EMBO/soda-nel huggingface dataset:
Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo (2021)
Figure 2

Figure 2: A minority of CTL-tumor cell interactions results in target cell apoptosis in vitro

OVA+Eµ-myc B cells expressing a FRET-based reporter for caspase 3 activity were labeled with the calcium-sensitive dye Indo-1, incubated with OT-I CTLs and subjected to live imaging.

(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CTL (during the duration of the contacts). Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.

(C) The number of calcium fluxes in individual tumor cells is plotted for unconjugated tumors or conjugated tumors. Values were normalized to 100 min. Each dot represents one tumor cell. Bars represent mean values.

(D) The cumulated period of calcium signals was recorded for individual unconjugated or conjugated target cells and expressed as percentage of total time. Each dot represents one tumor cell. Bars represent mean values.

(E-F) Distinct tumor cell outcomes in response to CTL attack. Three categories were defined: no hit (no calcium response in the target cell), sublethal hit (transient calcium response and no caspase 3 activity), lethal hit (prolonged calcium response and induction of caspase 3 activity). (E) Pie chart representing the percentage of each category. (F) Time-lapse images (left) of a representative CTL-tumor cell conjugate in each of the three categories are shown together with the corresponding quantification (right) of calcium response and caspase 3 activity over time. T cells are delineated by white dashed lines. Scale bar, 10 µm. Data were pooled from three independent experiments. (**p<0.01, unpaired t‑test).
Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo (2021)
Figure 5

Figure 5: Extensive functional heterogeneity within tumor-infiltrating CTLs in vivo

(A-B) CTLs exhibit distinct capacities to trigger calcium responses in contacted tumor cells. Mice with established B cell lymphoma expressing OVA and the Twitch-2B calcium indicator were adoptively transferred with GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. (A) Left. Two-photon time-lapse image and corresponding scheme showing representative CTLs triggering either limited (upper images) or numerous (lower images) calcium signals in 5 contacted targets. Red arrows mark tumor cells with elevated calcium. Scale bar, 5 µm. Right. The periods of contact for the indicated targets are shown as white boxes and periods of calcium elevation are shown in magenta. (B) Total number of distinct calcium fluxes triggered by individual CTLs in all contacted tumor cells during an imaging period of one hour. For a rigorous comparison, only CTLs which activity could be monitored for at least 1 h were included in the analysis. Data are representative of 4 independent experiments.

(C-E) CTLs exhibit extensive differences in lytic activity in vivo. Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct-GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. The frequency of CTLs engaged in lytic activity (induction of apoptosis in at least one contacted tumor cell) is shown (C) together with the frequency of lytic contacts within all CTL-tumor cell contacts recorded (D). (E) Representative two-photon time-lapse images showing a representative CTL with high lytic capacity (killing 4 targets) and a CTL with no lytic activity for up to 4 hours. Blue arrows mark apoptotic tumor cells. Live and apoptotic tumors appear in grey and blue, respectively. CTLs appear in green. Scale bar, 10 µm. Representative of 3 independent experiments.
Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo (2021)
Figure 6

Figure 6: A minority of CAR T cell-tumors interactions results in target cell apoptosis in vitro

Eµ-myc B cells expressing a FRET-based reporter for caspase 3 activity were labeled with the calcium-sensitive dye Indo-1, incubated with GFP-expressing anti-CD19 CAR T cells and subjected to live imaging.

(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CAR T cell. Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.

(C) The number of calcium fluxes in individual tumor cells is plotted for unconjugated tumors or conjugated tumors. Values were normalized to 100 min. Each dot represents one tumor cell. Bars represent mean values. **p<0.01, unpaired t‑test.

(D) The cumulated period of calcium signals was recorded for individual unconjugated or conjugated target cells and expressed as percentage of total time. Each dot represents one tumor cell. Bars represent mean values. **p<0.01, unpaired t‑test.

(E) Distinct tumor cell outcomes in response to CAR T cell attack. Three categories were defined as previously described: no hit, sublethal hit, lethal hit. Pie chart representing the percentage of each category. Data were pooled from three independent experiments with 3 distinct image acquisitions in each experiment.
Histidine is selectively required for Myc-dependent dedifferentiation in the Drosophila CNS (2019)
Figure 1

Figure 1: Dietary histidine withdrawal selectively reduces nerfin-1 and NACT but not pros clonal growth

