EMBO/soda-nel huggingface dataset:
Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA (2020)
Figure 6

Figure 6. Genetic relationship of CST and BRCA2 in protecting genome stability.

Micronuclei (MN) formation in BRCA2- and STN1-deficient cells. Co-depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1. Representative DAPI staining images are shown. Arrows point to MN. Scale bars: 20 µm. Average percentages of nuclei containing MN from three independent experiments are presented. In each experiment, >300 nuclei were analyzed per sample. P values were calculated by one-way ANOVA analysis with post hoc Tukey. Error bars: SEM.

Anaphase bridges (arrows) in BRCA2- and STN1-deficient U2OS cells. Scale bar: 10 µm. Average percentages of anaphase bridges from three independent experiments are presented. P values were calculated by one-way ANOVA analysis with post hoc Tukey. Error bars: SEM.

γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells. Nuclei containing ≥ 5 foci were considered as positive γ-H2AX staining. Results were from three independent knockdown experiments. In each experiment, >80 cells were analyzed per sample. P values were calculated by one-way ANOVA analysis with post hoc Tukey. Error bars: SEM.

Co-depletion of STN1 and BRCA2 significantly impairs DNA replication. Scale bar: 50 µm. Results were from three independent knockdown experiments. In each experiment, >180 nuclei were analyzed per sample. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments. Error bars: SEM.

Co-depletion of STN1 and BRCA2 increases chromosome instabilities. U2OS cells with siBRCA2 and/or shSTN1 knockdown were treated with HU (2 mM, 3 h). Representative metaphase images show aberrant chromosomes (red arrows). Boxed areas are amplified and shown at the bottom of images. Scale bars: 20 µm. Two independent knockdown and chromosome spread experiments were performed. Results of the biological replicate are included in Appendix Fig. S6B. N, the number of metaphase spreads analyzed in each sample. P values were calculated by one-way ANOVA analysis with post hoc Tukey. *** P<0.001. Error bars: SEM.
Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA (2020)
Figure 7

Figure 7. CST deficiency reduces RAD51 binding to stalled forks.

SIRF analysis of RAD51 binding to forks. Representative RAD51 SIRF images at normal or stalled forks are shown. Scale bars: 10 µm. Two independent treatments were performed and scatter plot of RAD51-bound replication sites from one experiment is shown. Results from the biological replicate are included in Appendix Figure S7A. Mean values in each sample are listed at the top of the graph. N, number of cells analyzed in each sample in one experiment. P values were calculated by one-way ANOVA analysis with post hoc Tukey. *** P<0.001. Red lines indicate the mean values. Error bars: SEM.

RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi-resistant WT but not by ∆700N. Cells were treated with HU (2 mM, 16 h) and stained with anti-RAD51. Representative images are shown. Scale bars: 10 µm. Scatter plot of RAD51 foci number with and without HU treatment is shown. Mean values in each condition are listed at the top of the graph. P values were calculated by one-way ANOVA analysis with post hoc Tukey. *** P<0.001. N, the number of cells analyzed in each sample in one experiment. Red lines indicate the mean values. Error bars: SEM. Results from biological replicates are included in Appendix Figure S7B.

DNA fiber analysis of nascent-strand DNA degradation in U2OS cells with STN1 and RAD51 co-depletion. Three independent treatments were performed and results from one biological replicate are shown. Results from two additional replicates are shown in Appendix Figure S7C. N, the number of DNA fibers analyzed in each sample in one experiment. P values were calculated by one-way ANOVA analysis with post hoc Tukey. *** P<0.001. Red lines indicate the mean values. Error bars: SEM. Western blots showing protein knockdown in DNA fiber assays are included.

Model for CST in protecting reversal forks stability. Upon fork stalling, the stalled fork is reversed by SMARCAL1, ZRANB3, or others. CST binds to ssDNA (5' overhang) at reversed forks to directly block MRE11 access and may also recruit RAD51. Without CST, nascent-strand DNA is degraded by MRE11, leading to the generation of excessive ssDNA and genome instability.
An RDH-Plin2 axis modulates lipid droplet size by antagonizing Bmm lipase (2021)
Figure 2

Figure 2. Elevating the level of RDH/CG2064 in CSN RNAi increases lipid droplet size.

