Fig 1: TCP transfection induces apoptosis that is blocked by oncogenic KRas, Bcl-2 and Bcl-xL overexpression. (A) Specific oncogenic or anti-apoptotic signaling pathways were manipulated in the stable background of MCF10A cells. These cell lines were then transfected with sfGFP control, TCP-VASH1, or TCP-VASH2 for 24 h. After 24 h, the cells were fixed and stained with an anti-active Caspase-3 antibody and AF594-conjugated secondary to determine cells actively undergoing apoptosis. Using the HCA Nikon Analysis software, sfGFP-positive cells were identified and then average intensity of the AF549 signal for each sfGFP-positive object was determined. If the AF549 average intensity was greater than 10,000 RFU, that object was counted as positive for active Caspase-3. Cells transfected with TCP-VASH1 and TCP-VASH2 had a higher level of Caspase-3 activation compared to the sfGFP control in MCF10A parentals, PTEN−/−, Bcl-2- and Bcl-xL-overexpressing cells. Interestingly, MCF10A KRas and PTEN-/ KRas did not show an increase in Caspase-3 activation compared to sfGFP control. (B) For each TCP construct (VASH1 or VASH2), the level of Caspase-3 activation was compared to sfGFP. Oncogenic KRas provides the most protection against TCP-induced cell death, but Bcl-2 and Bcl-xL also provide a lesser level of protection. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, n = 4; triplicate.
Fig 2: VASH1/SVBP and VASH2/SVBP increase deTyr-Tub in a specific and time-dependent manner. (A) MCF10A cells transfected with a plasmid containing sfGFP, VASH1-sfGFP-IRES-SVBP, or VASH2-sfGFP-IRES-SVBP and immunostained for deTyr-Tub (red) as well as the nuclear stain Hoescht 33342 (blue). Scale bar = 20 µm. (B) MCF10As were transfected with the same constructs as in panel A and lysed at times 0, 16 h, 24 h, and 48 h post-transfection. Primary antibodies against deTyr-Tub, α-tubulin, GAPDH, and FLAG-tag were incubated with HRP-conjugated secondary antibodies. sfGFP, VASH1, and VASH2 expressed on the plasmids are FLAG-tagged, allowing for the simultaneous visualization of all three proteins at various molecular weights. (C) MCF7 cells were transfected with the same plasmids as in panel A and processed in the same manner for visualization of deTyr-Tub-rich microtubules. (D) MCF7 cells were transfected with the same constructs as in panel A, and probed for the protein targets as in panel B. The values under deTyr-Tub indicate the raw intensity/1000 for the band above without any normalization.
Fig 3: VASH1 is a target protein of miR-1269a. VASH1 (A) mRNA and (B) protein expression levels in CRC tissues and adjacent healthy tissues. Correlation between (C) miR-1269a and VASH1, and (D) circASS1 and VASH1 in CRC tissues. VASH1 (E) mRNA and (F) protein expression levels in CRC cell lines. (G) Transfection efficiency of miR-1269a inhibitor. VASH1 (H) mRNA and (I) protein expression levels in miR-1269a inhibitor-transfected HT29 cells. VASH1 (J) mRNA and (K) protein expression levels in Oe-circASS1-transfected HT29 cells. (J-K) The mRNA and protein levels of VASH1 in HT29 cells treated with Oe-circASS1. Binding sites between miR-1269a and VASH1 were (L) predicted and (M) confirmed. *P<0.05, **P<0.01 and ***P<0.001. VASH1, vasohibin 1; miR, microRNA; CRC, colorectal cancer; Oe, overexpression; circASS1, circular RNA argininosuccinate synthase 1; NC, negative control; WT, wild-type; MUT, mutant.
Fig 4: TCP expression induced cell death in MCF10s and MCF7s. (A) MCF10A cells transfected with a TCP-VASH1 construct was stained for deTyr-Tub (red, 1:1000) and the nuclei stained with Hoechst 33342 (blue, 1:5000). This image shows nuclear condensation and the formation of apoptotic-like bodies which are positive for the VASH1-sfGFP fusion protein as well as deTyr-Tub. Scale bar = 50 µm (B) MCF10A cells were imaged 16, 24, and 48 h post-transfection in both phase contrast and the GFP channel. Representative images of the GFP channel overlaid on phase contrast show cell rounding in the TCP-VASH1 and TCP-VASH2, but not in the sfGFP control. Scale bar = 100 µm (C) Using the HCA Nikon Analysis software, sfGFP-positive objects were identified. To compare condition, the number of objects, length and circularity of each object were quantified. TCP-transfected MCF10A cells showed a lower cell number, decrease in cell size, and an increase in circularity compared to the sfGFP control. (D) MCF7 cells showed a decrease in cell number, and size for TCP transfections compared to sfGFP control. There was no significant difference in cell circularity amount constructs in MCF7 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, n = 4; triplicate.
Fig 5: Components of the TCP across cell lines do not correlate with the level of deTyr-Tub. (A) Protein was isolated from MCF10As and the MCF10A variants using RIPA lysis buffer. DeTyr-Tub (1:10,000), total alpha-tubulin (1:1000), and the components of the TCP (VASH1(1:1000), VASH2 (1:1000), and SVBP (1:1000)) were probed. An increase in basal level of deTyr-Tub can be seen in the MCF10A cell lines with oncogenic KRas, that is not present in the MCF10A parental or anti-apoptotic MCF10A cell lines. The level of VASH1 and VASH2 appears to be consistent across all lines, with a small increase in SVBP in the KRas containing lines. (B) To better understand the presence of deTyr-Tub and TCP components in breast cancer, a number of commonly used breast cancer cell lines were lysed using RIPA buffer and subjected to immunoblotting. These breast cancer cell lines contain various basal amounts of deTyr-Tub, VASH1, VASH2, and SVBP with no clear connection between components of the TCP and level of deTyr-Tub. The values under deTyr-Tub indicate the raw intensity/1000 for the band above without any normalization.
Supplier Page from Abcam for Anti-VASH1 antibody [EPR17420]