EMBO/soda-nel huggingface dataset:
Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy (2019)
Figure 3

Figure 3 - Combining Apelin blockage and anti-angiogenic treatment improves survival and reduces mammary tumor growth.

(A) Experimental set up and (right) Kaplan Meier survival plot of NeuT;Apln+/+ and NeuT;Apln-/- mice with mammary cancer, left untreated (control) or treated with the indicate dose of the broad VEGFR blocker sunitinib. NeuT;Apln+/+ Control (n=8), NeuT;Apln-/- Control (n=11), NeuT;Apln+/+ Sunitinib (n=11), NeuT;Apln-/- Sunitinib (n=12); *P<0.05; Log rank test. Mice were sacrificed when the tumor size reached 1cm3, following ethical guidelines. The dotted line indicates 50% of survival. (B) Percentages (mean ± S.E.M.) of tumor burden in mammary glands of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 4 weeks after tumor onset. NeuT;Apln+/+ Control (n=6), NeuT;Apln-/- Control (n=8), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); *P<0.05; ***P<0.001; one-way ANOVA. Representative H&E images are shown for each genotype in the right panels. Scale bars = 1000 μm (large panels) and 50 μm (insets). (C) Tumor volumes of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mammary tumors, size-matched at 20-70mm3 and followed over time by MRI analysis; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=6) mice per group; Lines represent nonlinear fit of tumor growth. Box and arrow indicate the time point used for analysis in panel D. (D) Mitotic counts (mean ± S.E.M.) and representative H&E images of mammary tumors in untreated (control) ­and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after tumor onset.; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=6), NeuT;Apln+/+ Sunitinib (n=9), NeuT;Apln-/- Sunitinib (n=4) tumors per group; *P<0.05; one-way ANOVA to sunitinib-treated NeuT;Apln-/-. White arrows indicate mitotic figures. Scale bars = 50 μm (E) Tumor volumes, followed over the indicated time, of orthotopically injected E0771 shRenilla cells left untreated (control) or treated three times a week from day 8 after tumor injection with an Apelin antagonist alone (MM54, 0.4μg/g), sunitinib alone (60mg/kg) or a combination of both. Tumor volumes were measured using calipers and are shown as mean tumor volumes ± S.E.M. E0771 shRenilla Control (n=6), MM54 (n=5), Sunitinib (n=5), Sunitinib+MM54 (n=5); *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA.
Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy (2019)
Figure 4

Figure 4 - Combining Apelin blockage and sunitinib treatment reduces tumor angiogenesis and normalizes tumor blood vessels.

(A) Experimental set up for the MRI (non-invasive magnetic resonance imaging) experiments. (B) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of relative tumor blood volume (rTBV) over time after NeuT+ mammary tumors were size-matched at 20-70mm3 (0 weeks). Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); **P<0.01, and ***P<0.001 compared to untreated NeuT;Apln+/+ mice and #P<0.05 compared to untreated control NeuT;Apln-/- mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analyzed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus, we did not perform any statistical analysis on the 6 weeks timepoints. (D) Analysis (mean values ± S.E.M.) of CD31+ area (x104 μm2)/field, number of dilated tumor vessels and percentage of alphaSMA+ area per CD31+ blood vessels in mammary tumors of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=4); *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA and Kruskal-Wallis test. (E) Representative immunofluorescence and immunohistochemistry images from Fig. 5D quantification. Dilated blood vessels are marked by a red asterisk. DAPI (blue) is shown as a counterstain to visualize nuclei. Scale bars = 100 μm (upper panels), 50 μm (middel panels) and 20 μm (lower panels).
Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy (2019)
Figure 5

Figure 5 - Apelin inactivation reduces sunitinib therapy-induced hypoxia and vessel permeability.

(A) Percentages of CA9+ cells adjacent to CD31+ tumor blood vessels in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=4); 100-200 peri-vascular intra-tumoral regions per group were counted. ***P<0.001; one-way ANOVA. Right panels show representative immunofluorescent images. Areas limited by dotted white lines indicate CA9+ areas. Scale bars = 50 μm. (B) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of vessel permeability (K2) in NeuT+ mammary tumors followed over time. Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=5); ***P<0.001, compared to untreated control NeuT;Apln+/+ mice and §P<0.05 compared to sunitinib-treated NeuT;Apln+/+ mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analysed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus we did not perform any statistical analysis on the 6 weeks timepoints.
Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy (2019)
Figure 6

Figure 6 - Apelin inactivation reduces sunitinib therapy-induced metastasis and stratifies sunitinib therapy responses in patients.

