Journal: Scientific Reports

Article Title: Runx2 stimulates neoangiogenesis through the Runt domain in melanoma

doi: 10.1038/s41598-019-44552-1

Figure Lengend Snippet: Deletion of the Runt domain in A375 melanoma cells. ( A ) PCR products. 11 clones have been generated by applying the CRISPR/Cas9 technique. The PCR products, analysed by agarose gel electrophoresis, have been obtained by amplifying the Runt domain region. ( B ) Sanger sequencing chromatogram. The image shows the comparison between the WT and the 3G8 sequences with the deletion in genomic context.

Article Snippet: The above gRNAs were individually cloned in the PX459 V2.0 Cas9 expressing vector (Addgene), following the protocol described by Ran et al . .

Techniques: Clone Assay, Generated, CRISPR, Agarose Gel Electrophoresis, Sequencing, Comparison

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: The effect of anti-HBV pri-miR-31-mimic flanking sequence in ternary cassette. ( A ) A dual-luciferase assay was conducted to detect the production of functional miR-HBV from gRNA3-miR-HBV-gRNA2 cassettes with different lengths of anti-HBV pri-miR-31 mimic in HuH7 cells co-transfected with pGL3-HBV (1575-1604) or pGL3-control, PRL-TK and each of the plasmids containing gRNA3-miR-HBV-gRNA2 cassettes with different length of anti-HBV pri-miR-31 mimics. The vectors pGL3-control and PRL-TK were used as negative control and internal control, respectively. Data was shown as mean±SD of 4 independent experiments. ( B ) A polyA tailing reaction and a quantitative reverse transcription-PCR (qRT-PCR) were conducted to detect the production of mature miR-HBV in HuH7 cells transfected with the plasmids containing gRNA3-miR-HBV-gRNA2 cassettes with different length of anti-HBV pri-miR-31 mimics. ( C ) Schematic illustration of gRNA-gRNA binary cassette. ( D ) The expression plasmid of 1.2×HBV was co-transfected with the expression plasmid containing 3-2 binary cassette or gRNA3-miR-HBV-gRNA2 ternary cassette with different length of anti-HBV pri-miR-31 mimic flanking sequence in HuH7 cells at the ratio of 3:1 and 1:3. HBsAg levels in the cell culture supernatant were measured using a time-resolved fluoroimmunoassay at 72 hours after transfection. Data was shown as mean±SD of 5 independent experiments. (* indicated P <0.05, Mann-Whitney U test). PX458 plasmid was used as a vector control. 5S rRNA was used as the internal control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Sequencing, Luciferase, Functional Assay, Transfection, Control, Negative Control, Reverse Transcription, Quantitative RT-PCR, Expressing, Plasmid Preparation, Cell Culture, MANN-WHITNEY

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: The function of miR-HBV in gRNA-miR-HBV-gRNA ternary cassettes. ( A ) A dual-luciferase assay was conducted to analyze the effect of 3-M38-2 ternary cassette in the target sequence of miR-HBV. The 3-M38-2 ternary cassette was used as a positive control. Data was shown as mean±SD of 5 independent experiments. ( B ) The expression plasmids of 1.2×HBV (genotype C) and miR-HBV or mutant miR-HBV (miR-HBVm) were co-transfected into HuH7 cells. The HBsAg levels in the cell and culture supernatant of HuH7 cells were measured using a time-resolved fluoroimmunoassay at 48 hours after transfection. Data was shown as mean±SD of 4 independent experiments. ( C ) The expression plasmids of 1.2×HBV and 3-2 binary cassette, 3-M38-3 or 3-H38-2 ternary cassette were co-transfected into HuH7 cells. The levels of HBsAg and HBeAg in the culture supernatant were measured using a time-resolved fluoroimmunoassay at 72 hours after transfection. Data was shown as mean±SD of 5 independent experiments. (D) The level of gRNA2 in the HuH7 cells transfected with the expression plasmid of vector control, 3-2 binary or 3-H38-2 ternary cassette was detected by qRT-PCR (SYBR Green). The level of (E) gRNA3 carrying flanking sequence of anti-HBV pri-miR-31 mimic and (F) mature miRNA (miR-HBV or mutant miR-HBV) in the HuH7 cells transfected with the expression plasmid of vector control, 3-2 binary or 3-H38-2 ternary cassette was detected by qRT-PCR (SYBR Green). Data was shown as mean±SD of 4 independent experiments. (** indicated P <0.01, ***indicated P <0.001, Mann-Whitney U test). PX458 plasmid was used as a vector control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Luciferase, Sequencing, Positive Control, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, SYBR Green Assay, MANN-WHITNEY

