Journal: Oncology Letters

Article Title: Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma

doi: 10.3892/ol.2017.6203

Figure Lengend Snippet: Ang-2 expression was investigated by reverse-transcription polymerase chain reaction and western blot analysis in colorectal cancer cell lines, HUVEC and normal cell line (CCD-841 CoN). (A) Among 5 colorectal cancer cell lines, 3 cell lines expressed Ang-2 mRNA, as did the HUVEC cell line. (B) LoVo cells expressed a markedly higher level of Ang-2 protein compared with other colorectal cancer cell lines, as detected by western blotting. (C) The LoVo cell line transfected with Ang-2 target shRNA did not express Ang-2 mRNA. (D) The LoVo cell transfected with Ang-2 target shRNA exhibited markedly decreased expression levels of Ang-2 protein. Ang-2, Angiopoietin-2.

Article Snippet: LoVo cells (1×10 6 ) were grown overnight in RPMI 1640 medium in six-well culture plates to 60–70% confluence prior to transfection with Ang-2 shRNA lentiviral particles (TRCN000005923, TRCN000005924, TRCN000005925, TRCN000005926, and TRCN000005927), which were purchased from Sigma-Aldrich (Merck Millipore) along with a non-target negative control shRNA (SHC002V).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Transfection, shRNA

Journal: Oncology Letters

Article Title: Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma

doi: 10.3892/ol.2017.6203

Figure Lengend Snippet: (A) The proliferation of LoVo cells following Ang-2-targeted shRNA transfection was significantly decreased compared with control LoVo cells and LoVo cell transfected with non-targeted shRNA. (B) The invasion ability of Ang-2 target shRNA transfected LoVo cells was decreased markedly compared with the control and non-target shRNA-transfected LoVo cells. ***P<0.05. Ang-2, Angiopoietin-2; shRNA, short hairpin RNA.

Article Snippet: LoVo cells (1×10 6 ) were grown overnight in RPMI 1640 medium in six-well culture plates to 60–70% confluence prior to transfection with Ang-2 shRNA lentiviral particles (TRCN000005923, TRCN000005924, TRCN000005925, TRCN000005926, and TRCN000005927), which were purchased from Sigma-Aldrich (Merck Millipore) along with a non-target negative control shRNA (SHC002V).

Techniques: shRNA, Transfection, Control

Journal: Oncology Letters

Article Title: Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma

doi: 10.3892/ol.2017.6203

Figure Lengend Snippet: The migration of Ang-2 target shRNA transfected LoVo cells was decreased compared with the control and non-target shRNA transfected LoVo cells. ***P<0.01. Ang-2, Angiopoietin-2; shRNA, short hairpin RNA.

Article Snippet: LoVo cells (1×10 6 ) were grown overnight in RPMI 1640 medium in six-well culture plates to 60–70% confluence prior to transfection with Ang-2 shRNA lentiviral particles (TRCN000005923, TRCN000005924, TRCN000005925, TRCN000005926, and TRCN000005927), which were purchased from Sigma-Aldrich (Merck Millipore) along with a non-target negative control shRNA (SHC002V).

Techniques: Migration, shRNA, Transfection, Control

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: siRNA screen to identify Rab proteins affecting cell migration (A) U2OS cells transfected with control siRNA or pools of four different siRNAs against human Rabs were grown to confluency, scratch-wounded, and imaged every 3 h. The graph shows the relative wound density at 18 h after wounding represented as mean ± SEM of four independent experiments. The red solid line indicates the relative wound density (%) and the dashed red lines the boundaries of the SEM for cells transfected with control siRNA. (B) Representative images are shown for time 0 and 18 h after wounding for the two Rabs whose depletion had the strongest effect on wound closure. Scale bar: 200 μm. (C) U2OS cells transfected with control siRNA or a pool of four different siRNAs targeting Rab33b were imaged for 48 h. The rate of proliferation (measured as percentage of cell confluence) over time is shown as mean ± SEM of three independent experiments. (D) U2OS cells were transfected with control siRNA or each of the different individual four siRNAs present in the pool targeting Rab33b (Rab33b siRNA_1, siRNA_2, siRNA_3, or siRNA_4), grown to confluency, scratch-wounded, and imaged every 3 h. Representative images for time 0 and 24 h after wounding are shown. Scale bar: 200 μm. (E) Graph showing the relative wound density (%) over time for each sample in (d). The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, n.s. not significant, for t = 24h (two-tailed paired Student′s t-test). (F) Lysates from U2OS cells transfected with control siRNA, or with each of the different individual four siRNAs present in the pool targeting Rab33b were subjected to Western blot analysis with the indicated antibodies.

