EMBO/sd-character-level-ner huggingface dataset:
Mitochondrial fission is required for neuronal cell death and mediated by Drp1 and Mfn1.Figure 7(A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25-35 versus Aβ35-25(10 μM, 6 h), as indicated. Scale bar, 20 μm. (B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining. (C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (*,** and *** significance at P0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments). (D) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4). (E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3). (F) Mitochondrial fission by Aβ35-25 or Aβ25-35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (††significance at P0.01 compared to pcDNA3 transfected, Aβ35−25 treated control; * and ** significance at P0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ25-35 treated neurons; n=3).
null embryos exhibit localised integrated stress response (A-L) mRNA in situ hybridization was performed at E9.Figure 5mRNA in situ hybridization was performed at E9.5 to detect Chac1 in (A-a6) wildtype and (B-b6) Slc7a5-null littermate embryos (n=3 for each condition); Chac1 expression at E10 in emerging limb buds assessed in (C) wildtype and (D) Slc7a5-null littermate embryos (n=2 for each condition) Scale bars 200 µm for whole embryo images and 100 µm for sections. mRNA in situ hybridization was performed at E9.5 to detect Trib3 mRNA detected in (E-e6) wildtype and (F-f6) Slc7a5-null littermate embryos (n=3 for each condition) and in emerging limb buds at E10.5 assessed in (G) wildtype and (H) Slc7a5-null littermate embryos (n=2 for each condition). Scale bars 200 µm for whole embryo images and 100 µm for sections. mRNA in situ hybridization was performed at E9.5 Chac1 and Trib3 transcripts were also detected in wildtype E9.5 embryos after >3 days in situ hybridisation reaction (n=3 and n=6 respectively). Lateral view (I and K), optic vesicles (white arrowheads), branchial arches (ba) and otic vesicles (ov). Dorsal view (J and L), neural tube (nt) and limb buds (white arrowheads). Scale bars 200 µm for whole embryo images and 100 µm for sections. (M) Schematic indicating regions (purple) in which high levels of Slc7a5 and ISR genes Trib3 and Chac1 are detected in wildtype embryos.
a) Anti-VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b–e.figf4(a) Anti-VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b-e. The cells were stimulated with 10% serum for 0, 1, 5, 15, 30 and 60 min, as indicated at the bottom of the figure. Equal amounts of total protein were loaded into each lane. (b-e) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation.
A.Figure 8A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast. B. Western blot detection of the myc-tagged sTgAtg3 protein (arrowed) in parasite lysates corresponding to the induction conditions described in A. Anti-SAG1 antibody was used as a loading control. C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control.
Figure 5A) VPS34 CI was phosphorylated in vitro by Ulk1 1-427. Coexpressed VPS34, VPS15, Beclin1 and ATG14-ZZ were immunopurified and incubated with Ulk1 1-427 in vitro. The samples were then separated by SDS PAGE and transferred onto PVDF membrane. In vitro phosphorylated proteins were visualised by autoradiography before VPS15 was visualised by immunoblotting, revealing that VPS15 and ATG14-ZZ were phosphorylated in this assay. B) All subunits for VPS34 CI containing ATG14-ZZ or CII containing UVRAG-ZZ were phosphorylated in vitro by WT or KI Ulk1 1-427 as indicated in A. Autoradiogram shows VPS15, ATG14-ZZ, UVRAG-ZZ and Ulk1 1-427 are phosphorylated. Phosphorylation occurred in reaction mixtures containing Ulk1 1-427 WT. The Coomassie-stained gel shows VPS15 levels in assay. C) WT VPS15 or VPS15 with 6SA, 2SA or individual phosphoacceptors mutated to alanine were incubated in vitro with Ulk1 1-427 and analysed as in B. The Coomassie-stained gel shows VPS15 levels in assay. Mean +/-SEM, n=3. D) VPS34 CI components expressed with Ulk1 1-427 WT or KI as indicated. After starvation for 1 hr in the presence or absence of MRT68921 (ULK inhibitor), cells were lysed and VPS34 CI coimmunoprecipitated via ATG14-ZZ. VPS15 S861 phosphorylation was increased in the presence of active Ulk1. VPS15 pS861/total VPS15 was quantified, mean +/- SEM, n=3. E) HEK293A were transfected with VPS34 CII components and Ulk1 1-427 WT or KI and treated as indicated before VPS34 CII co-immunopurification via UVRAG-ZZ. Samples were analysed by Western blot as in D, revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII. Quantifications show mean of 4 independent experiments +/-SEM, ***p<0.001 [one-tailed ANOVA]. F) Representative loading control samples from experiments in D and E were analysed by immunoblot. UVRAG and ATG14, each visualised using the affinity of the ZZ tag for 2° antibodies alone, underwent electrophoretic mobility shifts in the presence of active Ulk1 only, indicative of direct phosphorylation in cells.