(A) The number of proteins changed in CSN2 or CSN7 RNAi revealed by quantitative proteomics. Red: the number of up-regulated proteins; green: the number of down-regulated proteins. Candidate proteins from the quantitative proteomics are listed along with protein fold changes in CSN2 or CSN7 RNAi.

(B, F and I) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (B) RDH/CG2064 RNAi suppresses the enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression (ppl>DGAT1) or high-fat diet conditions.

(C) Quantification of the lipid droplet size in (B). HFD: high-fat diet.

(D) In Drosophila S2 cells expressing RDH/CG2064-GFP, treatment with the proteasome inhibitor MG132 increases the RDH/CG2064-GFP protein level detected by anti-GFP antibody in a western blot.

(E) The RDH/CG2064-GFP protein level is increased in ppl>DGAT1 larvae with CSN2 RNAi, as detected by anti-GFP antibody in a western blot.

(F) RDH/CG2064 overexpression increases lipid droplet size in ppl>DGAT1 larvae. (G) Quantification of the lipid droplet size in (F).

(H) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein.

(I) RDH/CG2064 and CG2065 deletion does not affect lipid droplet size under normal or high-fat diet conditions.

In (C), (G) and (H), data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.
An RDH-Plin2 axis modulates lipid droplet size by antagonizing Bmm lipase (2021)
Figure 3

Figure 3. The membrane protein RDH/CG2064 regulates lipid droplet size independent of its enzymatic activity.

(A) The protein domain structure of RDH/CG2064. Grey box: predicted transmembrane region; Pink box: short-chain dehydrogenase domain. The key enzymatic motif (YXXXK) and active site (NXXG) are shown.

(B) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Overexpressing wild-type or site-mutated RDH/CG2064 increases the lipid droplet size in larvae with DGAT1 overexpression (ppl>DGAT1). Scale bar represents 25 µm.

(C) Quantification of the lipid droplet size in (B).

(D) Immunofluorescent staining of RDH/CG2064-GFP with anti-GFP antibody in 3rd instar larval fat body. DAPI (blue) stains nuclei. RDH/CG2064 is enriched in the peri-nuclear region. Scale bar represents 25 µm.

(E) Immunofluorescence imaging of RDH/CG2064-GFP, detected with anti-GFP antibody, and the ER protein marker RFP-KDEL in 3rd instar larval fat body. RDH/CG2064 co-localizes with RFP-KDEL. Scale bar represents 10 µm.

(F) Whole lysates and cytosolic and membranous fractions were prepared from fat body samples of 3rd instar larvae expressing an RDH/CG2064-GFP knockin reporter. RDH/CG2064-GFP protein was detected by anti-GFP antibody in a western blot. RDH/CG2064-GFP is mainly present in the membranous fraction.
An RDH-Plin2 axis modulates lipid droplet size by antagonizing Bmm lipase (2021)
Figure 4

Figure 4. RDH/CG2064 interacts with Plin2.

(A-B) Lysates of 3rd instar larval fat body samples of different genetic backgrounds were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin2, but not Plin1 or Bmm, is specifically coimmunoprecipitated with RDH/CG2064 (A). Plin2 was coimmunoprecipitated with RDH/CG2064 in samples from larvae overexpressing DGAT1 (B).

(C) Schematic diagram of full-length RDH/CG2064 and TM domain-deleted RDH/CG2064 (∆TM). Lysates of Drosophila S2 cells expressing the different proteins were immunoprecipitated with anti-GFP antibody and detected by anti-GFP or anti-Flag antibody in a western blot. Both full-length and ∆TM RDH/CG2064 were coimmunoprecipitated with Plin2.

(D) Schematic diagram of Plin1, Plin2 and the Plin2-Plin1-C chimeric protein. Lysates of Drosophila S2 cells expressing the different proteins were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Neither Plin1 nor Plin2-Plin1-C chimeric protein coimmunoprecipitated with RDH/CG2064.