(A) Number of metastatic lung foci in untreated (control) and sunitinib-treated (60mg/kg, three times a week from tumor initiation) NeuT;Apln+/+ or NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. Data of individual lung sections and means (black bars) are shown. Right panels show representative H&E images, where black arrows and insets indicate metastatic foci. Scale bars = 1000 μm (large panels) and 50 μm (insets). *P<0.05, **P<0.01; ***P<0.001; Kruskal-Wallis test; n=3 mice per cohort and three sections per lung were analysed. (B) Kaplan Meier survival plot for distant metastasis free survival from the KM-plotter database (Data Ref: Győrffy et al, 2013) for high and low Apelin (APLN)-expressing groups in human breast cancer. Patients were split by the median. Affymetrix Apelin ID 222856_at. (C) Kaplan-Meier plot for progression-free survival stratifying RCC patients with high and low APELIN serum levels 3-5 months after the start date of sunitinib treatment. *P=0.0367; Log rank test. (D) Kaplan Meier plots for progression-free survival in RCC patients stratified into groups of high or low levels of APELIN and VEGF. Cut-off levels were set by the median. Serum was analysed 3-5 months after the start date of sunitinib treatment. *P<0.05, **P<0.01; Log rank test.

Expanded View Figure Legends
A ubiquitin-like system mediates protein lipidation (2000)
figf1

a, Phase partitioning of Apg8. Wild-type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning (see Methods). b, MALDI mass spectrum of His6–Apg8. Purified His6–Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4 (Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6–Apg8FG. c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.
A ubiquitin-like system mediates protein lipidation (2000)
figf2

a, Homology between Apg8 and Apg12 at the C termini. b, Interaction between Apg8 and Apg7. Δapg7Δapg8 cells were transformed with APG7–Myc and/or APG8 on 2µ plasmids. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc or anti-Apg8 antibody. Apg8 co-immunoprecipitated with Apg7–Myc. c, Effect of Apg8 C-terminal processing on the Apg8–Apg7 interaction. Δapg4 cells co-expressing Apg7–Myc and HA–Apg8 (FGR, FG and F) from 2µ plasmids. FGR, wild-type Apg8; FG, Apg8 lacking C-terminal arginine; F, Apg8 lacking C-terminal glycine and arginine. Cell lysates were immunoprecipitated with anti-HA antibody, and immunoblotted with anti-HA and anti-Myc antibody. d, Thioester formation between Apg8 and Apg7. Cell extracts of Δapg7Δapg8 cells expressing Apg7–Myc and/or HA–Apg8 from 2µ plasmids were immunoprecipitated with anti-HA antibody, and immunoblotted (IB) with anti-Myc and anti-HA antibody. DTT–, no DTT; DTT+, 100 mM DTT; asterisk, Apg8–Apg7 conjugate.
A ubiquitin-like system mediates protein lipidation (2000)
figf3

a, Homology between Apg3, Apg10 and their homologues. Cys 234 of Apg3 corresponds to the active centre Cys 133 of Apg10 (arrowhead). b, Interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing APG3 and/or Myc–APG8 from 2µ plasmids were immunoprecipitated with anti-Apg3 antibody, and immunoblotted with anti-Apg3 and anti-Myc antibody. c, Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing Myc–APG8 and either wild-type (wt) APG3 or mutant APG3 (C234S and C234A) from 2µ plasmids were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc and anti-Apg3 antibodies. d, Thioester formation between Apg8 and Apg3. Δapg3, Δapg7 and Δapg10 cells were transformed with both Myc–APG8 and Apg3, or only Apg3, on 2µ plasmids. Cell extracts were immunoprecipitated with anti-Myc antibody, and subjected to immunoblot (IB) using anti-Myc and anti-Apg3 antibody. DTT–, no DTT; DTT+, 100 mM DTT; asterisk, Apg8–Apg3 conjugate; diamond, possible Myc–Apg8 dimer.
A ubiquitin-like system mediates protein lipidation (2000)
figf4

a, Apg7 and Apg3 are necessary for attachment of Apg8 to PE. Δapg cell lysates were subjected to SDS–PAGE containing 6 M urea, and immunoblotted with anti-Apg8 antibody. b, Autophagic activity in apg3 mutants. Wild-type, Δapg3 and apg3C243A cells were grown in rich medium (open bar) and subjected to nitrogen starvation for 4 h (filled bar). The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity. The apg3C243A mutant showed defective autophagy.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 1

Figure 1. PDS5A distribution in mouse spermatocytes.

