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Cell Signaling Technology Inc anti akt3
Anti Akt3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+akt3/Akt3+Rabbit+mAb/pm41547950-303-27-28
Average 93 stars, based on 53 article reviews

anti akt3 - by Bioz Stars, 2026-10

93/100 stars

Images

Confocal Microscopy:

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape
Article Snippet: Transfections were performed in 12-well plates at a density of 2 × 10 5 cells per well using 1 μg of plasmid DNA and Lipofectamine 3000 (Thermo Fisher Scientific, #L3000008). .. Cells were treated with PROTAC peptides (0.1–10 μM) for 24 hours, followed by assessment of AKT3 degradation using confocal microscopy (Zeiss LSM 880) and western blotting with anti-AKT3 (Cell Signaling Technology, #14982) and anti-GAPDH (Sigma-Aldrich, G8795) antibodies. .. A SynNotch reporter system was developed using SynNotch plasmids obtained from Addgene, as described previously .

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape.
Article Snippet: Transfections were performed in 12-well plates at a density of 2×105 cells per well using 1 μg of plasmid DNA and Lipofectamine 3000 (Thermo Fisher Scientific, #L3000008). .. Cells were treated with PROTAC peptides (0.1–10 μM) for 24 hours, followed by assessment of AKT3 degradation using confocal microscopy (Zeiss LSM 880) and western blotting with anti-AKT3 (Cell Signaling Technology, #14982) and anti-GAPDH (Sigma-Aldrich, G8795) antibodies. .. A SynNotch reporter system was developed using SynNotch plasmids obtained from Addgene, as described previously40.

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape
Article Snippet: Transfections were performed in 12-well plates at a density of 2×10 5 cells per well using 1 μg of plasmid DNA and Lipofectamine 3000 (Thermo Fisher Scientific). .. Cells were treated with PROTAC peptides (0.1–10 μM) for 24 hours, followed by assessment of AKT3 degradation using confocal microscopy (Zeiss LSM 880) and western blotting with anti-AKT3 (Cell Signaling Technology, #14982) and anti-GAPDH (Sigma-Aldrich, G8795) antibodies. .. A SynNotch reporter system was developed using SynNotch plasmids obtained from Addgene (Plasmid #79125 and #79130), as described previously .

Western Blot:

other:

Article Title: Erratum: PA-MSHA exerts potent activity against cetuximab-resistant colorectal cancer through the miR-7-5p/Akt3/Wnt-β-catenin pathway
Article Snippet: Transl Cancer Res 2024;13(8):4441-4458 | https://dx.doi.org/10.21037/tcr-23-2211 nonphosphorylated β-catenin (#19807), anti-E-cadherin (#14472), anti-survivin (#2808), anti-Akt3 (#4059) and antiGAPDH (glyceraldehyde-3-phosphate dehydrogenase) (#2118), were purchased from Cell Signaling Technology (CST, Massachusetts, USA).

Article Title: Phosphorylation of plectin by Akt3 promotes triple-negative breast cancer cell invasive migration
Article Snippet: Anti-phospho-Akt substrate (pAkt-motif) (9614S), anti-phospho-Akt Ser473 (4060L), anti-Akt (4691S), anti-phospho-PRAS40 Thr246 (2997S), anti-PRAS40 (2691S), anti-phospho-GSK3β (9336S), anti-GSK3β (9315), anti-Akt1 (2938S), anti-Akt2 (3063S), anti-Akt3 (8018S), and anti-actin (3700S) were purchased from Cell Signaling Technology.

Incubation:

