Tks5 expression in breast tissue and breast cancer.

<p>A) 293 cells were transfected with empty vector, or vectors expressing Tks4 or Tks5. Cells were pelleted, fixed, embedded in paraffin and processed for immunohistochemistry with an anti-Tks5 antibody. B) Representative images of normal breast lobules and duct, and ductal carcinoma in situ, stained with anti-Tks5 antibodies. Red arrows indicate areas with intense Tks5 staining. Scale bar represents 100μm. C) Representative images of primary invasive breast cancer specimens, at low and high magnification, to illustrate the range of Tks5 expression observed. Scale bars represent 100μm. Quantification of 163 specimens is shown on the right. D) Kaplan-Meier survival curves for patients with high (red) and low (black) Tks5α mRNA levels. E) Kaplan-Meier survival curves for stage I/II patients with high (red) and low (black) Tks5α mRNA levels. F) Kaplan-Meier survival curves for stage III/IV patients with high (red) and low (black) Tks5α mRNA levels.</p

Tks5 is required for tumor progression.

<p>MDA-MB-231-Luc cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 shRNA are under the control of the tetracycline promoter (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#sec002" target="_blank">Materials and methods</a> for the experimental procedure). A) Immunoblot demonstrating Tks5 expression reduction in the MDA-MB-231-Luc cell line in dose- and time-dependent fashion in response to doxycycline exposure. B) TetOn/D6 were injected orthotopically (blue, red, and green lines) compared to SCR and no doxycycline controls (grey and purple lines, respectively) under three conditions: when Tks5 was already reduced by <i>in vitro</i> exposure of the cells to doxycycline for 10 days (DOX A) (blue line), when unexposed cells were injected into the animal and the animal received doxycycline starting at the day of injection (DOX B) (red line), as well as when the animals received doxycycline in the drinking water for the first time after the tumor has been growing for 7 days (DOX C) (green line). C) Animals were given doxycycline 15 days after tumor cell injection and after randomization of the mice. TetOn/D6 mice were divided up in 3 groups where 2 groups received doxycycline in the drinking water at different time points and 1 group received doxycycline-free drinking water. Tumor volumes were measured at different time points as described in Materials and methods, and tumors were allowed to grow to a final volume of approximately 2cm³. N = 4 mice per tumor type. Experiments were performed in duplicate. D-I) Tumors from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g004" target="_blank">Fig 4C</a> were analyzed for vascularization by CD31 (panel D-E), for apoptosis by TUNEL (panel F-G) and for proliferation using Ki-67 (panel H-I) immunofluorescence staining. Scale bar: 100μm. Images are representative for all experiments performed. J-K) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immunopositive cells for apoptosis (TUNEL) (panel J) and proliferative (Ki67) (panel K) markers at the day of dissection (endpoint of experiment). Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate <i>p</i> values.</p

Reduced expression of Tks5 leads to decreased growth of tumor cells <i>in vitro</i> and <i>in vivo</i>.

<p>A) MDA-MB-231-Luc infected with either a scrambled (Scr) or Tks5 (D6) shRNA via lentivirus were subjected to growth assays in 3D conditions for 7–10 days. Immunoblot shows shRNA Tks5 knockdown (KD) compared to scrambled control in the cells used to evaluate growth. D6 = Tks5 knockdown clone. Experiments were performed in triplicate. B) Representative images of day 1, 5, and 8 MDA-MB-232-Luc [Scr and Tks5 KD (D6)] acini in 3D matrigel assays. Shown are low magnification phase-contrast images. Experiment was performed in triplicate. Scale bar: 100μm. C) Immunoblot: D6 and D7 represent different shRNAs tested for effective Tks5 knockdown in MDA-MB-231-Luc cells. These cell lines, as well as Scr and rescue (D6+hTks5-GFP) lines, were used for orthotopic injections in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g001" target="_blank">Fig 1D and 1E</a>. D) Control (Scr) and Tks5 (D6) KD clones were inoculated in the mammary fat pad of SCID-Beige mice. Tumor volumes were measured every 2–3 days as described in Materials and Methods and tumors were allowed to grow to a final volume of approximately 2cm³. n = 20 mice per tumor type. Experiments were performed in triplicate (at least). Inset: Photographs of 6 tumors per group at the day of dissection (endpoint of experiment). Scale bar: 1cm. Tumor images are representative for all experiments performed. E) Control (Scr), Tks5 (D6 and D7) KD clones, and a Tks5 rescue clone (D6+ hTks5-GFP) were inoculated in the mammary fat pad of SCID-Beige mice. Tumor volumes were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g001" target="_blank">Fig 1D</a>. n = 5 mice per tumor type. Experiments were performed in triplicate (at least). Data are expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate <i>p</i> values.</p

Reduction of Tks5 expression in tumor cells is associated with decreased angiogenesis.

<p>Tumors from Scr KD and Tks5 KD MDA-MB-231-Luc-orthotopic mouse models in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g002" target="_blank">Fig 2</a> were analyzed via immunohistochemistry and immunofluorescence in size-matched tumors. A-B) Vessel morphology and density was examined by staining tumor samples with CD31 (quantification panels K and L). C-D) Hematoxylin and eosin staining revealed altered vessel morphology and hemorrhaging in Tks5 KD tumors as compared to Scr KD tumors. Tumors were also analyzed for FITC-Dextran leakage (panels E-F, red = CD31; Green = FITC-dextran; quantification panel M), VEGF expression (red; panels G-H), and hypoxic areas (pimonidazole staining in panel I-J, quantification panel N). Red dashed lines delineate borders for areas of hypoxia. Scale bar: 100 μm, except panels C and D where scale bar: 50μm. Images are representative for all experiments performed. Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate <i>p</i> values.</p

Tumor proliferation and apoptosis is affected by Tks5 knockdown.