A) Schematic depicting wild type NBs (left panel) which express Mira (red), undergoing asymmetric division to generate a GMC, which divides only once to give rise to two postmitotic neurons. The cell fate determinant Pros (blue) is inherited by the GMC, where it translocates to the nucleus to promote differentiation. Postmitotic neurons express Pros (blue) and Nerfin-1 (green). Upon the loss of Nerfin-1 (nerfin-1-, middle panel), neurons undergo step-wise reversion, by increasing cellular growth, and switching on stem cell genes while maintaining the expression of neuronal specific markers such as Pros, before their complete reversion to NBs, giving rise to clones consisting a mixture of NBs and neurons. In pros- clones, GMCs fail to differentiate, and revert to NBs, giving rise to clones consisting mostly of Mira+ NBs. Schematic depicting type II wild type NB lineages (left) and dedifferentiation of INP to NBs upon Notch over-activation, which gives rise to clones consisting of mostly NBs (right).

B-E) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.

F-H) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his), quantified in (F) (n=28,22,7), scale bar = 50 μm

I-K) Representaitve pictures showing that adult nerfin-1 clonal growth was significantly reduced on -his diet compared to CDD (measured at day 0, and day 9) quantified in (K) (n= 27, 110, 136). Scale bar = 75μm. ***p<0.001

L-N) Representaitve pictures showing that larval nerfin-1 clonal growth was significantly reduced on 25% his diet compared to CDD (after 6 days), quantified in (N) (n=53,36). Scale bar = 50 μm.

O-Q) Representaitve pictures showing that adult pros clonal growth was not significantly altered on -his diet compared to CDD (after 7 days), quantified in (Q) (n = 12, 23,16). Scale bar = 75μm.

R-T) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction (25% his) compared to CDD (after 6 days), quantified in (T) (n=19,13). Scale bar = 50 μm.

U-W) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction (25% his) compared to CDD, quantified in (W) (n=27,19). Scale bar = 100 μm.

Data information: In all graphs, the key indicates that green bars represent a significant increase (p<0.05), red bars a significant decrease (p<0.05), and gray bars no significant change (p>0.05) in t tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC: fold change. See also Figures EV1 and EV4.
Histidine is selectively required for Myc-dependent dedifferentiation in the Drosophila CNS (2019)
Figure 2

Figure 2: Histidine metabolic perturbations affect nerfin-1 neuron to NB reversion

A) Schematic depicting the metabolic reaction between histidine and its downstream metabolites histamine and 3-methyl-L-histidine.

B) Concentration of histidine and 3-methyl-L-histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or -his diet (n=3,3). Both compounds are reduced to below detection levels (<0.3mM) upon dietary his depletion.

C) Dietary supplementation of 3-methyl-L-his for 4 days significantly increased adult nerfin-1 clonal growth, and rescued nerfin-1 growth inhibition due to his dietary withdrawal (n =18,14, 7,9).

D) Dietary supplementation of histamine does not significantly increase adult nerfin-1 clone size, but can rescue nerfin-1 clonal growth inhibition due to his dietary withdrawal (n =42,14,23,25).

E) Larval nerfin-1 clonal growth is significantly decreased upon overexpression of HDC RNAi (n=23,36).

F), Schematic depicting stepwise neurons to NBs reversion in nerfin-1 type I lineages where neurons express Elav, NBs express Dpn, and reverting neurons express both markers.

G) Hdc inhibition significantly reduced the percentage of Dpn+ NBs (n=16,17), and significantly increased the percentage of Elav+ neurons per clone (n=16,17).

H) HdcRi overexpression reduced cellular growth of nerfin-1 NBs (n=17,19) and neurons (n=36,19) measured as the ratio of nucleolus/nuclear volume.

Data information: In all graphs, the key indicates that green bars represent a significant increase (p<0.05), red bars a significant decrease (p<0.05), and gray bars no significant change (p>0.05) in t tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC: fold change. See also Figures EV2 and Figure EV3.
Histidine is selectively required for Myc-dependent dedifferentiation in the Drosophila CNS (2019)
Figure 3

Figure 3: histidine depletion slows down nerfin-1 dedifferentiation via decreasing cellular growth

A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm. Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav+ neurons (red) quantified in (D) (n = 16,17) and significantly fewer Mira+ NBs (blue), quantified in (E) (n = 12,22), without significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7).

F-G) Larval nerfin-1 clones consisted of significantly greater proportion of differentiated neurons (n=10,7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n=9,11).