Article Title: The Distinct Biological Effects of 6-Hydroxy-L-Nicotine in Representative Cancer Cell Lines
Article Snippet: A549, MCF7, and U87 cells were exposed to 50 nM NIC/6HLN for 48 h and their corresponding controls were fixed with 4% paraformaldehyde (A11313, Thermo Scientific Chemicals, Waltham, MA, USA), washed with PBS to remove traces of fixation solution, and permeabilized using a permeabilization mix of 0.3% digitonin (G9441, Promega, Madison, WI, USA), 0.3% Triton X 100 (X100, Sigma—Aldrich, Darmstadt, Germany), and 0.3% saponin (558255, Merck, Darmstadt, Germany). .. The cells were incubated with anti-AKT3 (E1Z3W, Cell Signaling Technology, Danvers, MA, USA), anti-MMP9 (SC-21733, Santa-Cruz Biotechnology, Dallas, TX, USA), anti-vimentin (M0725, Dako REAL, Santa Clara, CA, USA), anti-IL-1β (D3U3E, Cell Signaling Technology, Danvers, MA, USA), and anti-IL6 primary antibodies (SC-130326, Santa-Cruz Biotechnology, Dallas, TX, USA), diluted into antibody diluent (S202230-2, Dako REAL, Glostrup, Denmark) (1:25), and incubated at 4 °C for 72 h. Following two washing steps with PBS, the cells were further incubated with Alexa Fluor 546 Goat anti-Mouse IgG (A-11030, ThermoFisher Scientific, Waltham, MA, USA) or fluorescein goat anti-rabbit IgG (F2765, ThermoFisher Scientific, Waltham, MA, USA) secondary antibodies (diluted 1:200 into antibody diluent) at 4 °C for 24 h. Then, the cells were washed twice with PBS to remove the unbound secondary antibodies and incubated overnight with Prolong Gold antifade (P36935, ThermoFisher Scientific, Waltham, MA, USA) with DAPI at 4°C. .. All pictures were acquired at 20× magnification using a Zeiss Axio Observer Z1 Microscope (Hamamatsu C11440-22C camera) from TissueGnostic rig and TissueFAXS 6.146 software.

Article Title: The Distinct Biological Effects of 6-Hydroxy-L-Nicotine in Representative Cancer Cell Lines.
Article Snippet: A549, MCF7, and U87 cells were exposed to 50 nM NIC/6HLN for 48 h and their corresponding controls were fixed with 4% paraformaldehyde (A11313, Thermo Scientific Chemicals, Waltham, MA, USA), washed with PBS to remove traces of fixation solution, and permeabilized using a permeabilization mix of 0.3% digitonin (G9441, Promega, Madison, WI, USA), 0.3% Triton X 100 (X100, Sigma—Aldrich, Darmstadt, Germany), and 0.3% saponin (558255, Merck, Darmstadt, Germany). .. The cells were incubated with anti-AKT3 (E1Z3W, Cell Signaling Technology, Danvers, MA, USA), anti-MMP9 (SC-21733, Santa-Cruz Biotechnology, Dallas, TX, USA), anti-vimentin (M0725, Dako REAL, Santa Clara, CA, USA), anti-IL-1β (D3U3E, Cell Signaling Technology, Danvers, MA, USA), and anti-IL6 primary antibodies (SC-130326, Santa-Cruz Biotechnology, Dallas, TX, USA), diluted into antibody diluent (S202230-2, Dako REAL, Glostrup, Denmark) (1:25), and incubated at 4 ◦C for 72 h. Following two washing steps with PBS, the cells were further incubated with Alexa Fluor 546 Goat anti-Mouse IgG (A-11030, ThermoFisher Scientific, Waltham, MA, USA) or fluorescein goat anti-rabbit IgG (F2765, ThermoFisher Scientific, Waltham, MA, USA) secondary antibodies (diluted 1:200 into antibody diluent) at 4 ◦C for 24 h. Then, the cells were washed twice with PBS to remove the unbound secondary antibodies and incubated overnight with Prolong Gold antifade (P36935, ThermoFisher Scientific, Waltham, MA, USA) with DAPI at 4◦C. .. All pictures were acquired at 20× magnification using a Zeiss Axio Observer Z1 Microscope (Hamamatsu C11440-22C camera) from TissueGnostic rig and TissueFAXS 6.146 software.