<p>Tumors from Scr KD and Tks5 KD MDA-MB-231-Luc-orthotopic mouse models in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g002" target="_blank">Fig 2</a> were analyzed via immunohistochemistry and immunofluorescence, using size-matched tumors. A-D) TUNEL staining was used to visualize cell death in small (panels A-B) and large (panels C-D) tumors from Scr (panels A, C) and Tks5 (panels B, D) KD mice. Scale bar: 100μm. E-H) Ki-67 staining (nuclear protein marker) was used to visualize cell proliferation in small (panels E-F) and large (panels G-H) tumors from Scr (panels E, G) and Tks5 (panels F, H) KD mice. Scale bar: 50μm. Images are representative for all experiments performed. I-J) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immune-positive cells for apoptosis (TUNEL) (panel I) and proliferative cells (Ki67) (panel J) at the day of dissection (endpoint of experiment). Data are expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate <i>p</i> values.</p

Correlation between clinical parameters and development of broadly neutralizing antibody responses.

<p>(A) Bivariate and multivariable GLM correlation analyses between the listed variables, and the best neutralization score for the M48+ subset of Protocol C participants. Number of participants in each subgroup (N) is indicated. Estimated coefficients (EstCoef), p-values, q-values, odd ratios (ExpEst) upper (U95) and lower (L95) values of the 95% confidence interval are indicated. P-values are color coded as follows: 0.01< p-value < 0.05, in green; 0.001< p-value < 0.01, in yellow; 2E-16 < p-value <0.001, in red. Q-values below 0.1 are indicated in bold. (B) Kaplan Meier curves recording the time for Protocol C neutralizers (best neutralization score ≥ 0.5, N = 157) within the indicated subgroups to reach a neutralization score ≥ 0.5. Log-Rank test p-values are indicated. DC: Discordant couple, OHS: Other Heterosexual transmission, HSM: Women to Men Heterosexual transmission, HSW: Men to Women Heterosexual transmission, MSM: Men who have Sex with Men.</p

Correlation between total and antigen-specific IgG responses and development of broadly neutralizing antibody responses.

<p>Correlations were assessed by Spearman analyses: p-values and r-values are indicated; (ns) not significant. Linear, semi-Log or Log-log regressions are also shown as dotted lines. Neutralization score corresponds to a participant’s best neutralization score on the 6-virus panel across all tested time points. (A,C) IgG binding activity to recombinant MN gp41 (Subtype B), BG505 gp120 (subtype A) and IAVIC22 gp120 (Subtype B) was assessed, by ELISA, in plasma samples of Protocol C participants (N = 61) from the M48+ subset, at visits matching development of bnAb responses (M24-72, mean = 36.6 mpi). (B) Avidity index for IAVIC22-gp120 IgG titers were calculated from high salt (1.5M or 3M NaSCN) ELISA experiments. (C) Total IgG titers were assessed by ELISA. (D) Total IgG titers in pre-infection (N = 27), ~4mpi (N = 56, M00, mean = 4.0 mpi) and ~36mpi (N = 61) M24-72) samples. (E) Total IgG titers in ~36mpi samples (M24-72). (F) ELISA binding ID50 and neutralization score from (A) were standardized to a reference concentration of 20mg/mL of total plasma IgG.</p

Evolution of broadly neutralizing antibody responses in plasma from Protocol C participants.

<p>(A-C) Plasma from HIV-1 infected participants collected at various time points post infection were assessed for neutralizing activity on a predictive 6v-panel [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.ref016" target="_blank">16</a>]. Neutralization score on the 6v-panel was calculated as indicated in Material and Methods (A) Best neutralization score across all time points tested for Protocol C participants. (B) Fraction of Protocol C individuals with the indicated plasma neutralization score at the indicated visits. Neutralization score is color-coded as indicated in (A). (C) Detailed evolution of neutralization score over time (months) for individual Protocol C best neutralizers (N = 46), organized by time to reach neutralization score ≥ 1 in months post infection (MPI). NT: Not Tested. ART: Participants was on Anti-Retroviral Therapy at this visit, OFF: Participant was off-study at this visit.</p

Specificities mediating neutralization breadth and potency in the top 42 Protocol C neutralizers.

<p>(A) Samples are ranked by their neutralization score on the 37-virus panel (37vP) (S4 Fig in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.s001" target="_blank">S1 Text</a> and S2 Table in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.s001" target="_blank">S1 Text</a>). VC: Visit Code (months post infection). Symbols recapitulate the strength of the phenotypes tested using the different approaches detailed in this manuscript (S7-S11 Figures in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005369#ppat.1005369.s001" target="_blank">S1 Text</a>) to determine the Env epitope region targeted by the plasma broadly neutralizing activity: gp120 absorption of bnAb activity, effect of b6 competition in gp120 absorption experiments, RSC3 binding and neutralization competition, HIV-2 chimera neutralization, viruses produced in presence of kifunensine or bearing mutations. Absent (-), very weak (+/-), weak (+), moderate (++), strong (+++), phenotype was attributed based on i) the median fold or average percent decrease in ID50 and ii) the fraction of viruses which neutralization ID50 was decreased <2 fold, <10 <50 fold or <20%, <40%, <60%, <80%. Blank = not tested, NB: not binding. A dominant specificity was attributed for each sample based on results from all these experiments. UD: Undefined. (B) Overall distribution of dominant nAb specificities mediating plasma neutralization breadth in the top 42 Protocol C neutralizers as detailed in (A).</p