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: The efficiency of gRNA-miR-HBV-gRNA ternary cassettes in suppressing HBV replication. The expression plasmids of each genotype (A, B or C) HBV and 3-H38-2 or 4-H38-1 cassette were co-transfected into HuH7 cells, and the levels of HBsAg ( A ) and HBeAg ( B ) in the cell culture supernatant or cells were measured using a time-resolved fluoroimmunoassay at 72 hours after transfection. Data was shown as mean±SD of 5 independent experiments. ( C ) The level of HBcAg in the HuH7 cells co-transfected with the expression plasmids of each genotype (A, B or C) HBV and 3-H38-2 or 4-H38-1 cassette were measured by Western Blot. The right figures were the statistical graphs of Western Blot, which was analyzed by Image J software (NIH, Bethesda, USA) at least in triplicate. HBc: HBcAg. PX458 plasmid was used as a vector control. (** indicated P<0.01, ***indicated P<0.001, Mann-Whitney U test).

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Transfection, Cell Culture, Western Blot, Software, Plasmid Preparation, Control, MANN-WHITNEY

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: Detection on the HBV-specific gRNAs-mediated destruction of HBV genome. ( A ) The expression plasmids of 1.2×HBV and 3-2 binary, 3-M38-2, 3-H38-2 or 4-H38-1 ( B ) ternary cassette were co-transfected into HuH7 cells. Cellular DNA was extracted at 72 hours after transfection, and PCR amplifications were performed using the primers beyond the cleavage sites of two gRNAs. ( C ) Sequencing analysis of the smaller fragment cleft by gRNA2 and 3. ( D ) Sequencing analysis of the smaller fragment cleft by gRNA1 and 4. PX458 plasmid was used as a vector control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Transfection, Sequencing, Plasmid Preparation, Control

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: Detection on the HBV-specific gRNAs-mediated destruction of HBV cccDNA. ( A ) HepAD38 cells were seeded into 10 cm dish. The expression plasmid of gRNA3-RNA2 binary or gRNA-miRNA-gRNA ternary cassette was co-transfected into HepAD38 cells. Transfection efficiency of HepAD38 cells was evaluated by observing the enhanced green fluorescent protein (EGFP) expressing cells under immunofluorescence microscopy. ( B ) HBsAg and HBeAg levels in the cell culture supernatant of HepAD38 cells were measured using a time-resolved fluoroimmunoassay. Data was shown as mean±SD of 6 independent experiments. ( C ) HBsAg levels in the cell culture supernatant of HepG2-NTCP-tet cells were measured using a time-resolved fluoroimmunoassay. Data was shown as mean±SD of 5 independent experiments. ( D ) HBV cccDNA levels in EGFP positive HepAD38 cells selected by Flow cytometry were measured using a KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR (SYBR green) combined method. Data was shown as mean±SD of 4 independent experiments. ( E ) PCR amplification of cccDNA was performed using the primers beyond the cleavage sites of two gRNAs following above rolling circle amplification. HepAD38 cells were transfected with 3-2 binary cassette expression plasmid, and then treated with or without lamivudine (3TC). The HBV DNA levels ( F ) in HepAD38 cells were detected by quantitative PCR, and HBV cccDNA levels ( G ) in EGFP positive HepAD38 cells selected by Flow cytometry were measured as above. Data was shown as mean±SD of 4 independent experiments. (* indicated P <0.05, ** indicated P <0.01, Mann-Whitney U test). PX458 plasmid was used as a vector control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Plasmid Preparation, Transfection, Immunofluorescence, Microscopy, Cell Culture, Flow Cytometry, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay, MANN-WHITNEY, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