Image Search Results

a Schematic timeline of in vivo lymphoma model for isolation of CAR T cells for RNA seq analysis. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bar graph of RNA seq showing pathway analysis of dysregulated genes associated with OXPHOS, mitochondrial metabolism, FOXO signaling, and autophagy in TR group CAR T cells. c Representative flow cytometry histogram showing the fluorescence intensity of FITC-labeled AKT3 (FITC-AKT3) in T cells from different groups. d Bar graph showing the mean fluorescence intensity (MFI) of AKT3 in T cells from the CON and TR groups ( n = 6 mice). e Illustration of the plasmids showing pLenti-AKT3 lentiviral vector used for co-transduction of CAR T cells with CAR vectors. f Schematic representation of the 17-day in vitro experimental timeline for evaluating T cell responses in TR model. T cells were isolated, activated, and transduced on Day 0 and further expanded until Day 7, followed by tumor challenge with Raji WT cells on Day 7. g Line graph comparing the survival of CAR T cells transduced with b20/19 CAR and either a control vector (b20/19 pVEC , blue circles) or an AKT3-expressing vector (b20/19 pAKT3 , orange squares) over 15 days ( n = 5 biologically independent samples). h Histogram analysis and pie chart showing distribution of T em and T cm with PD-1 low or PD-1 high within these subsets ( n = 5 biologically independent samples) at day 17. i Line graph showing Extracellular Acidification Rate (ECAR, mpH/min) over 100 min day 17. j Bar graph of basal ECAR (mpH/min, normalized) between b20/19 pVEC (blue) and b20/19 pAKT3 (orange) groups ( n = 6 biologically independent samples). k , l Similarly line graph showing Oxygen Consumption Rate (OCR, pMoles/min) and corresponding bar graph ( n = 6 biologically independent samples). m Schematic of the lentiviral vector delivering shRNA against AKT3 (shAKT3). The lower panel depicts the lentiviral particle transduced target CAR T cells. n Line graph comparing the survival of CAR T cells over 15 days for three groups. Statistical analysis was done for day 15 data points ( n = 5 biologically independent samples). o Bar graph and pie charts showing the percentage of various subsets of CD8 + CAR T cells across three groups ( n = 5 biologically independent samples). p , q Bar graph of ECAR, and OCR across three groups ( n = 8). Data represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For g , h , and o , a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic timeline of in vivo lymphoma model for isolation of CAR T cells for RNA seq analysis. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bar graph of RNA seq showing pathway analysis of dysregulated genes associated with OXPHOS, mitochondrial metabolism, FOXO signaling, and autophagy in TR group CAR T cells. c Representative flow cytometry histogram showing the fluorescence intensity of FITC-labeled AKT3 (FITC-AKT3) in T cells from different groups. d Bar graph showing the mean fluorescence intensity (MFI) of AKT3 in T cells from the CON and TR groups ( n = 6 mice). e Illustration of the plasmids showing pLenti-AKT3 lentiviral vector used for co-transduction of CAR T cells with CAR vectors. f Schematic representation of the 17-day in vitro experimental timeline for evaluating T cell responses in TR model. T cells were isolated, activated, and transduced on Day 0 and further expanded until Day 7, followed by tumor challenge with Raji WT cells on Day 7. g Line graph comparing the survival of CAR T cells transduced with b20/19 CAR and either a control vector (b20/19 pVEC , blue circles) or an AKT3-expressing vector (b20/19 pAKT3 , orange squares) over 15 days ( n = 5 biologically independent samples). h Histogram analysis and pie chart showing distribution of T em and T cm with PD-1 low or PD-1 high within these subsets ( n = 5 biologically independent samples) at day 17. i Line graph showing Extracellular Acidification Rate (ECAR, mpH/min) over 100 min day 17. j Bar graph of basal ECAR (mpH/min, normalized) between b20/19 pVEC (blue) and b20/19 pAKT3 (orange) groups ( n = 6 biologically independent samples). k , l Similarly line graph showing Oxygen Consumption Rate (OCR, pMoles/min) and corresponding bar graph ( n = 6 biologically independent samples). m Schematic of the lentiviral vector delivering shRNA against AKT3 (shAKT3). The lower panel depicts the lentiviral particle transduced target CAR T cells. n Line graph comparing the survival of CAR T cells over 15 days for three groups. Statistical analysis was done for day 15 data points ( n = 5 biologically independent samples). o Bar graph and pie charts showing the percentage of various subsets of CD8 + CAR T cells across three groups ( n = 5 biologically independent samples). p , q Bar graph of ECAR, and OCR across three groups ( n = 8). Data represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For g , h , and o , a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: The top-performing peptide, P2, was initially tested in GFP-expressing CAR T cells and endogenous AKT3 was detected using rabbit anti-AKT3 primary antibody (Cell Signaling Technology, #14982) followed by staining with Alexafluor 546-conjugated secondary anti-rabbit antibody (ThermoFischer Scientific, # A-11035).