H-P) Representative images of adult nerfin-1 (I-J', green ) and pros clones (K-L', green), scale bar = 5μm. In nerfin-1 clones, the size of the NBs (Ase+, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n=33,20). Cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was also significantly reduced, quantified in (M) (n=33,20). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n=9,8) and (O) (n=9,7). Cellular growth measured as the ratio of nucleolus /nuclear volume was significantly reduced in larval nerfin-1 NBs after 6 days his dietary reduction (25% his compared to CDD, n=14,6) quantified in (N). Panel (P) shows that the larval nerfin-1 neuron to NB reversion requires 15-fold increase in cellular volume (n=10) and pros GMC to NB reversion requires approximately 3.5-fold increase in cellular volume (n=31).

Data information: In all graphs, the key indicates that green bars represent a significant increase (p<0.05), red bars a significant decrease (p<0.05), and gray bars no significant change (p>0.05) in t tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC: fold change. See also Figures EV2.
Histidine is selectively required for Myc-dependent dedifferentiation in the Drosophila CNS (2019)
Figure 4

Figure 4: Histidine deprivation inhibits nerfin-1 clones via Myc and eIF4E

A-D) Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in C (n=25,31,21,16). pros NBs exhibit similar nucleolus /nuclear ratio (white arrows, nucleolus marked by Fib) compared to wild type and pros;TorDN NBs, quantified in D (n=10,18,15).

E-F) Representative pictures showing pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F), nucleolus labelled with Fib (red) [quantified in G (n=44,43)], clone size [quantified in H (n=41,34)], or the proportion of Dpn+ NBs and Elav+ neurons per clone [quantified in I (n=15,15,15,15)].

J-K) Representative pictures showing that nerfin-1 clones (green) generated in myc hypomorphic background (J-K) exhibited significantly reduced nucleolus/nuclear ratio, [nucleolus labelled with Fib (red)] and reduced clone size, quantified in L (n=34,27) and M (n= 106,75), and displayed an increased proportion of differentiated Elav+ neurons, and reduced proportion of Dpn+ NBs per clone, quantified in N (n=20,20,20,20). scale bar =10μm

O) Reducing Myc dosage with the hypomorphic Myc allele, mycP0, abolished the increase in nerfin-1 clone size mediated by 2x histidine dietary intake, 2x his (n=41,18,10,57).

P) Overexpression of myc restored the growth of nerfin-1 clones inhibited by histidine dietary reduction, 25% his (n=51,36,39,29).

Q, R) pros (Q) and nerfin-1 (R) clones are reduced upon Tor inhibition (n=22,63) compared to control (n=51,65), but their growth differentially depend on amino acid transporter Slif ( n=12,32) and downstream growth regulators eIF4E (n= 21,47) and S6K (n=39,37).

Data information: In all graphs, the key indicates that green bars represent a significant increase (p<0.05), red bars a significant decrease (p<0.05), and gray bars no significant change (p>0.05) in t tests with the relevant paired controls (black bar). In all graphs, error bars represent 1 standard error of the mean (SEM). FC: fold change. See also Figure EV3.

Expanded View Figure Legend:
BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites (2020)
Figure 1

Figure 1. BGL3 directly binds BARD1/PARP1.

A Proteins captured by BGL3 and RMRP antisense purifications. Proteins with an absolute log2 (fold change) > 1 and -log10 (p-value) > 1.3 (compared with control purification) were considered as proteins enriched by BGL3 specifically.

B Protein-protein interaction network of the DDR-related proteins captured by lncRNA-BGL3.

C&D RNA pull-down assay for BGL3-BARD1/PARP1 interactions before and after irradiation. Biotinylated in vitro-transcribed LncRNA-BGL3 sense or antisense transcripts were incubated with HEK-293T cell lysates with or without ionizing radiation (10 Gy) with 1 hr recovery for in vitro streptavidin RNA pull-down assays, followed by Western blots using the indicated antibodies.

E-H Schematic representation of BARD1 (E) or PARP1(G)-truncated mutants used in this study. HA-tagged full-length or deletion mutants of BARD1 (F) or PARP1(H) were transfected into 293T cells. 48 hr later, BGL3 RNA pull-down assay were performed, and arrowheads indicate the bands for HA-tagged full-length or deletion mutants of BARD1 (F).
BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites (2020)
Figure 2

Figure 2. BGL3 is recruited to DNA damage sites, and regulates genome stability.

A BGL3 is recruited to DNA damage sites. U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. The relocation kinetics of BGL3 to DSBs was monitored by RNA fluorescence in situ hybridization (FISH) in a time course as indicated. BGL3 intensities at the laser line were normalized into a numerical value using Nikon NIS-Elements AR software (version 4.40.00). Data are presented as mean ±SD of four biological replicates.