A-N Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptotene (A-B), zygotene (C-D), early pachytene (E-F), mid pachytene (G-H), diplotene (I-L) and diakinesis (M-N). Arrows in (I-L) indicate the position of some chromocenters.

O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte.

Q, R Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte.

S, T Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte.

Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes. Insets included in (O, Q and S) correspond to a 300% magnification. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 2

Figure 2. PDS5B distribution in prophase I spermatocytes.

A-F Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages.

G-L Immunolabeling of PDS5B (green) and either REC8 (pseudocolored in blue, G-H) or SYCP3 (red) and TRF1 (pseudocolored in yellow, I-J) or ACA (pseudocolored in purple, K-L) onto a spread pachytene spermatocyte.

M-P Double immunolabeling of PDS5B (green) and SYCP3 (red) on diplotene (M-N) and diakinesis (O-P) spread nuclei.

Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. Insets included in C-P correspond to a 300% magnification. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 3

Figure 3. PDS5B distribution in condensed meiotic chromosomes.

A-F Double immunolabeling of PDS5B (green) and SYCP3 (red) in a metaphase I spermatocyte (A-B) and selected bivalents (C-F). The sex bivalent (XY) is indicated.

G-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.

Data information: Insets included in (I) correspond to a 300% magnification. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 4

Figure 4. Reduced AEs/LEs length in Pds5AB cKO spermatocytes.

A-J Double immunolabeling of either PDS5A (green, A-E) or PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.

K-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red).

N Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed T-test function (****, p < 0.0001).

Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 5

Figure 5. Synapsis progression in Pds5AB cKO spermatocytes.

A-H Double immunolabeling of SYCP1 (green) and SYCP3 (red) in wild-type (A-D) and Pds5AB cKO (E-H) spread spermatocytes at the indicated stages.

I-L Double immunolabeling of HORMAD2 (pseudocolored in white) and SYCP3 (red).

Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 6

Figure 6. Distribution of cohesin subunits and regulators is not altered in Pds5AB cKO spermatocytes.

A-O Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC3 (green, A-C), REC8 (green, D-F) and SMC1β (green, G-I) and cohesin cofactors Sororin (green, J-L) and WAPL (green, M-O) in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 7

Figure 7. PDS5 proteins affect meiotic recombination.

A-H Double immunolabeling of SYCP3 (red) and either γH2AX (pseudocolored in blue, A-D) or RAD51 (green, E-H) in Pds5AB cKO spread spermatocytes at the indicated stages.

I Scatter dot-plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype. Bars indicate mean and standard error and statistical significance was assessed with an unpaired two-tailed T-test function (****, p < 0.0001).

J-L Double immunolabeling of MLH1 (pseudocolored in white) and SYCP3 (red) in Pds5AB cKO spread spermatocytes.

Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 8

Figure 8. Altered distribution of telomeric proteins in Pds5AB cKO spermatocytes.

A-H Double immunolabeling of SYCP3 (red) and either TRF1 (green, A-D) or RAP1 (green, E-H) in Pds5AB cKO spread spermatocytes at the indicated stages.

I Selected zygotene-like and pachytene-like bivalents presenting altered TRF1 distribution as (from left to right) (i) telomere stretches, (ii) multiple telomere, (iii) distant telomeres and (iv) telomere-less. Arrowheads in (I) indicate the position of altered telomeres.

J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages (n=1646 telomeres in zygotene-like spermatocytes and 738 in pachytene-like ones).

K, L As in (I, J), respectively, but for RAP1 staining (n=1376 telomeres in zygotene-like spermatocytes and 328 in pachytene-like ones). Arrowheads in (K) indicate the position of altered telomeres.