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Migration, Transfection, Two Tailed Test, Western Blot

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b depletion promotes cell migration (A) U2OS cells transfected with either control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b or transiently transfected with GFP, or GFP-Rab33b T47N, were scratch-wounded and imaged every 3 h. Representative images for time 0 and 23 h after wounding are shown. Scale bar: 200 μm. (B) Graph showing relative wound density (%) for each sample in (a) over time. The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, n.s., not significant, for t = 24h (two-tailed paired Student′s t-test). (C) Cell lysates from cells treated with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b were subjected to Western blot analysis with antibodies against Rab33b and tubulin (as loading control). (D) Representative track plots of the single-cell distances of migration for cells transfected with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b. Individual tracks are shown so that each starts at the origin (distance 0). (E) Quantification of the mean ± SEM of the single cell speed from at least three independent experiments. n > 30 cells per condition and per experiment. ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figures S1 A–S1B

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Migration, Transfection, Two Tailed Test, Western Blot

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b is involved in the regulation of focal adhesion dynamics (A) U2OS cells silenced with control siRNA, Rab33b siRNA_1, or Rab33b siRNA_2, were fixed and stained with DAPI, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 5 μm. (B–C) Quantification of FA number per 100 μm 2 cell area (b) and size (c). The graphs represent the mean ± SEM for three independent experiments (n > 90 cells). ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (D) Control or Rab33b-depleted cells transfected with RFP-vinculin were imaged every 10 min for 40 min. Arrows show FA disassembly, and arrowheads show FA assembly. Scale bar: 10 μm. (E) Rainbow color representation of FA assembly and disassembly over time from cells shown in panel (d). Each time point is shown in a different color, as indicated in the bar. Insets show magnifications of the boxed areas. Scale bar: 10 μm. (F) Quantification of assembly and disassembly rates of FAs. The assembly and disassembly rate is shown as percentage of focal adhesion formation or disassembly per minute. The values represent the mean ± SEM from three independent experiments, in which 15 FAs were analyzed per cell (n > 5), per condition, and per experiment. ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (G) Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and RFP-vinculin (red). Cells were imaged every 5 s with a Zeiss LSM880 confocal microscope. Magnifications of the boxed areas in the side panels show Rab33b-positive vesicles moving to focal adhesion sites. Scale bar: 5μm, inset: 1μm. See also Figure S2 and Video S1. Rab33b-positive vesicles are delivered to FAs, Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnification of the boxed area 1 in Figure 4g is shown and illustrates two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S2. Rab33b-positive vesicles are delivered to FAs,Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnifications of the boxed area 2 in Figure 4g is shown and illustrate two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S3. Rab33b-positive vesicles are delivered to growing FAs during membrane protrusion,U2OS cells transiently transfected with GFP-Rab33b (green) and vinculin-RFP (red) were imaged every 30 s using a TIRF microscope with a penetration depth of 90 nm. The movie shows the recruitment of GFP-Rab33b-positive vesicles during membrane protrusion. Related to Figure 4. .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Staining, Two Tailed Test, Transfection, Live Cell Imaging, Microscopy

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: VSV-G transport to the cell surface is inhibited by Rab33b depletion (A) U2OS cells silenced with control siRNA, siRNA against Rab33b, or silenced with Rab33b siRNA and transfected with RFP-Rab33b (red), were transfected with YFP-VSV-G (green) and incubated at 39°C for 16 h. Cells were then fixed either immediately (T0), 20 min (T20), or 90 min (T90) after a shift to 32°C. Scale bar: 10 μm. (B) Quantification of the VSV-G distribution 90 min after the shift to 32°C. 200 cells were analyzed from three independent experiments and the percentage of cells in which YFP-VSV-G was located at the Golgi, post-Golgi vesicles, and plasma membrane was determined. The graph shows the mean ± SEM ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figure S4 .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transfection, Incubation, Two Tailed Test

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet:

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transduction, Recombinant, Two Hybrid Screening, Sequencing, Plasmid Preparation, Software