(E) Lysates of Drosophila S2 cells expressing full-length Plin1 or Plin1 with a C-terminal deletion (Plin1ΔC) were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin1ΔC coimmunoprecipitated with RDH/CG2064.

(F) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Under high-fat conditions, RDH/CG2064 overexpression increases the lipid droplet size, and Plin2 RNAi suppresses the enlargement induced by RDH/CG2064 overexpression. Scale bar represents 25 µm.
An RDH-Plin2 axis modulates lipid droplet size by antagonizing Bmm lipase (2021)
Figure 5

Figure 5. The CSN-RDH/CG2064 axis changes the sub-cellular localization of Plin2.

(A, B, C, E and F) Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm. (A) CSN2 RNAi or RDH/CG2064 overexpression reduces the peripheral localization of Plin2-GFP and increases the localization of Plin2-GFP on small peri-nuclear lipid droplets (boxed). DAPI (blue) stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets.

(B) The peripheral localization of Plin2-GFP is greatly reduced in fat body cells from larvae with CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells.

(C) In fat body cells from ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, the localization of Plin2-GFP is greatly reduced at the periphery and is dramatically increased on large medial lipid droplets. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets.

(D) Fluorescence intensity line plots of the images in (C). GFP intensity along the line across a lipid droplet was measured by ImageJ. In ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, Plin2-GFP localization on large medial lipid droplets is represented by two peaks. More than 30 lipid droplets were measured for each genotype. The plot shown is a typical image curve for the indicated genotype.

(E) The peripheral localization of Plin2-GFP is greatly reduced in fat body cells from ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells.

(F) The localization of Plin2-GFP is greatly reduced in peripheral lipid droplets and increased in medial lipid droplets in CSN2 RNAi larvae fed on a 30% high-fat diet. DAPI (blue) stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets.

(G) The peripheral lipid droplets in larval fat body cells from different genetic backgrounds were stained by BODIPY (green). CSN3 RNAi or RDH/CG2064 overexpression reduces the number of peripheral lipid droplets in both wild-type and ppl>DGAT1 larvae.
An RDH-Plin2 axis modulates lipid droplet size by antagonizing Bmm lipase (2021)
Figure 6

Figure 6. The CSN-RDH/CG2064-Plin2 axis affects the level and lipid droplet localization of Bmm lipase.

(A, D, E and F) Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (A) Knocking down CSN2 or overexpressing RDH/CG2064 in animals with DGAT1 overexpression (ppl>DGAT1) decreases the level and lipid droplet localization of Bmm-GFP.

(B) The GFP intensity along the line across a lipid droplet in (A) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, is clearly visible in fat cells from ppl>DGAT1 larvae, but it is lost in fat cells from ppl>DGAT1 larvae with CSN2 RNAi or overexpression of RDH/CG2064. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.

(C) The level of Bmm-GFP in 3rd instar larval fat bodies of different genotypes was detected by anti-GFP antibody in a western blot. In ppl>DGAT1 larvae, CSN2 RNAi or overexpression of RDH/CG2064 decreases the level of Bmm.

(D) CSN2 RNAi decreases the level and lipid droplet localization of Bmm-GFP in fat cells from larvae fed on a 30% high-fat diet.

(E) The level and lipid droplet localization of Bmm-GFP are similar in fat cells from larvae overexpressing DGAT1 or mRFP (control).

(F) Overexpression of Plin2 decreases the level and lipid droplet localization of Bmm-GFP in fat cells from larvae with DGAT1 overexpression.

(G) GFP intensity along the line across a lipid droplet from (F) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, was evident in fat cells from ppl>DGAT1 larvae, but is lost in ppl>DGAT1 larvae with overexpression of Plin2. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.

(H) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. bmm RNAi increases the lipid droplet size in fat cells from larvae with DGAT1 overexpression. Scale bar represents 25 µm.

(I) Quantification of the lipid droplet size in (H). Data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body. At least 30 cells were analyzed from each fat body. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant.