Techniques: In Vivo, Isolation, RNA Sequencing, Flow Cytometry, Fluorescence, Labeling, Plasmid Preparation, Transduction, In Vitro, Control, Expressing, shRNA

a Table listing the sequences of peptides designed using RFdiffusion for PROTAC application, targeting AKT3 (AKT3_P1 to AKT3_P5, AKT3_PC) and a non-targeting peptide (NTP), with AKT3_P2 highlighted as the selected peptide. b Schematic illustration of mCherry-AKT3 CAR T cells induced with PROTAC. The PROTAC consists of the AKT3-targeting peptide (AKT3-P) fused to a cell-penetrating peptide (CPP) and via a linker connected to an E3 ligase ligand. c Line graph showing the dose-dependent effect of PROTAC peptide P2 (blue circles) compared to a non-targeting peptide (NTP, orange circles) on the ratio of endogenous AKT3 to GFP fluorescence in CAR T cells ( n = 4 biologically independent samples). The AKT3 was detected using primary anti-AKT3 antibody (raised in rabbit) followed by detection with secondary anti-rabbit antibody (Alexafluor 546; red). d Representative fluorescence microscopy images of CAR T cells treated with NTP or P2, showing GFP (green), anti-AKT3 (Alexafluor 546; red), and merged channels with regions of interest (ROIs). ROIs highlight reduced AKT3 signal in P2-treated cells compared to NTP-treated cells. e Violin plot comparing the integrated density of AKT3 in P2 and NTP-treated CAR T cells ( n = 15 images). f Schematic of the b20/19 CAR construct, including a signal peptide (SP), furin cleavage (FC), and AKT3 PROTAC (P2A linker), designed to target AKT3 for degradation in CAR T cells. Timeline of the 15-day experiment for evaluating b20/19 CAR T cell responses. g Jittered dot plot showing the relative fluorescence units (RFU) of AKT3 in b20/19 CAR T cells treated with NTP or AKT3 PROTAC ( n = 25 data points from three independent experiments). h Western blot analysis of b20/19 CAR T cells treated with NTP PROTAC or AKT3 PROTAC , probing for endogenous AKT3 using anti-AKT3, and GAPDH as a loading control. i Bar graph showing the densitometry analysis of the blots ( n = 6 biologically independent samples). j Representative fluorescence microscopy images of b20/19 CAR T cells treated with non-targeting peptide (NTP PROTAC ) or AKT3 PROTAC , showing GFP (green), anti-AKT3 (Alexafluor 546; red), and merged channels. k Violin plot comparing the integrated density of AKT3 in NTP PROTAC and AKT3 PROTAC treated b20/19 CAR T cells ( n = 10 images). l Survival curve showing percentage of RajiWT cell survival after co-culture with CAR T cells at different target-to-effector (T:E) ratios ( n = 4 biologically independent samples). m Percentage of patient-derived cancer cell survival after 24-h co-culture with CAR T cells at a target-to-effector (T:E) ratio of 1:2.5. b20/19-AKT3 PROTAC exhibits higher cytotoxicity in patient derived cells ( n = 5 biologically independent samples). n In vitro tumor rechallenge model showing survival of b20/19-AKT3 PROTAC CAR T cells over time ( n = 5 biologically independent samples). o Flow cytometry histogram showing various T cell subsets ( n = 5). p , q Dot plot showing showing ECAR and OCR analysis ( n = 12 data points from three independent experiments). Data represent mean ± SEM. ** p < 0.01; *** p < 0.005; **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For o , Two-way ANOVA followed by post-hoc testing was applied. Scale bar; d : 200 μm; j : 100 μm. Source data are provided as a file.

