Multicolor Microscopy and Spectroscopy Reveals the Physics of the One-Photon Luminescence in Gold Nanorods

This work demonstrates for the first time that one-photon luminescence in gold nanorods is an entirely plasmon-induced process. To achieve this goal, we used confocal microscopy in combination with higher order laser modes as well as light spectroscopy. We show that gold nanorod luminescence can be produced in three different ways: first, by directly exciting their longitudinal and transversal plasmon modes; second, by an energy transfer from the transversal to the longitudinal plasmon mode promoted by electron–holes pairs; and third, by conversion of directly excited electron holes pairs into the longitudinal plasmon mode

Minitumour spheroids have a different drug response than spheroids consisiting of only HUVEC and fibroblasts.

<p>A – Incubation of EndoFib spheroids with different anti-angiogenic agents for 40 h shows inhibition of sprouting with Thalidomide, Endostatin and Galardin. B – Incubation of Minitumour spheroids with different anti-angiogenic agents for 40 h shows a response to Galardin but a non-significant effect of both Thalidomide and Endostatin.</p

MT1-MMP gene silencing in both HUVECs and fibroblasts decreases endothelial sprout formation.

<p>Cells were infected with lentiviral particles expressing 2 different shRNAs against MT1-MMP, selected with puromycin and used to make spheroids. A – Representative images of pre-dyed endothelial cell sprouting from Minitumour spheroids made with HUVECs transduced with the different lentiviral derived shRNAs and controls. B – Quantification of endothelial cell sprouting showing a decrease in sprout formation with HUVECs expressing MT1-MMP shRNAs. C - Western Blots confirming MT1-MMP knock down levels of about 50% in HUVECs, adjusted relative intensity was determined through the ImageJ software follwed by normalization against the loading and mock controls. D – Representative images of pre-dyed endothelial cell sprouting from Minitumour spheroids made with Fibroblasts transduced with different lentiviral derived shRNAs and controls. E – Quantification of endothelial cell sprouting showing a decrease in sprout formation with from Minitumour spheroids with Fibroblasts expressing MT1-MMP shRNAs. F - Western blots confirming MT1-MMP knock down levels of about 30% in the fibroblasts, adjusted relative intensity was determined through the ImageJ software followed by normalization against the loading and mock controls.</p

Minitumour spheroids growth factor dependency.

<p>A – Direct incubation of function blocking antibodies for VEGF or PDGF within the collagen-I gel decreases endothelial cell sprouting from the Minitumour spheroids. B – Minitumour spheroids incubated with function blocking antibodies to IL6 and IL8 show similar levels of sprout formation to EndoFib spheroids. C - Minitumour and EndoFib spheroids show a differential response to inhibition of growth factor signaling using small molecule growth factor receptor inhibitors. D – Increase in endothelial cell sprouting in both Minitumour and EndoFib spheroids after 40 h incubation with the gamma-secretase inhibitor DAPT. E – Representative images from Minitumour and EndoFib spheroids incubated in collagen-I for 40 h with the addition of different growth factor receptor inhibitors.</p

Bioluminescence imaging of Minitumour spheroids reveals no difference in cancer cell proliferation with MMP inhibition.

<p>A – Quantification of bioluminescence levels from MB231luc21H4 cell dilutions showing a linear relation between cell number and luciferase signal. B – Representative images showing the bioluminescence signals from sequential concentration dilutions of MB231luc21H4 cells. C – Quantification of total luminescence signal of Minitumour spheroids including MDA-MB-231-luc2 after 40 h incubation in collagen-I with galardin, a vector control and Nocodazole as a positive control for proliferation inhibition (p-value<0.05). D – Quantification of bioluminescence signal from Minitumour spheroids made with MB231luc21H4 and fibroblasts expressing lentiviral derived shRNAs for MT1-MMP and non-targeting controls.</p

Characterization of the Minitumour spheroid model.

<p>A - Fluorescent (left) and phase contrast (right) images of HUVEC, EndoFib and Minitumour spheroids before incubation in the collagen gel; endothelial cells pre-dyed with a CMFDA Green CellTracker dye are seen in each different spheroid type. B – Representative fluorescent images of spheroids after 48 h incubation in collagen gels, in the presence of complete medium, showing pre-dyed endothelial cells organized into pre-capillary sprouts. C – Quantification of endothelial sprout length from different spheroids show that MDA-MB-231 cells stimulate sprout formation even in the absence of exogenous growth factors VEGF and bFGF. D – Confocal (upper) and phase contrast (lower) images of MDA-MB231 cells pre-dyed with the green CellTracker dye in the Minitumour spheroid after 48 h incubation in complete medium. E - A 3D reconstruction of a Minitumour spheroid where the HUVECs have been dyed with a CMRA Orange CellTracker dye and the fibroblasts with a CMFDA Green Cell Tracker side panels show optical x and y sections of sprouts showing the deposition of HUVECs and Fibroblasts relative to sprout formation.</p

Multiphoton microscopy images of Minitumour spheroids after 40 h or 7 days culture.