doi: 10.3389/fcell.2022.968164

Figure Lengend Snippet: Plasma membrane repair is defective in S100A11-KO EA.hy926 cells. (A) Representative images of FM4-64 influx following membrane damage in S100A11-KO, clone #1 and #3, or WT EA.hy926 cells (see ). Cells kept in Tyrode’s buffer supplemented with 2.5 mM Ca 2+ and 5 μg/ml FM4-64 were injured by laser ablation at 820 nm (near infrared) directed at the plasma membrane on the lateral edge (white triangles represent the wound sites). Wounding occurred at t = 0; pre-wounding and post-wounding time points are shown. Calibration bar of fluorescence intensity is provided in the upper right panel. Scale bars = 10 µm. (B) Time course of whole-cell FM4-64 fluorescence normalized to fluorescence before injury in WT (left) or S100A11-KO cells (clone #1: middle and clone #3: right) laser-ablated in the presence of extracellular Ca 2+ or EGTA. (C) AUC values of FM4-64 fluorescence in WT or S100A11-KO cells damaged in the presence of extracellular Ca 2+ . S100A11-KO, n = 27 cells per clone; WT, n = 18 cells. Results were pooled from three independent experiments. Statistical analysis was performed with ordinary one-way ANOVA. (D) Exemplary fields of mechanically wounded WT or S100A11-KO cells stained with FITC-Dextran, TRITC-Dextran and Hoechst. The protocol (see Materials and Methods) permits the distinction between wounded and repaired cells (that had taken up only FITC-Dextran) and wounded but non-repaired cells (that had taken up FITC-Dextran and TRITC-Dextran). Hoechst was included as a marker for all cells. Scale bars = 50 μm. Quantification of non-repaired WT or S100A11-KO cells, represented as a percentage of total wounded cells (right). Results were pooled from three independent experiments ( n = 75 fields). Statistical analysis was carried with two-tailed Student’s t test. Data are mean ± SD.

Article Snippet: Therefore, a ready-to-use transfection-based S100A11 gene specific gRNA-Cas9 expression plasmid vector (S100A11 CRISPR gRNA 4_PX459 V2.0) from GenScript was utilized ( ).

Techniques: Fluorescence, Staining, Marker, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

doi: 10.3389/fcell.2022.968164

Figure Lengend Snippet: S100A11 translocates to the injury site in response to wounding. (A) Time-lapse images of wave-like S100A11 translocation to laser-induced plasma membrane wounds in WT or S100A11-KO cells, respectively . YFP-S100A11 transfected cells kept in Tyrode’s buffer supplemented with 2.5 mM Ca 2+ and 5 μg/ml FM4-64 were injured at the lateral membrane edge (white triangles represent the wound sites). Wounding occurred at t = 0; pre-wounding and post-wounding time points are shown. Scale bars = 10 µm. Insets highlight the immediate vicinity of the wound. Scale bars = 2 μm. (B) Time course of YFP-S100A11 fluorescence in WT or S100A11-KO cells, normalized to fluorescence before injury. Mean fluorescent intensity was measured in the area next to the wound site (a circular ROI of 10 μm around the wound) for each acquisition time point. YFP-S100A11 AUCs for WT and S100A11-KO cells are also shown. Results were pooled from four independent experiments ( n = 24). Statistical analysis was performed with two-tailed Student’s t test. (C) Time course of whole-cell FM4-64 fluorescence in WT or S100A11-KO cells ectopically expressing YFP-S100A11, normalized to fluorescence before injury. Control experiments carried out in the absence of extracellular Ca 2+ (EGTA containing medium) showed no resealing under the experimental condition used here, indicative of a still Ca 2+ -dependent repair process. Statistical comparison between FM4-64 AUCs was performed with two-tailed Student’s t test. Data are mean ± SD.