(J) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. Error bars represent ± SEM. **: P < 0.01; ***: P < 0.001.
An RDH-Plin2 axis modulates lipid droplet size by antagonizing Bmm lipase (2021)
Figure 7

Figure 7. The CSN-RDH-Plin2 axis functions under normal conditions and is required for prolonged starvation.

(A) S2 cells expressing RDH/CH2064-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of RDH/CH2064-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control.

(B) Quantification of the relative RDH/CH2064-GFP protein levels in (A). Error bars represent ± SEM.

(C) S2 cells expressing Bmm-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of Bmm-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control.

(D) Quantification of the relative Bmm-GFP protein levels in (C). Error bars represent ± SEM.

(E) The RDH/CG2064-GFP protein level in 3rd instar larval fat cells is increased in CSN2 or CSN7 RNAi, as detected by anti-GFP antibody in a western blot. Tubulin was used as loading control.

(F) Oil red staining of 3rd instar larval oenocytes in different genetic backgrounds under fed or starvation conditions.

(G) The survival rate of control and CSN2 RNAi larvae at various fasting time points. Error bars represent ± SEM. *: P < 0.05; **: P < 0.01; ***: P < 0.001; NS: non-significant.

(H) Schematic model showing how the CSN-RDH/CG2064-Plin2 axis inhibits Bmm and promotes the formation of large lipid droplets under high-fat/increased TAG conditions.

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Urinary proteome profiling for stratifying patients with familial Parkinson's disease (2020)
Figure 4

Figure 4. Pathogenic LRRK2-dependent lysosomal dysregulation is reflected in the urinary proteome and distinct from pathogenic GBA-induced alterations

A) Proteins that differ significantly differ between pathogenic LRRK2 carriers and controls using an ANCOVA analysis with sex, age, PD status and GBA status as confounders and an FDR of 5%.

B) Mean fold-changes for each of the 33 proteins that were LRRK2-dependently regulated in both cohorts using a pairwise t-test comparing the four subgroups (HC, NMC, iPD and LRRK2 PD).

C) Correlation of mean fold-changes of the proteins changing LRRK2-dependently in the Columbia and LCC cohorts. Only proteins identified in both cohorts are shown (n=237). The colors match to the GO terms shown in (D). All proteins associated with the GO-term 'glycosphingolipid metabolic process' labeled in purple are also associated with the lysosomal-related GO-terms. Proteins overlapping between the two cohorts are labeled with their name.

D) Fisher exact test was performed to identify significantly enriched GO-terms in the LRRK2-dependently regulated proteins in urine. All GO-terms that were significant in either cohort are displayed (FDR < 5%).

E) Proteins that differ significantly between pathogenic GBA carriers and controls or pathogenic LRRK2 carriers and controls using an ANCOVA analysis with sex, age, PD status and LRRK2/GBA status as confounders and an FDR of 5%. In total, 237 proteins were differentially expressed in these two comparisons with 166 and 74 regulated proteins in the LRRK2 carriers and GBA carriers, respectively, only three of which were common between both mutations.

F) Correlation of mean fold-changes of the proteins changing LRRK2-dependently and GBA-dependently (n=237) in the Columbia cohort. Carriers of pathogenic variants in both GBA and LRRK2 were excluded from the analysis. The colors match to the GO terms shown in (D).
Urinary proteome profiling for stratifying patients with familial Parkinson's disease (2020)
Figure 5

Figure 5. Correlations with clinical parameters

A) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the MoCA total score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included in the analysis. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.

B) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the UPDRS-III score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 1

Fig. 1. PTPN2-deletion in T cells increases tumour immunosurevillance.

a-b) 12 month old Ptpn2fl/fl;p53+/- and Lck-Cre;Ptpn2fl/f;p53+/- mice were assessed for a) disease and tumour incidence. b) Lymphocyte subsets from 12 month old Ptpn2fl/fl;p53+/- and Lck-Cre;Ptpn2fl/f;p53+/- mice were analyzed by flow cytometry. Significance in (a) was determined using two-sided Fisher's exact test.

c-f) AT-3-OVA breast cancer cells were injected into the fourth inguinal mammary fat pads of female Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl mice and (c) tumour growth monitored over 26 days. d) At day 26 (d26) the numbers of activated tumour infiltrating lymphocytes (TILs) were determined. e) The proportion of IFNγ+ versus IFNγ+TNF+ d26 TILs was determined by flow cytometry. f) d26 TILS were incubated with AT-3-OVA tumour cells isolated from tumour bearing C57BL/6 mice and the proportion of IFNγ+ T cells was determined.

