: CDC1011 induces the browning of white adipocytes. a effect of CDC1011 treatment on browning markers in preadipocytes and differentiated SW872 adipocytes. b effect of CDC1011 treatment on mitochondrial electron transport chain subunit protein expression in preadipocytes, differentiated adipocytes, and isoproterenol-induced differentiated adipocytes. Presented results are representative of biological replicates ( N = 3). c cAMP content in control and CDC1011-treated preadipocyte and differentiated adipocyte. The values were measured by ELISA and normalized to the protein content. d effect of CDC1011 treatment on basal and forskolin-induced PKA activity in preadipocytes and differentiating adipocytes. The values were normalized to the protein content. e effect of CDC1011 treatment on basal and isoproterenol/forskolin-induced intracellular glycerol levels in differentiating adipocytes. The results of the replicates and independent experiment analyses ( N = 3) were presented as mean ± S.D. Statistical analyses have been performed using Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Human SW872 white preadipocytes (ATCC® HTB-92TM, RRID: CVCL_1730) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 medium (Thermo Fisher Scientific, 11330032), supplemented with 8% fetal bovine serum (FBS) (Thermo Fisher Scientific, 16000044), 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122), and 15 mM HEPES (Thermo Fisher Scientific, 15630080).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: A small‑molecule HDAC/PDE modulator activates human adipocyte UCP1 and resolves inflammatory signaling

doi: 10.1186/s12967-026-07970-0

Figure Lengend Snippet: CDC1011 treatment enhances mitochondrial respiration and glycolytic flux. a Oxygen consumption rates (OCR) diagrams of Seahorse Mito stress assay results on differentiated SW872 treated with vehicle and CDC1011 in real time under basal and starvation conditions and in response to oligomycin, FCCP and antimycin A/rotenone injected at the indicated time points with the quantification of basal and maximal respiration, proton leak, spare capacity, non-mitochondrial respiration and ATP production. Assay results were normalized to cell count in a representative field. b Normalized OCR diagrams for FAO stress test obtained in BSA- and palmitate- treated SW872 adipocytes, with or without Etomoxir. Measurements were taken for differentiated adipocytes treated with vehicle or CDC1011. c Extracellular acidification rates (ECAR) and normalized glucoPER diagram for glycolysis stress test conducted to evaluate responses of vehicle and CDC1011 treated adipocytes to glucose and the glycolytic inhibitor 2-DG. Basal and induced glycolysis are calculated and shown. d Basal and insulin induced glucose uptake in preadipocyte and differentiating SW872 cells upon treatment with CDC1011 compared to vehicle. All data is presented as mean ± S.D. and representative of independent replicates ( N = 3). p values were calculated by unpaired two-tailed Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Human SW872 white preadipocytes (ATCC® HTB-92TM, RRID: CVCL_1730) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 medium (Thermo Fisher Scientific, 11330032), supplemented with 8% fetal bovine serum (FBS) (Thermo Fisher Scientific, 16000044), 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122), and 15 mM HEPES (Thermo Fisher Scientific, 15630080).

Techniques: Injection, Cell Characterization, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: A small‑molecule HDAC/PDE modulator activates human adipocyte UCP1 and resolves inflammatory signaling

doi: 10.1186/s12967-026-07970-0

Figure Lengend Snippet: CDC1011 attenuates inflammation in human white adipocytes. a cytokine array analysis of secreted factors in the supernatants from vehicle- and CDC1011-treated SW872 cells at both the preadipocyte and differentiated adipocyte stages. Downregulated cytokines are marked with red boxes and upregulated ones with green boxes. b Fold change in THP-1 cell migration towards conditioned media derived from pre-differentiated and differentiated SW872 treated with CDC1011 compared to vehicle. c Fold change in basal and forskolin-induced cGMP concentration measured by ELISA in CDC1011-treated preadipocyte and differentiating SW872 cells relative to control and normalized to the protein content. d and e Western blot analysis of (d) PDE5A and (e) phosphor-p65 (Ser536) expression measured in both preadipocytes and mature adipocytes treated with CDC1011 or DMSO. ANG1, Angiopoietin 1; OPN, Osteopontin; GDF15, Growth/differentiation factor 15; BAFF, B-cell activating factor; CXCL5, C-X-C motif chemokine ligand 5; CXCL1, C-X-C motif chemokine ligand 1; IL6, interleukin 6. Presented results are representative of biological replicates ( N = 3). p values were calculated by unpaired two-tailed Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Human SW872 white preadipocytes (ATCC® HTB-92TM, RRID: CVCL_1730) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 medium (Thermo Fisher Scientific, 11330032), supplemented with 8% fetal bovine serum (FBS) (Thermo Fisher Scientific, 16000044), 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122), and 15 mM HEPES (Thermo Fisher Scientific, 15630080).