Article Snippet: Therefore, a ready-to-use transfection-based S100A11 gene specific gRNA-Cas9 expression plasmid vector (S100A11 CRISPR gRNA 4_PX459 V2.0) from GenScript was utilized ( ).

Techniques: Translocation Assay, Transfection, Fluorescence, Two Tailed Test, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

doi: 10.3389/fcell.2022.968164

Figure Lengend Snippet: S100A11 interacts with ANXA1 and ANXA2 via its C-terminal extension at elevated Ca 2+ conditions. (A) Domain structures of wild type S100A11, S100A11 ΔCTM and S100A11 CM. The scheme shows N-terminus on the left and C-terminus on the right. (B) Representative immunoblots of samples (input fraction, non-bound fraction and immunoprecipitates/beads) from S100A11-KO cells transfected with empty GFP vector, YFP-S100A11, YFP-S100A11 ΔCTM or YFP-S100A11 CM. Lysates of the respective cells were subjected to IP reactions in the presence of 2 mM Ca 2+ . Immunoblots were probed with anti-GFP antibodies to detect GFP alone (27 kDa) and YFP-S100A11, YFP-S100A11 ΔCTM or YFP-S100A11 CM (∼39 kDa), respectively, and with anti-ANXA1 (39 kDa) or anti-ANXA2 (38 kDa) antibodies to detect co-immunoprecipitated endogenous proteins. Representative blots from three to six independent experiments are shown. Note that ANXA1 and ANXA2 show some unspecific binding to the beads in control experiments carried with the empty GFP vector. This background binding is also observed in the YFP-S100A11 ΔCTM and YFP-S100A11 CM immunoprecipitates, but is significantly lower than the Co-IP in case of YFP-S100A11. (C) IP efficiencies for GFP, YFP-S100A11, YFP-S100A11 ΔCTM, and YFP-S100A11 CM, respectively (left), and Co-IP efficiencies for endogenous ANXA1 (middle) or ANXA2 (right) quantified in three to six independent blots. Statistical comparisons between groups were performed with ordinary one-way ANOVA (Tukey’s multiple comparison test). Data are mean ± SD.

Article Snippet: Therefore, a ready-to-use transfection-based S100A11 gene specific gRNA-Cas9 expression plasmid vector (S100A11 CRISPR gRNA 4_PX459 V2.0) from GenScript was utilized ( ).

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Co-Immunoprecipitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

doi: 10.3389/fcell.2022.968164

Figure Lengend Snippet: Absence of S100A11 affects the wound site recruitment of ANXA2 but not ANXA1. (A) Time-lapse images of ANXA1 recruitment to laser-induced plasma membrane wounds in WT or S100A11-KO cells, respectively . ANXA1-GFP expressing cells kept in Tyrode’s buffer supplemented with 2.5 mM Ca 2+ and 5 μg/ml FM4-64 were injured at the lateral membrane edge (white triangles represent the wound sites). Wounding occurred at t = 0; pre-wounding and post-wounding time points are shown. Scale bars = 10 µm. Insets highlight the immediate vicinity of the wound. Scale bars = 2 μm. (B) Time course of ANXA1-GFP fluorescence in WT or S100A11-KO cells, normalized to fluorescence before injury. Mean fluorescent intensity was measured in the area next to the wound site (a circular ROI of 10 μm around the wound) for each acquisition time point. ANXA1-GFP AUCs for WT and S100A11-KO cells are also shown. Results were pooled from four independent experiments ( n = 24). Statistical analysis was performed with two-tailed Student’s t test. (C) Time course of whole-cell FM4-64 fluorescence in WT or S100A11-KO cells ectopically expressing ANXA1-GFP, normalized to fluorescence before injury. Control experiments carried out in the absence of extracellular Ca 2+ (EGTA containing medium) showed no resealing under the experimental condition used here, indicative of a still Ca 2+ -dependent repair process. Statistical comparison between FM4-64 AUCs was performed with two-tailed Student’s t test. (D–F) Same as in (A–C) , but WT or S100A11-KO cells were transfected with ANXA2-GFP . Data are mean ± SD.