<p>A - HUVECs dyed with a CMFDA Green CellTracker dye were imaged within the Minitumour spheroid immediately following their embedding into the type-I collagen matrix using a Multiphoton microscope with a 20× objective. B – Immediately after collagen embedding, the collagen-I gel emits a weak homogenous Second Harmonic Generation (SHG) signal. C – Multiphoton imaging from of spheroids after 40 h incubation in the collagen-I gel shows the formation of green endothelial sprouts into the collagen matrix. D - The SHG signal from the collagen reveals an increase in matrix intensity around the endothelial sprouts. E – Merged image between CMFDA Green CellTracker dye and SHG signals after 40 h incubation. F – A higher amplification (40×) image of an endothelial cell sprout from a Minitumour spheroid after 40 h shows the alignment of collagen fibrils along the endothelial cell sprout (white arrows). G – Phase contrast images after 7 days incubation in the collagen-I gel showing a homogenous layer of cells. H – Multiphoton imaging after 7 days incubation shows the formation of a network of pre-dyed endothelial cells within the layer of cells. I – SHG signal from the collagen matrix after 7 days spheroid incubation. Scale bars represent 50 µm in F and 100 µm in all others.</p

Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA

<div><p>Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion <em>in vivo</em> using degron tagging in budding yeast. The previously described DHFR–based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 “core” subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.</p> </div

Interaction of Pds5, Scc3 and Wpl1 with cohesin ring.

<p>Lysates of nocodazole/benomyl arrested yeast cultures were incubated with IgG sepharose to precipitate Scc1-TAP or Smc3-TAP. The presence of different proteins on the IgG beads was analysed by Western blot probed with anti-HA (12CA5), anti-MYC (71D10) and PAP (P1291, Sigma). The strains were in (A): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 1829 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>), 1958 (<i>SCC3-MYC18, PDS5-HA6, SCC1-TAP</i>); in (B): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 1834 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>), 1956 (<i>PDS5-MYC18, SCC3-HA6, SCC1-TAP</i>); in (C): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2014 (<i>WPL1-MYC18, PDS5-HA6, SCC1-TAP</i>), 2016 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>); in (D): 1880 (<i>WPL1-MYC18, SCC3-HA6</i>), 2012 (<i>WPL1-MYC18, SCC3-HA6, SCC1-TAP</i>), 2018 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>); in (E): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 2251 (<i>SCC3-MYC18, PDS5-HA6, SMC3-TAP</i>), 2290 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (F): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 2249 (<i>PDS5-MYC18, SCC3-HA6, SMC3-TAP</i>), 2264 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>); in (G): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2253 (<i>WPL1-MYC18, PDS5-HA6, SMC3-TAP</i>), 2265 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (H): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2261 (<i>WPL1-MYC18, SCC3-HA6, SMC3-TAP</i>), 2271 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>).</p

Depletion of Scc3 and Pds5 with a “conventional” temperature-sensitive degron.

<p>(A–C) Strains 2395 (<i>SCC1-HA6</i>), 2452 (<i>SCC1-HA6</i>, degron-<i>MYC18-PDS5</i>), 2455 (<i>SCC1-HA6</i>, degron-<i>MYC18</i>-<i>SCC3</i>) and 2456 (<i>SCC1-HA6</i>, degron-<i>MYC18- PDS5, degron-MYC18-SCC3</i>) were arrested with nocodazole in YEP raffinose at 30°C for 2 hours, resuspended in YEP galactose containing nocodazole and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were shifted to 37°C in YEP galactose containing nocodazole and doxycycline to deplete Pds5 and/or Scc3. (A) Chromosomal spreads were prepared at the indicated time points and stained with DAPI for DNA, anti-HA (mouse, 16B12) and anti-MYC (rabbit, 71D10) antibodies. The secondary antibodies were Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit. Protein fluorescence was quantified using Metamorph software. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. (B) Western blot of TCA protein extracts probed with anti-HA (16B12), anti-MYC (71D10) and anti-Cdc28 (sc-28550, Santa Cruz). (C) FACS analysis of cellular DNA content. (D–F) Strains were staged in G1 with <i>α</i>-factor in YEP raffinose at 30°C, resuspended in YEP galactose containing <i>α</i>-factor and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were then shifted to 37°C in YEP galactose containing doxycycline and <i>α</i>-factor, incubated for 90 minutes to deplete Pds5 and/or Scc3 and subsequently released in YEP galactose containing nocodazole and doxycycline at 37°C. Chromosomal spreads (D), Western blot (E), and FACS analysis of cellular DNA content (F) are shown.</p