M-P Double immunolabeling of SUN1 (green) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. 0, 1 and 2 in (N and O) indicate the number of chromosome ends labelled by SUN1 in a given bivalent.

Q As in (I), but for SUN1 staining. Arrowheads in (Q) indicate the position of altered telomeres.

R Selected zygotene-like and early pachytene-like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 (red) and SUN1 (green), and their respective percentage of appearance.

S Quantification of telomeres with a regular or altered disposition of SUN1 (n=715 telomeres in zygotene-like spermatocytes and 410 in pachytene-like ones).

Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. For further details on quantification of telomere aberrations see Appendix Table S1.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 9

Figure 9. Altered organization of telomeric DNA in Pds5AB cKO spermatocytes.

A-C Immunolabeling of SYCP3 (red) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages.

D 300% magnification of selected zygotene-like and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals.

E Quantification of telomeres with a regular or altered disposition of telomeric DNA in Pds5AB cKO spermatocytes at the indicated stages (n=1198 telomeres in zygotene-like spermatocytes and 574 in pachytene-like ones).

F-X Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks on (H, M, R and W) indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification in (I, J, N, O, S, T, and X), respectively, displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. Yellow arrowheads in (I) indicate telomeres displaying a distant telomere configuration observed both by telomere FISH and RAP1 immunolabeling. White arrowheads in (J, N, O, S and T) indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with (N) or without RAP1 signals (J, and T), and telomere stretches (O and S) without RAP1 signals. Pink arrowheads in (T) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal.

Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.
PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis (2020)
Figure 10

Figure 10. Altered attachment of telomeres to the NE in Pds5AB cKO spermatocytes.

A-P Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type (A-D, I-L) or Pds5AB cKO mice (E-H, M-P). The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in (F and N) indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.

Q-T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches (Q), multiple telomere (R), distant telomere (S) and telomere-less (T).

Expanded View figure legends
Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion (2018)
Figure 1

Figure 1. Loss of Vgll4 in tumor cells inhibits tumor growth in syngeneic mouse tumor models.

(A) VGLL4 protein expression levels in 6 murine tumor cell lines. Total cell lysates were analyzed by western blot.

(B) VGLL4 protein levels were reduced in Vgll4 depleted cells. Total cell lysates of Vgll4-knockdown LLC and MB49 cells were analyzed by western blot.

(C) Control or shVgll4 LLC cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=12 tumors for each group.

(D) Quantification of tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL/6 mice from (C). n=12 tumors for each group. *** p<0.001, two-tailed Student's t-test. The solid line represents the average weight ± SEM.

(E) Control or shVgll4 LLC cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group.

(F) Quantification of tumor weights 15 days after transplantation of control or shVgll4 LLC cells into nude mice from (E). n=8 tumors for each group. n.s. p>0.05, two-tailed Student's t-test. The solid line represents the average weight ± SEM.

(G) Control or shVgll4 MB49 cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=9 tumors for each group.

(H) Quantification of tumor weights 30 days after transplantation of control or shVgll4 MB49 cells into C57BL/6 mice. n=9 tumors for each group. *** p<0.001, two-tailed Student's t-test. The solid line represents the average weight ± SEM.

(I) Control or shVgll4 MB49 cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group.

(J) Quantification of tumor weights 15 days after transplantation of control or shVgll4 MB49 cells into nude mice from (I). n=8 tumors for each group. n.s. p>0.05; two-tailed Student's t-test. The solid line represents the average weight ± SEM.

(K) Immunofluorescent staining of CD3, CD8 and CD45 (red) counterstained with DAPI for DNA (blue) using control or shVgll4 LLC tumors from C57BL/6 mice. Statistical analysis of the percentage of CD3, CD8 and CD45 positive cells in the tumors is shown in the right panel respectively. n=3 tumors for each group. *** p<0.001, two-tailed Student's t-test, mean±SEM. Scale bar 100 μm.

(L) Quantification of tumor weights 30 days after transplantation of shVgll4 MB49 cells into C57BL/6 mice receiving anti-CD4 alone, anti-CD8 alone, or both anti-CD4 and anti-CD8 blocking antibodies. n=20 tumors for each group. The solid line represents the average weight ± SEM.