Techniques: Migration, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: FLIP(C1orf112)-FIGNL1 complex regulates RAD51 chromatin association to promote viability after replication stress

doi: 10.1038/s41467-024-45139-9

Figure Lengend Snippet: a 293 T cells were transfected with RAD51-HA and either empty vector (EV), or the indicated 3X-FLAG-tagged proteins for 48 h prior to coIP using FLAG beads. Lysates were digested with benzonase prior to coIP, n = 4 independent experiments. b 293 T cells were transfected with EV, FLAG-tagged FLIP or HA-tagged RAD51 as indicated for 48 h prior to coIP using HA beads. Lysates were digested with benzonase prior to coIP, n = 3 independent experiments. c 293 T cells were reverse transfected with the indicated siRNAs. The next day, cells were transfected with EV, FLAG-tagged FLIP or HA-tagged RAD51 as indicated for 48 h prior to coIP using FLAG beads, n = 2 independent experiments. d Schematic of the experiment in ( e , f , g ). Cisplatin was added at 0.5 μM. e Control gRNA expressing and ΔFLIP U2OS cells were processed as shown in ( d ). Representative IF images show RAD51 and γH2AX foci. f , g Quantification of the experiment in ( d , e ). N = 3 independent experiments, mean ± SD shown. ND – no drug. Statistics represent unpaired t tests (two-tailed) for each time point, ns – not significant; * P = 0.0436; ** P = 0.0075; *** P ≤ 0.001. h Control gRNA-expressing (WT) or ΔFLIP U2OS cells were treated with vehicle or 2.5 μM cisplatin for 18 h then fractionated prior to blotting for the indicated proteins. CE – cytoplasmic extract. Black arrowhead – FLIP. Blue arrowhead – FIGNL1, n = 3 independent experiments. i Control gRNA-expressing (WT) or ΔFLIP U2OS cells stably expressing empty vector (EV) or GFP-FLIP (FLIP) were treated with vehicle or 1.5 μM cisplatin for 18 h. Samples were processed for chromatin fraction prior to blotting for the indicated proteins, n = 2 independent experiments. CE – cytoplasmic extract. RAD51 numbers are quantified related to ORC2 loading control. Scale bars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this work are as follows: Anti-HA 1:1000 (Sigma, H3663-200), c1orf112 1:1000 (Sigma, HPA023778), FANCD2 F17 1:200 (Santa Cruz Biotechnology, sc-20022), FANCA 1:1000 (Bethyl, A301-980A), tubulin 1:1000 (Sigma, T4026-.2 ML), FANCI 1:1000 (Bethyl, A301-254A), Vinculin 1:1000 (Sigma, V9131-.2 ML), ATM 1:1000 (Abcam, ab81292), γH2AX (JBW301) 1:1000 (Sigma, 05-636), mouse M2 anti-FLAG 1:1000 (Sigma, F1804-200UG), Rabbit anti-GFP antibody 1:1000 (Abcam, ab6556), RPA32-P-S4/8 1:1000 (Bethyl, A300-245A), RPA32 9H8 1:200 (Santa Cruz Biotechnology, sc-56770), CHK1-P-S317 1:1000 (Cell Signaling, 2344 S), CHK1 G4 1:200 (Santa Cruz Biotechnology, sc-8408), FIGNL1 1:1000 (Proteintech, 17604-1-AP-150UL), GAPDH 1:8000 (Santa Cruz Biotechnology, sc-47724), ORC2 1:1000 (Abcam, ab68348), RAD51 1:1000 (Abcam, ab63801), Mouse monoclonal RAD51 1:1000 (Millipore-Sigma, 05-530-I), Rad51 Antibody (H-92) 1:1000 (Santa Cruz Biotechnology, sc-8349), Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 1:400 (Invitrogen, A32731), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 1:400 (Invitrogen, A32723), Goat anti-Rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:400 (Invitrogen, A48262), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 594 1:400 (Invitrogen, A32742), Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 594 1:400 (Invitrogen, A32740).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Two Tailed Test, Stable Transfection