B Chromosome aberrations induced by BGL3 depletion. Wild-type or BGL3 depleted BJ-5ta cells were used in the metaphase spread analysis for spontaneous DNA breaks. Representative image (left panel) and the percentage of cells containing at least one DNA break (right panel). Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, ** P<0.01.

C MCF-7 cells were treated as indicated, and cell response to ionizing radiation were analyzed by colony formation assays (left panel). Data are presented as mean ± SEM of four independent experiments, analyzed by two-way analysis of variance (ANOVA), **P < 0.01. Knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-Actin (middle panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01. Knock-down efficiency of CtIP siRNA was examined by blotting (right panel).

D MCF-7 cells were transfected with the indicated siRNAs. Cell sensitivity to camptothecin (CPT), hydroxyurea (HU), etoposide (ETO), or ionizing radiation was determined by MTS assays. Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.

E BGL3 deficiency inhibits DNA damage repair. Quantification of γ-H2AX foci at indicated times after irradiation (2Gy) are presented. Data shown are results of three independent experiments (100 cells for each experiment), presented as mean ± SD, two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.
BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites (2020)
Figure 3

Figure 3. BGL3 regulates HR and BRCA1/BARD1 accumulation at DSBs.

A&B HEK293 cells integrated with HR or NHEJ reporter were transfected with the indicated siRNAs and subjected to the HR (A) or NHEJ assay (B) as described in Methods. Data presented as mean ±SD of four biological replicates, and positive cell percentages as compared with control group. Two-tailed Student's t test, **P < 0.01. NS: no significant difference.

C-F Wild-type (CTRL) or BGL3 knockdown U2OS cells were subjected to micro-irradiation; 1 hr later, cells were fixed and processed for immunostaining with indicated antibodies. Representative images of BRCA1 (C), BARD1 (D), RAD51 (E), and RPA70 (F) accumulation at sites of laser-induced DNA damage are shown.
BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites (2020)
Figure 4

Figure 4. BGL3 and BARD1 function together to regulate HR.

A-C Parental or BGL3-deficient U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. DDR factor recruitment were examined 2 hr later. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, **P < 0.01.

D U2OS cells were transfected with the indicated siRNAs, and 48 hr later, HR efficiency were determined using the reporter assay, as described in the Methods. Data shown are the average of three independent experiments. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference.

E U2OS cells were transfected with the indicated siRNA, and RAD51 accumulation at sites of laser-induced DNA damage were examined. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference. Knock-down efficiency of BARD1 siRNA was examined by blotting (bottom left panel), and knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-actin (bottom right panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01.
BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites (2020)
Figure 5

Figure 5. BGL3 enhances molecular interactions between BARD1 and its partners.

A BGL3 promotes BARD1-HP1γ interactions. 293T cells expressing the indicated siRNAs were irradiated (10 Gy), and BARD1-HP1γ interactions examined at the indicated time points.

B BGL3 promotes RAP80 binding to K63 ubiquitin chain in cells. 293T cells expressing the indicated siRNAs and plasmids were irradiated (10 Gy). Cell lysates were immunoprecipitated with HA beads and subjected to immunoblot with the indicated antibodies.

C BGL3 promotes BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions in vitro. GST-HP1γ, GST-RAD51, and FLAG-BARD1 were overexpressed and purified from cells as indicated in Methods. FLAG-BARD1 was divided into two equal parts, one part was used for BARD1-HP1γ interaction analysis (up panel), and another part was used for BARD1-RAD51 interaction analysis (middle panel). BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions with or without BGL3 were respectively detected by GST pull-down assays, here GST was used as a pull-down control.
BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites (2020)
Figure 6

Figure 6. BGL3 is recruited to DNA damage sites at an early time point by PARP1.

A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs. U2OS cells were treated with DMSO, PARP inhibitor (olaparib), ATM inhibitor (KU-55933), ATR inhibitor (VE-821) or caffeine. BGL3 recruitment to DNA damage sites was assessed by RNA-FISH assays.

B Quantification of the positive cells. For each group, 100 randomly selected cells were counted. Data are presented as mean ±SD of three biological replicates. Two-tailed Student's t test, **P < 0.01, N: no significant difference.

C PARP inhibitor mainly affected BGL3 recruitment at an early time point. U2OS cells were treated with DMSO or olaparib, and BGL3 recruitment was examined following laser micro-irradiation at indicated time point. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.

D U2OS cells were transfected with the indicated siRNAs, and BGL3 recruitment to DSBs were monitored at the indicated time point by RNA FISH. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.

E A model demonstrating how the BGL3 lncRNA mediates BRCA1/BARD1 complex retention at DNA damage sites.

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