Article Snippet: Therefore, a ready-to-use transfection-based S100A11 gene specific gRNA-Cas9 expression plasmid vector (S100A11 CRISPR gRNA 4_PX459 V2.0) from GenScript was utilized ( ).

Techniques: Expressing, Fluorescence, Two Tailed Test, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

doi: 10.3389/fcell.2022.968164

Figure Lengend Snippet: The C-terminal extension is dispensable for the function of S100A11 in PM repair. (A) Time-lapse images of S100A11 ΔCTM dynamics in response to laser-induced plasma membrane wounds in WT or S100A11-KO cells, respectively . YFP-S100A11 ΔCTM expressing cells kept in Tyrode’s buffer supplemented with 2.5 mM Ca 2+ and 5 μg/ml FM4-64 were injured at the lateral membrane edge (white triangles represent the wound sites). Wounding occurred at t = 0; pre-wounding and post-wounding time points are shown. Scale bars = 10 µm. Insets highlight the immediate vicinity of the wound. Scale bars = 2 μm. (B) Time course of YFP-S100A11 ΔCTM fluorescence in WT or S100A11-KO cells, normalized to fluorescence before injury. Mean fluorescent intensity was measured in the area next to the wound site (a circular ROI of 10 μm around the wound) for each acquisition time point. YFP-S100A11 ΔCTM AUCs for WT and S100A11-KO cells are also shown. Results were pooled from four independent experiments ( n = 24). Statistical analysis was performed with two-tailed Student’s t test. (C) Time course of whole-cell FM4-64 fluorescence in WT or S100A11-KO cells ectopically expressing YFP-S100A11 ΔCTM, normalized to fluorescence before injury. Control experiments carried out in the absence of extracellular Ca 2+ (EGTA containing medium) showed no resealing under the experimental condition used here, indicative of a still Ca 2+ -dependent repair process. Statistical comparison between FM4-64 AUCs was performed with two-tailed Student’s t test. (D–F) Same as in (A–C) , but WT or S100A11-KO cells were transfected with YFP-S100A11 CM . Data are mean ± SD.

Article Snippet: Therefore, a ready-to-use transfection-based S100A11 gene specific gRNA-Cas9 expression plasmid vector (S100A11 CRISPR gRNA 4_PX459 V2.0) from GenScript was utilized ( ).

Techniques: Expressing, Fluorescence, Two Tailed Test, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

doi: 10.3389/fcell.2022.968164

Figure Lengend Snippet: S100A11 associates with E-Syt1 in endothelial cells. (A) Representative time-lapse images of injured HUVEC expressing GFP plus mApple-S100A11 (upper panel) or EGFP-E-Syt1 plus mApple-S100A11 (lower panel) . Transfected cells kept in Tyrode’s buffer supplemented with 2.5 mM Ca 2+ were injured at the lateral membrane edge (white triangles represent the wound sites). Wounding occurred at t = 0; pre-wounding and post-wounding time points are shown. Scale bars = 10 µm. Insets highlight the immediate vicinity of the wound. Scale bars = 2 μm. (B) Time course of mApple-S100A11 fluorescence in injured HUVEC expressing GFP or EGFP-E-Syt1, normalized to fluorescence before injury. mApple-S100A11 AUCs for empty GFP vector or EGFP-E-Syt1 transfected HUVEC are also shown. Results were pooled from three independent experiments ( n = 18). Statistical analysis was performed with two-tailed Student’s t test. (C) Domain structures of wild type E-Syt1 as well as E-Syt1 ΔC2C and E-Syt1 ΔTM mutants. The scheme shows N-terminus on the left and C-terminus on the right. (D,E) Time course of mApple-S100A11 fluorescence in EGFP-E-Syt1 ΔC2C (D) or EGFP-E-Syt1 ΔTM (E) expressing HUVEC, normalized to fluorescence before injury. mApple-S100A11 AUCs for EGFP-E-Syt1 ΔC2C or EGFP-E-Syt1 ΔTM transfected HUVEC are also shown. Results were pooled from three independent experiments ( n = 18). Statistical analyses were performed with two-tailed Student’s t test. (F) Representative immunoblots of samples (input fraction, non-bound fraction and immunoprecipitates/beads) from non-transfected HUVEC or HUVEC transfected with GFP or YFP-S100A11 plasmids, which were subjected to IP reactions in the presence of 2 mM Ca 2+ . Immunoblots were probed with anti-GFP antibodies to detect GFP alone (27 kDa) and YFP-S100A11 (39 kDa), and with anti E-Syt1 (123 kDa) antibodies to detect co-immunoprecipitated endogenous protein. Representative blots from seven independent experiments are shown. (G) IP efficiencies for empty GFP vector, YFP-S100A11 (left) and Co-IP efficiencies for endogenous E-Syt1 (right) quantified in seven independent blots. Statistical comparisons between groups were performed with ordinary one-way ANOVA (Tukey’s multiple comparison test). Data are mean ± SD.