Figure 4A FRAP images of CAF-1 bodies to indicate the internal diffusion property. GFP-tagged CHAF1A was overexpressed in HEK293T cells. Twenty-four hours later, live cells were proceeded to time series imaging. One of the CAF-1 bodies was bleached with intense laser pulse. Images of live cell were captured every 4 seconds. One of the unbleached CAF-1 bodies was used as control. Data represented eight time points. At least three cells of each sample were treated with intense laser pulse to obtain bleached CAF-1 bodies. B Representative images of two CAF-1 bodies fusing into one. C Representative images of one CAF-1 body splitting into two. D Live cells were imaged every 2 seconds in time series. 1,6-hexanediol was added into the cell incubation well carefully at time point of 5th second. Images were captured for 3 minutes. E GFP-tagged CHAF1A-N-NLS and CHAF1A-C were imaged with RFP-tagged CHAF1A respectively. N: CHAF1A N-terminal. NLS: nuclear localization signal of CHAF1A, which was presented within C-terminal. C: CHAF1A C-terminal. F Upper panel showed WT-CHAF1A bodies. Lower panel showed dLLPS-CHAF1A which was CHAF1A-Q34A-I116A-K395D-R402D-K409D. G-I GFP-tagged CHAF1A-IDR and CHAF1A-mIDR were purified in vitro. Ten micromole of each protein was incubated with gradient droplet formation buffer. The droplets of CHAF1A-IDR appeared in NaCl concentration of 125 mM or lower (G). No CHAF1A-mIDR droplet was detected within any NaCl concentration (I). The volumes of CHAF1A-IDR droplets increased with the increase of protein concentration (H). Left subpanels within each panel represented images of protein droplets in different buffers. Right subpanels within each panel represented statistical analysis results of droplets areas and numbers.
inhibition on lung metastasis(A) Schematic depiction of the AMD treatment protocol before sacrifice and analysis of the lungs.Figure 6(A) Schematic depiction of the AMD treatment protocol before sacrifice and analysis of the lungs. Intra peritoneal (ip) injection of 5 mg/kg/day AMD into NeuNT mice (daily for 2 weeks, week 27 - 29). (B) IF tissue staining identifying blood vessel invasions (BVI), surrounded by an EC monolayer (arrow) and expanded EC (asterisk), scale bar, 50µm. (C) Quantification of BVI with normal (EC monolayer) and aberrant (expanded EC) phenotype expressed as ratio. N = 5 mice (PBS), n = 3 sections, n = 15 BVI, n = 7 PM (parenchymal metastasis); N = 5 mice (AMD), n = 3 sections, n = 12 BVI, n = 2 PM, ****, p < 0.0001, Fisher's exact test. ( (D) Schematic depiction of the AMD treatment protocol before sacrifice and analysis of the lungs. Tail vein (iv) tumor cell engrafted mice (WT/shC, WT/shTNC) were ip injected (daily for one week, week 4-5) with AMD 7.5 mg/kg/day. Metastasis assessment in the 5 weeks iv orthotopic grafting model (D-G). (E) Ratio of mice with and without lung metastasis determined by H&E tissue staining. N = 8 mice (shC, PBS), N = 7 mice (shC, AMD), N = 7 mice (shTNC, PBS), N = 7 mice (shTNC, AMD). p < 0,0001, Chi square test. Fisher's exact test was used to compare the conditions pairwise *, p < 0,05, *** p < 0,001. p = 0,0109 (shC/PBS versus shTNC/PBS), p = 0.0001 (shC/AMD versus shTNC/AMD). (F, G) Ratio of BVI and PM in lungs from shC (F) and shTNC (G) engrafted mice. N = 8 mice (shC, PBS), n = 4 BVI, n = 13 PM; N = 7 mice (shC, AMD), n = 15 BVI, n = 21 PM; N = 7 mice (shTNC, PBS), n = 8 BVI, n = 9 PM; N = 7 mice (shTNC, AMD). n = 5 BVI, n = 1 PM, *** p < 0,001, **** p < 0,0001. p = 0.0009 (shC/PBS versus shC/AMD) and p = 0.0001 (shTNC/PBS versus shTNC/AMD), Fisher's exact test. (H) Schematic depiction of the AMD treatment protocol before sacrifice and analysis of the lungs. Mice with mammary gland (MG) engrafted tumor cells (WT/shC) were daily injected peritumorally (pt) for 5 weeks with 5 mg/kg/day (week 2-7). Metastasis assessment in the 7 weeks orthotopic grafting model (H-L). (I-L) qRT-PCR assessment of Neu (I), Cd8a (J), Ifnγ (K) and Gzmb (L) by qRT-PCR. PBS (N = 16) and AMD (N = 19) treated mice, *, p = 0.0364, Mann whitney test (I), **, p = 0.0086 unpaired t-test (J), **, p = 0.0055, unpaired t-test (K), ***, p = 0.0003 unpaired t-test (L), respectively. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum.
Subcellular localization of the Dfmrp variants in sLNv neurons.Figure 7A-D Subcellular localization of transgenically expressed dfmr1wt, dfmr1ΔCt, dfmr1wt+NLS, and dfmr1ΔCt+NLS proteins in LNv neurons. DfmrpΔCt+NLS shows nuclear localization (A), colocalizing with the genetically encoded nuclear marker RedStinger, while Dfmrpwt, DfmrpΔCt, and Dfmrpwt+NLS are predominantly cytoplasmic (B-D). These results suggest that an intact C-terminus can mediate the nuclear export of the Dfmrp protein. Scale bar represents 5 μm.
Figure 5A, B PPL-C99-TMD (A) and PPL-C99FL (B) were in vitro translated in RRL from mRNAs lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and ε-TDBA-Lys-tRNA analogue. Reactions were irradiated with the UV light to induce photo-cross-linking, RNCs isolated and adducts immunoprecipitated with mouse anti-SRP54, mouse anti-SGTA or a control antibody as described in Materials and Methods. Samples were resolved by SDS-PAGE and results visualised by phosphorimaging. C-terminal 40 amino acid residues located in the ribosomal exit tunnel are indicated. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively. Arrows indicate unmodified translation products. C PPL-C99FL variants lacking a stop codon and carrying the indicated Lys to Arg substitutions were in vitro translated in the presence of 2 μM HisTrx-SGTA. Reactions were processed using chicken anti-SGTA antibody for immunoprecipitation of SGTA adducts. D Cross-linking adducts between ribosome-stalled PPL-C99FL lysine mutants and recombinant HisTrx-SGTA shown in panel (C) were quantified, any differences in translation efficiency accounted for and cross-linking efficiency expressed relative to the wild-type (WT) protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.