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Table listing the sequences of peptides designed using RFdiffusion for PROTAC application, targeting AKT3 (AKT3_P1 to AKT3_P5, AKT3_PC) and a non-targeting peptide (NTP), with AKT3_P2 highlighted as the selected peptide. b Schematic illustration of mCherry-AKT3 CAR T cells induced with PROTAC. The PROTAC consists of the AKT3-targeting peptide (AKT3-P) fused to a cell-penetrating peptide (CPP) and via a linker connected to an E3 ligase ligand. c Line graph showing the dose-dependent effect of PROTAC peptide P2 (blue circles) compared to a non-targeting peptide (NTP, orange circles) on the ratio of endogenous AKT3 to GFP fluorescence in CAR T cells ( n = 4 biologically independent samples). The AKT3 was detected using primary anti-AKT3 antibody (raised in rabbit) followed by detection with secondary anti-rabbit antibody (Alexafluor 546; red). d Representative fluorescence microscopy images of CAR T cells treated with NTP or P2, showing GFP (green), anti-AKT3 (Alexafluor 546; red), and merged channels with regions of interest (ROIs). ROIs highlight reduced AKT3 signal in P2-treated cells compared to NTP-treated cells. e Violin plot comparing the integrated density of AKT3 in P2 and NTP-treated CAR T cells ( n = 15 images). f Schematic of the b20/19 CAR construct, including a signal peptide (SP), furin cleavage (FC), and AKT3 PROTAC (P2A linker), designed to target AKT3 for degradation in CAR T cells. Timeline of the 15-day experiment for evaluating b20/19 CAR T cell responses. g Jittered dot plot showing the relative fluorescence units (RFU) of AKT3 in b20/19 CAR T cells treated with NTP or AKT3 PROTAC ( n = 25 data points from three independent experiments). h Western blot analysis of b20/19 CAR T cells treated with NTP PROTAC or AKT3 PROTAC , probing for endogenous AKT3 using anti-AKT3, and GAPDH as a loading control. i Bar graph showing the densitometry analysis of the blots ( n = 6 biologically independent samples). j Representative fluorescence microscopy images of b20/19 CAR T cells treated with non-targeting peptide (NTP PROTAC ) or AKT3 PROTAC , showing GFP (green), anti-AKT3 (Alexafluor 546; red), and merged channels. k Violin plot comparing the integrated density of AKT3 in NTP PROTAC and AKT3 PROTAC treated b20/19 CAR T cells ( n = 10 images). l Survival curve showing percentage of RajiWT cell survival after co-culture with CAR T cells at different target-to-effector (T:E) ratios ( n = 4 biologically independent samples). m Percentage of patient-derived cancer cell survival after 24-h co-culture with CAR T cells at a target-to-effector (T:E) ratio of 1:2.5. b20/19-AKT3 PROTAC exhibits higher cytotoxicity in patient derived cells ( n = 5 biologically independent samples). n In vitro tumor rechallenge model showing survival of b20/19-AKT3 PROTAC CAR T cells over time ( n = 5 biologically independent samples). o Flow cytometry histogram showing various T cell subsets ( n = 5). p , q Dot plot showing showing ECAR and OCR analysis ( n = 12 data points from three independent experiments). Data represent mean ± SEM. ** p < 0.01; *** p < 0.005; **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For o , Two-way ANOVA followed by post-hoc testing was applied. Scale bar; d : 200 μm; j : 100 μm. Source data are provided as a file.

Techniques: Fluorescence, Microscopy, Construct, Western Blot, Control, Co-Culture Assay, Derivative Assay, In Vitro, Flow Cytometry

a Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. b Relative mRNA expression levels (normalized to beta actin; ACTB) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR T ( n = 6 biologically independent samples). c Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR T cells after TR with RajiCD19 −/− cells. d Histogram analysis of the flow cytometry plots ( n = 10 biologically independent samples). e Bar graph shows the percentage of CD8 + CAR T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions ( n = 5 biologically independent samples). f Survival of CAR T cells over 15 days under various conditions ( n = 4 biologically independent samples). g Violin plots showing the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown elevated mTOR activity ( n = 6 biologically independent samples). h Bar plots show the percentage of MFI of autophagy from autophagic flux assay ( n = 8 data points from three independent experiments). i Dot plot showing ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from OXPHOS to glycolysis ( n = 12 data points from three independent experiments). j Similarly, OCR with FOXO4 knockdown decreases mitochondrial respiration. Individual data points are shown for each condition ( n = 12 data points from three independent experiments). k Box-and-whisker plot showing percentage of expression of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker ( n = 63 patient samples). l Bar graph showing the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. m Schematic illustration of K562 WT and CD20 expressing K562 stable cells transduced with different MOIs to obtain three populations: CD20 L (low), CD20 M (medium), and CD20 H (high), which were further FACS sorted. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . n Violin plots showing the percentage of CD20 expression (% CD20 expression) in the sorted CD20-expressing K562 cell populations, confirming distinct expression levels ( n = 10 data flow cytometry points from three independent experiments). o Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show CD20 (red) in K562-CD20 L (low), K562-CD20 M (medium), and K562-CD20 H (high) cell. p–r Survival curves of K562 cells expressing varying CD20 expression levels under CAR T cell treatments. The line graph shows the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) ( n = 4 biologically independent samples). s Survival of CAR T cells with varying CD20-targeting CAR constructs over 15 days ( n = 5). Data represents mean ± SEM. **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For e , f and s , a Two-way ANOVA followed by post-hoc testing was applied. Scale bar: 5 μm. Source data are provided as a file.

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. b Relative mRNA expression levels (normalized to beta actin; ACTB) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR T ( n = 6 biologically independent samples). c Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR T cells after TR with RajiCD19 −/− cells. d Histogram analysis of the flow cytometry plots ( n = 10 biologically independent samples). e Bar graph shows the percentage of CD8 + CAR T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions ( n = 5 biologically independent samples). f Survival of CAR T cells over 15 days under various conditions ( n = 4 biologically independent samples). g Violin plots showing the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown elevated mTOR activity ( n = 6 biologically independent samples). h Bar plots show the percentage of MFI of autophagy from autophagic flux assay ( n = 8 data points from three independent experiments). i Dot plot showing ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from OXPHOS to glycolysis ( n = 12 data points from three independent experiments). j Similarly, OCR with FOXO4 knockdown decreases mitochondrial respiration. Individual data points are shown for each condition ( n = 12 data points from three independent experiments). k Box-and-whisker plot showing percentage of expression of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker ( n = 63 patient samples). l Bar graph showing the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. m Schematic illustration of K562 WT and CD20 expressing K562 stable cells transduced with different MOIs to obtain three populations: CD20 L (low), CD20 M (medium), and CD20 H (high), which were further FACS sorted. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . n Violin plots showing the percentage of CD20 expression (% CD20 expression) in the sorted CD20-expressing K562 cell populations, confirming distinct expression levels ( n = 10 data flow cytometry points from three independent experiments). o Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show CD20 (red) in K562-CD20 L (low), K562-CD20 M (medium), and K562-CD20 H (high) cell. p–r Survival curves of K562 cells expressing varying CD20 expression levels under CAR T cell treatments. The line graph shows the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) ( n = 4 biologically independent samples). s Survival of CAR T cells with varying CD20-targeting CAR constructs over 15 days ( n = 5). Data represents mean ± SEM. **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For e , f and s , a Two-way ANOVA followed by post-hoc testing was applied. Scale bar: 5 μm. Source data are provided as a file.

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Whisker Assay, Marker, Transduction, Super-Resolution Microscopy, Construct