Journal: Nature Communications

Article Title: FLIP(C1orf112)-FIGNL1 complex regulates RAD51 chromatin association to promote viability after replication stress

doi: 10.1038/s41467-024-45139-9

Figure Lengend Snippet: a Western blots show FLIP levels in knockout (Δ), U2OS (WT) and FLIP-GFP expressing (OE, green arrow) cells, n = 2 independent experiments. b Control, ΔFLIP or FLIP-GFP overexpressing (OE) U2OS cells were processed for IF. Representative IF images show RAD51 and γH2AX foci. c Quantification of ( b ), mean ± SD shown, n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey’s, ns – not significant ( P = 0.9897), **** P ≤ 0.0001. d U2OS cells were reverse transfected with control or the indicated siRNAs for 48 h. Cells were plated on coverslips, exposed to 100 nM camptothecin (CPT) for 20 h and processed for RAD51 IF, mean ± SD shown, n = 3 independent experiments. Ordinary one-way ANOVA followed by Dunnett’s, ** P = 0.0072. e U2OS cells expressing control sgRNA or FLIP KO U2OS cells were reverse transfected using the indicated siRNAs for 48 h, plated on coverslips, exposed to 0.5 μM cisplatin for 24 h and released for 0 h or 24 h prior to processing for RAD51 IF. Mean ± SD shown, n = 3 independent experiments, two-way ANOVA followed by Tukey’s multiple comparison test, ns – not significant ( P = 0.3567); *** P ≤ 0.001. f Quantification of percent EdU positive cells after pulsing for the indicated durations, mean ± SD shown, n = 3 independent experiments. g FLIP KO U2OS cells were pulsed with EdU for 3 h. Cells were pre-extracted and stained for EdU (via Click-iT reaction) and RAD51. Pink arrows show RAD51 positive cells. Merged images show overlap between EdU staining (red) and RAD51 positive cells. h Plot showing observed overlap between RAD51 and EdU staining. Mean ± SD shown, n = 3 independent experiments. Expected values were obtained by multiplying EdU and RAD51 positive fractions. Statistics represent unpaired t tests (two-tailed) of expected vs observed values for each time point. * P = 0.0109; ** P = 0.0015; **** P ≤ 0.0001. Scale bars = 10 μm. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this work are as follows: Anti-HA 1:1000 (Sigma, H3663-200), c1orf112 1:1000 (Sigma, HPA023778), FANCD2 F17 1:200 (Santa Cruz Biotechnology, sc-20022), FANCA 1:1000 (Bethyl, A301-980A), tubulin 1:1000 (Sigma, T4026-.2 ML), FANCI 1:1000 (Bethyl, A301-254A), Vinculin 1:1000 (Sigma, V9131-.2 ML), ATM 1:1000 (Abcam, ab81292), γH2AX (JBW301) 1:1000 (Sigma, 05-636), mouse M2 anti-FLAG 1:1000 (Sigma, F1804-200UG), Rabbit anti-GFP antibody 1:1000 (Abcam, ab6556), RPA32-P-S4/8 1:1000 (Bethyl, A300-245A), RPA32 9H8 1:200 (Santa Cruz Biotechnology, sc-56770), CHK1-P-S317 1:1000 (Cell Signaling, 2344 S), CHK1 G4 1:200 (Santa Cruz Biotechnology, sc-8408), FIGNL1 1:1000 (Proteintech, 17604-1-AP-150UL), GAPDH 1:8000 (Santa Cruz Biotechnology, sc-47724), ORC2 1:1000 (Abcam, ab68348), RAD51 1:1000 (Abcam, ab63801), Mouse monoclonal RAD51 1:1000 (Millipore-Sigma, 05-530-I), Rad51 Antibody (H-92) 1:1000 (Santa Cruz Biotechnology, sc-8349), Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 1:400 (Invitrogen, A32731), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 1:400 (Invitrogen, A32723), Goat anti-Rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:400 (Invitrogen, A48262), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 594 1:400 (Invitrogen, A32742), Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 594 1:400 (Invitrogen, A32740).

Techniques: Western Blot, Knock-Out, Expressing, Control, Transfection, Comparison, Staining, Two Tailed Test