Global splicing effects upon Matrin3 knockdownWestern blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel).Figure 3A Western blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel). Lanes 1-4 contain a twofold dilution of the control C2 sample (lane 1-12.5%, lane 2-25%, lane 3-50%, lane 4-100%). Lanes 4-7 contain equal amount of proteins, as can be confirmed by the anti‐actin (lower panel), of control sample (lane 4), double‐PTB and nPTB siRNA‐treated sample (lane 5), Matrin3 siRNA‐treated sample (lane 6) and triple knockdown of Matrin3, PTB and nPTB (lane 7). Lane 7 is from the same gel and exposure, but some lanes present in the original gel were cropped for clarity, and a black line to indicate cropping was placed. B Pie chart of the different categories of Matrin3‐regulated alternative splicing events (ASEs), 330 cassette exons (50%), 116 alternative promoter usage (17%), 78 terminal exons (11%), 32 alternative 5′ (5%) and 34 3′ (5%) splice site and 18 intron retention (IR; 3%) events. C Gene ontology (GO) analysis of the Matrin3‐regulated cassette exons. The x‐axis represents the P‐value in a logarithmic scale as shown. D Pie chart of the activated and repressed cassette exons by Matrin3. E Venn diagram of the overlap between the PTB‐ (blue) and Matrin3‐ (yellow) regulated cassette exons, showing the 813 events regulated only by PTB, 270 by Matrin3 only and the 61 that overlap. F RT-PCR validation of Matrin3‐regulated alternative spicing events in the ST7, ACSL3, PLEKHA3, TCF12, VWA5A, PTBP2, PTBP3, C3orf17, ZMYND8, VEZT, PIGX and DMD genes. In each case, triplicates for each condition (C-control, M-Matrin3, PTB/nPTB and Matrin3/PTB/nPTB siRNA transfection samples) were analysed and exon inclusion (EI) percentage is shown beneath the corresponding lane, along with the standard deviation (s.d.).
Figure 7A, Peroxiredoxin levels in peroxiredoxin 1 (PRDX1 KO) and peroxiredoxin 2 (PRDX2 KO) knockout cells. The upper subpanel depictes the guides used to generated the KO cell lines. Lysates of the indicated cell lines grown in glucose-containing medium were analysed by immunoblot B. Peroxiredoxin levels in peroxiredoxin 1 (PRDX1 KO) and peroxiredoxin 2 (PRDX2 KO) knockout cells. quantitative proteomics (n = 4, technical replicates) The right subpanel of B lists the identified proteins belonging to cellular antioxidative systems which are not altered for PRDX1 KO or PRDX2 KO, respectively. In both quantitative proteomics experiments 4403 proteins were detected in total. C. HyPer7 steady state ratios of the indicated cell lines. HyPer7 probes were targeted to the indicated compartments of the respective cell lines. Cells were grown in glucose-containing medium. In the boxplot, the central band is the median. The lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 times the inter-quartile range, whereas the lower whisker extends from the hinge to the smallest value no further than 1.5 times the inter-quartile range. D. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1 KO; blue, PRDX2 KO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001 E. Response of HyPer7 probes targeted to indicated compartments to treatment with 25 µM of exogenous H2O2. Indicated cell lines (black, wild type; red, PRDX1 KO; blue, PRDX2 KO) were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001
Figure 5A-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. EB1::GFP expression was driven under Gal44-77. In the control ddaC neurons (A, D), EB1 comets move predominantly in retrograde direction. EB1 comets were largely diminished in msps810/mspsP mutant neurons (B). Interestingly, in msps810/mspsP18 mutants (C), tacc RNAi (E) and tacc59/tacc74 mutant (F), increased anterograde comets were detected in ddaC dendrites. G-J Quantitative analysis of the percentages of anterograde EB1::GFP comets, the track length, the number (per 30 µm) and the velocity of the comets in each neuron imaged.