a Schematic timeline of the experiment showing Raji WT cell injection, CAR T cell administration, and Raji CD19 −/− TR. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b In vivo bioluminescence imaging of mice treated with NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 , showing tumor burden (red indicates high tumor load, blue indicates low) over 84 days, with “X” marking deceased mice. c Tumor radiance over 84 days, demonstrating reduced tumor burden in the AKT3 PROTAC+scram group. d Kaplan-Meier survival curves, with the AKT3 PROTAC+scram group exhibiting the highest survival rate. e Line graph shows the percentage of CAR T cells detected in blood (% CAR T cells) over time. f Bar graph displays the percentage of CAR T cells in blood at day 84, with the AKT3 PROTAC+scram group showing detectable levels (~3%), while other groups were not analyzable due to the absence of surviving mice, indicated as not determined (ND) ( n = 5 mice). g Tumor burden assessment till day 56. The line graph shows the number of Raji cells over time. The AKT3 PROTAC+scram group exhibits no detectable Raji cell burden by day 56, while other groups with some surviving mice show some detectable cells ( n = 5 mice). h Bar graph shows the percentage of CD8 CAR T cells (% CD8 T cells) expressing different phenotypes on day 28 post-infusion with corresponding pie charts illustrating the proportional distribution ( n = 5 mice). i , j Dot plot showing ECAR and OCR under various conditions on day 28 ( n = 12 data points). Data represent mean ± SEM. *** p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups. The comparison was made between NTP PROTAC+Scram with NTPP ROTAC+shFOXO4 and AKT3 PROTAC+Scram with AKT3 PROTAC+shFOXO4 . For h , a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic timeline of the experiment showing Raji WT cell injection, CAR T cell administration, and Raji CD19 −/− TR. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b In vivo bioluminescence imaging of mice treated with NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 , showing tumor burden (red indicates high tumor load, blue indicates low) over 84 days, with “X” marking deceased mice. c Tumor radiance over 84 days, demonstrating reduced tumor burden in the AKT3 PROTAC+scram group. d Kaplan-Meier survival curves, with the AKT3 PROTAC+scram group exhibiting the highest survival rate. e Line graph shows the percentage of CAR T cells detected in blood (% CAR T cells) over time. f Bar graph displays the percentage of CAR T cells in blood at day 84, with the AKT3 PROTAC+scram group showing detectable levels (~3%), while other groups were not analyzable due to the absence of surviving mice, indicated as not determined (ND) ( n = 5 mice). g Tumor burden assessment till day 56. The line graph shows the number of Raji cells over time. The AKT3 PROTAC+scram group exhibits no detectable Raji cell burden by day 56, while other groups with some surviving mice show some detectable cells ( n = 5 mice). h Bar graph shows the percentage of CD8 CAR T cells (% CD8 T cells) expressing different phenotypes on day 28 post-infusion with corresponding pie charts illustrating the proportional distribution ( n = 5 mice). i , j Dot plot showing ECAR and OCR under various conditions on day 28 ( n = 12 data points). Data represent mean ± SEM. *** p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups. The comparison was made between NTP PROTAC+Scram with NTPP ROTAC+shFOXO4 and AKT3 PROTAC+Scram with AKT3 PROTAC+shFOXO4 . For h , a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a Source Data file.

Techniques: Injection, In Vivo, Imaging, Expressing, Comparison

a Schematic of CLDN scFv and EGFR scFv CAR constructs with ICOS, 4-1BB, CD3ζ, and AKT3 PROTAC domains. b , c Survival curves showing cytotoxicity of CLDN and EGFR CAR T cells with NTP PROTAC or AKT3 PROTAC against AGS (K) and A549 (L) cells at varying T:E ratios ( n = 5 biologically independent samples). d Line graph showing CAR T cell proliferation over 15 days, showing enhanced expansion with AKT3 PROTAC . e Bar graph and pie charts analysis of EGFR-CAR and CLDN-CAR T cells with NTP PROTAC or AKT3 PROTAC , showing the percentage of various T cell subsets ( n = 5 biologically independent samples). Data represent mean ± SEM. ****p < 0.001. A two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic of CLDN scFv and EGFR scFv CAR constructs with ICOS, 4-1BB, CD3ζ, and AKT3 PROTAC domains. b , c Survival curves showing cytotoxicity of CLDN and EGFR CAR T cells with NTP PROTAC or AKT3 PROTAC against AGS (K) and A549 (L) cells at varying T:E ratios ( n = 5 biologically independent samples). d Line graph showing CAR T cell proliferation over 15 days, showing enhanced expansion with AKT3 PROTAC . e Bar graph and pie charts analysis of EGFR-CAR and CLDN-CAR T cells with NTP PROTAC or AKT3 PROTAC , showing the percentage of various T cell subsets ( n = 5 biologically independent samples). Data represent mean ± SEM. ****p < 0.001. A two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Techniques: Construct