Data information: Representative flow cytometry profiles and results (means ± SEM) from at least two independent experiments are shown. In (c) significance was determined using 2-way ANOVA Test and in (d-f) significance determined using 2-tailed Mann-Whitney U Test. **p<0.01, ***p<0.001, ****p<0.0001.
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 2

Figure 2. PTPN2-deletion enhances CD8+ T cell-mediated immunosurveillance.

a-d) AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. Tumour-bearing Ly5.1+ mice were monitored for a) tumour growth over 21 days and d) for survival over 86 days. b) After 21 days TILs were processed for flow cytometry and donor T cell numbers (Ly5.1-Ly5.2+) determined. c) After 9 days the proportion of Ly5.2+IFNγ+TNF+ versus Ly5.2+GrzB+ TILs was determined.

e) Gene expression in tumours from mice treated with Ly5.2+;OT-1;Ptpn2fl/fl T cells 21 days post-adoptive transfer versus those re-emerging in mice treated with Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl T cells.

f-i) B16.F10-OVA melanoma cells (1x105) were engrafted onto the abraded skin in the flanks of Ly5.1+ mice. 24 h after tumour cell engraftment naïve CD8+CD62LhiCD44lo lymph node T cells from Ly5.2+ OT-1;Ptpn2fl/fl versus Ly5.2+ OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred and tumour incidence monitored. g) Epidermal lymphocytes from tumour-free mice were stained for CD69hiCD103hi and donor-derived (Ly5.2+vα2+) tissue resident memory T cells (TRMs) determined by flow cytometry. h) Tumour sizes in B16.F10-OVA melanoma bearing mice were determined between day 21 and 23. i) TILs were assessed for Ly5.2+;OT-1;Ptpn2fl/fl and Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl donor T cell numbers by flow cytometry.

Data information: Representative flow cytometry profiles and results (means ± SEM) from at least two independent experiments are shown. In (a) significance was determined using 2-way ANOVA Test and in (b, c, e, g, h, i) significance determined using 2-tailed Mann-Whitney U Test. In (d, f) significance was determined using Log-rank (Mantel-Cox) test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 3

Figure 3. PTPN2-deletion enhances the LCK-dependent activation of CAR-T cells.

a) CD8+ HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes were stained for intracellular p(Y418)-SFK and p(Y418)-SFK MFIs were determined by flow cytometry.

b) HER-2-specific Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with HER-2 expressing 24JK sarcoma cells (24JK-HER-2) or HER-2 negative 24JK sarcoma cells and CD44, CD25, PD-1 and LAG-3 MFIs on CD8+ CAR-T cells were determined by flow cytometry.

c) Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 24JK-HER-2 or 24JK sarcoma cells and the proportion of CD8+IFNγ+ versus CD8+IFNγ+TNF+ CAR-T cells determined by flow cytometry.

d) Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 5 μM CTV-labelled (CTVbright) 24JK-HER-2 and 0.5 μM CTV-labelled (CTVdim) 24JK sarcoma cells. Antigen-specific target cell lysis was monitored for the depletion of CTVbright 24JK-HER-2 cells by flow cytometry.

e) HER-2-specific Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with plate-bound α-CD3 and CD25 MFIs on CD8+CD44hiCD62Llo versus CD8+CD44hiCD62Lhi CAR-T cells determined by flow cytometry.

Data information: Representative histograms and results (means ± SEM) from at least two independent experiments are shown. In (a-c, e) significance determined using 2-tailed Mann-Whitney U Test. In (d) significance was determined using 2-way ANOVA Test. *p<0.05, **p<0.01, ***p<0.001.