Figure 6(a) WT and Smc5cKO cortices at E14.5 stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). Insets showing zoomed in view of 2 representative cells. Scale bar: 25 μm. (B and C) Quantification of the number of (B) Centrin foci and (C) γ-Tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Smc5cKO n = 134 cells, N = 3 embryos. #, chi-square test. WT data are from Figures EV1F and EV1G and shown alongside for comparison. (D) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the VZ and SVZ. Scale bar = 100 μm. (E) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Smc5cKO N = 5; two-tailed Mann-Whitney t-test. WT data are from Figure 1H and shown alongside for comparison. (F Quantification of the number of (F) apoptotic cells (CC3+) within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison. G) Quantification of the number of (G) cells with TP53 activation within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison. (H) Representative whole mount images of WT, Smc5cKO, Smc5cKO;Usp28cKO, Smc5cKO;Trp53-/- brains at P21. Scale bar = 0.2 cm. (I) Telencephalon area of P21 brains of the indicated genotypes. WT littermates N = 6, Smc5cKO N = 5, Smc5cKO;Usp28cKO N = 4, Smc5cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis. (J) Model for two TP53-dependent pathways underlying primary microcephaly. In the first model, NPCs with mutations in centrosome genes take longer to assemble their spindle in mitosis. This delay triggers the activation of the mitotic surveillance pathway, which activates TP53 through 53BP1 and USP28. By contrast, in microcephaly caused by mutations in genes required for DNA repair or genome stability, centrosome function and mitotic duration are unaffected. Instead, accumulated DNA lesions trigger DNA damage signaling in NPCs to activate TP53. In both models, increased TP53 activity leads to apoptosis, thus depleting the NPC pool and decreasing the final number of neurons.
Figure 7A. Survival assay of Myc/Bcl2+GMP- and DN2-derived leukemia cells that were isolated from diseased mice and treated for 24 h with 0.15 μM daunorubicine or 1 μM doxorubicine in liquid culture. Cell death was measured by FACS of 7-AAD+cells and was normalized to the solvent controls. The graphs show the mean+SEM of three different DN2- and five GMP-leukemias. **=p≤0.01. B. Experimental procedure of the shRNA library in vivo screen. Blasts from leukemic mice that were transplanted with Myc/Bcl2+ DN2-cells were infected with a retroviral shRNA library consisting of ~300 GFP- or YFP-tagged shRNAs targeting a total of 142 different genes. Cells were harvested 72 h post infection, the GFP+ and YFP+ fractions were flow-sorted and 6104cells were transplanted into secondary recipients. The residual cells were expanded for 5 days on OP9:OP9Dll1 1:1 layers for amplification (T0 reference samples). Secondary recipients were sacrificed after development of leukemia, and T1 samples were generated from BMs, spleens and thymi, which confirmed BM infiltration >20%). T1 and T0 samples were deep sequenced and changes in individual shRNAs were calculated. C. Scatterplot depicting the averaged quantitative changes (log10) of each shRNA within the library in the T1 spleen and BM samples in reference to T0. The changes in both organs were concordant (R2=0.72). T1 samples from 11 mice were compared to 3 T0 reference samples. Blue dots represent shRNAs with a significant depletion by ≥10-fold. D. Top 5 genes with ≥10-fold shRNA depletion in BM, spleen and thymus in comparison to the reference. E. Percent cell growth of 5 different DN2-derived leukemic cell lines transduced with 2 different shRNA constructs targeting Jak2. The cells were sorted for the GFP or YFP reporter and normalized to the control shRNA targeting the lacZ gene (lacZ). Four of five DN2-derived leukemias show reduced growth with both shRNAs after the second round of sorting within 8 days in culture. The lacZ control was set to 100%. F. Proliferation curves of 3 Myc/Bcl2+ DN2- and 3 GMP-derived leukemia lines during treatment with 0.5 μM ruxolitinib. 1.5105 cells were seeded in culture and counted at the indicated time points. Results are shown as fold growth of the starting cell number which was set to 1. *=p≤0.05, **=p≤0.01, Student's t-test. G. Western blot indicating active Jak/Stat signaling in DN2-derived leukemia with Stat3 as well as pStat3 protein expression, that diminishes after treatment with the Jak-inhibitor ruxolitinib. H. GSEA indicating significant enrichment of genes of the JAK/STAT signaling pathway of the KEGG pathway database in the DN2-like M2 AML samples as compared to the residual M2 AML samples using the Verhaak AML data set (Verhaak et al., 2009).
Figure 6Scatter plots of PD-1/LAG-3-expressing CD8 T cells after activation by A459-SC3 cells in a sample of G1 and G2 patients within CD28+ and CD28negative populations as indicated in the figure. Dot-plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated. Dot-plot representing the percentage of proliferating cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated. for CD8 T cells.