a Schematic of the strategy for trispecific CAR T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). b Correlation of expression of nbCD3, nbCD22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin, and data were obtained using intracellular flow cytometry ( n = 7 data points from three independent experiments). c Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. d Dose-dependent T cell activation (CD69 expression) in response to culture supernatants (used at various ratios with culture media) with nbCD3/22, using db coated with CD3 for validation ( n = 6 data points from three independent experiments). e Line graph of HEK293T synNotch reporter assay showing dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR T cell supernatants, confirming BiTE functionality under two condition 1 and condition 2. f Experimental timelines for in vitro T cell engineering, transduction, and co-culture with Raji cells (WT or knockout for CD19, CD20, or CD22). Anti-tumor assays were performed on days 9, 11, and 13. g , h Functional assay of CAR T cells against Raji cells (WT or knockout for CD19, CD20, or CD22) demonstrates that b20/19AKT3 PROTAC CAR T cells co-expressing nbCD3/22 exhibit stronger antitumor activity compared to b20/19-AKT3 PROTAC or mCD19 CAR T cells at Day 7 and Day 14. Data represent mean ± SEM. **** p < 0.001; ns: not significant. A nonparametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic of the strategy for trispecific CAR T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). b Correlation of expression of nbCD3, nbCD22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin, and data were obtained using intracellular flow cytometry ( n = 7 data points from three independent experiments). c Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. d Dose-dependent T cell activation (CD69 expression) in response to culture supernatants (used at various ratios with culture media) with nbCD3/22, using db coated with CD3 for validation ( n = 6 data points from three independent experiments). e Line graph of HEK293T synNotch reporter assay showing dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR T cell supernatants, confirming BiTE functionality under two condition 1 and condition 2. f Experimental timelines for in vitro T cell engineering, transduction, and co-culture with Raji cells (WT or knockout for CD19, CD20, or CD22). Anti-tumor assays were performed on days 9, 11, and 13. g , h Functional assay of CAR T cells against Raji cells (WT or knockout for CD19, CD20, or CD22) demonstrates that b20/19AKT3 PROTAC CAR T cells co-expressing nbCD3/22 exhibit stronger antitumor activity compared to b20/19-AKT3 PROTAC or mCD19 CAR T cells at Day 7 and Day 14. Data represent mean ± SEM. **** p < 0.001; ns: not significant. A nonparametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vitro, Transduction, Co-Culture Assay, Knock-Out, Functional Assay, Activity Assay

a Experimental timeline for in vivo study in Raji WT or NALM6 WT model followed by CAR T cell administration and TR with double knockout Raji CD19/CD20−/− or double knockout NALM6 CD19/CD20−/− cells. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bioluminescence imaging of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 CAR T cells, monitored from Day 7 to Day 84. c Line graph of quantified tumor radiance over time, showing sustained tumor control in Raji and NALM6 models with b20/19-AKT3 PROTAC+nbCD3/22 . d Line graph of percentage of CAR T cells in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days. e Bar graph of CAR T cell populations in blood at Day 56. f Levels of nbCD3/22 (pg/mL) in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days, showing sustained secretion. g Kaplan-Meier survival curves demonstrating improved survival with nbCD3/22-modified CAR T cells. h Bar graph and pie charts compare b20/19-AKT3 PROTAC and b20/19-AKT3 PROTAC+nbCD3/22 , showing various memory T cell subsets over time ( n = 5 mice) in all conditions. Data represent mean ± SEM. **** p < 0.001. A two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Experimental timeline for in vivo study in Raji WT or NALM6 WT model followed by CAR T cell administration and TR with double knockout Raji CD19/CD20−/− or double knockout NALM6 CD19/CD20−/− cells. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bioluminescence imaging of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 CAR T cells, monitored from Day 7 to Day 84. c Line graph of quantified tumor radiance over time, showing sustained tumor control in Raji and NALM6 models with b20/19-AKT3 PROTAC+nbCD3/22 . d Line graph of percentage of CAR T cells in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days. e Bar graph of CAR T cell populations in blood at Day 56. f Levels of nbCD3/22 (pg/mL) in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days, showing sustained secretion. g Kaplan-Meier survival curves demonstrating improved survival with nbCD3/22-modified CAR T cells. h Bar graph and pie charts compare b20/19-AKT3 PROTAC and b20/19-AKT3 PROTAC+nbCD3/22 , showing various memory T cell subsets over time ( n = 5 mice) in all conditions. Data represent mean ± SEM. **** p < 0.001. A two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Techniques: In Vivo, Double Knockout, Imaging, Control, Modification

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