Article Snippet: Therefore, a ready-to-use transfection-based S100A11 gene specific gRNA-Cas9 expression plasmid vector (S100A11 CRISPR gRNA 4_PX459 V2.0) from GenScript was utilized ( ).

Techniques: Expressing, Transfection, Fluorescence, Plasmid Preparation, Two Tailed Test, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

doi: 10.3389/fcell.2022.968164

Figure Lengend Snippet: E-Syt1 regulates translocation of S100A11 to plasma membrane wounds. (A) Immunoblot analysis of endogenous E-Syt1 (124 kDa) and E-Syt2 (102 kDa) expression in HUVEC transfected with control or E-Syt1 and E-Syt2-specific siRNAs (siControl or siE-Syt1/2, respectively). β-actin (42 kDa) is shown as the loading control. Representative blots from five independent experiments are shown. Efficiencies of E-Syt1 and E-Syt2 knockdown quantified in five independent blots are also shown. Statistical comparisons between non-targeted and targeted knockdowns were performed with two-tailed Student’s t test. (B) Time-lapse images of S100A11 recruitment to laser-induced plasma membrane wounds in siControl or siE-Syt1/2 treated HUVEC . Cells kept in Tyrode’s buffer supplemented with 2.5 mM Ca 2+ and 5 μg/ml FM4-64 were injured at the lateral membrane edge (white triangles represent the wound sites). Wounding occurred at t = 0; pre-wounding and post-wounding time points are shown. Scale bars = 10 µm. Insets highlight the immediate vicinity around the wound. Scale bars = 2 μm. (C) Time course of YFP-S100A11 fluorescence in siControl or siE-Syt1/2 treated HUVEC, normalized to fluorescence before injury. Mean fluorescent intensity was measured in the area next to the wound site (a circular ROI of 15 μm around the wound) for each acquisition time point. YFP-S100A11 AUCs for siControl or siE-Syt1/2 treated HUVEC are also shown. Results were pooled from four independent experiments ( n = 40). Statistical analysis was performed with two-tailed Student’s t test. (D) Representative images of FM4-64 infiltration following membrane damage (white triangles represent the wound sites) in HUVEC transfected with control or E-Syt1 and E-Syt2-specific siRNAs . Calibration bar of fluorescence intensity is provided on the upper right panel. Scale bars = 10 µm. (E) Time course of whole-cell FM4-64 fluorescence in HUVEC transfected with control or E-Syt1 and E-Syt2-specific siRNAs, normalized to fluorescence before injury. Data were compared for AUC values of FM4-64 fluorescence. Control experiments carried out in the absence of extracellular Ca 2+ (EGTA containing medium) showed no resealing under the experimental condition used here, indicative of a still Ca 2+ -dependent repair process. Results were pooled from three independent experiments ( n = 36). Statistical analysis was performed with two-tailed Student’s t test. Data are mean ± SD.

Article Snippet: Therefore, a ready-to-use transfection-based S100A11 gene specific gRNA-Cas9 expression plasmid vector (S100A11 CRISPR gRNA 4_PX459 V2.0) from GenScript was utilized ( ).

Techniques: Translocation Assay, Western Blot, Expressing, Transfection, Two Tailed Test, Fluorescence