Journal: bioRxiv

Article Title: Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT

doi: 10.64898/2026.04.02.715982

Figure Lengend Snippet: (A) Model depicting organoid monolayer preparation. Patient colonic tissue is digested, single celled, and plated in a bubble of Matrigel. Organoids are supplemented with media containing surplus of Wnt, EGF, R-spondin, and Noggin. Once organoids are fully grown in 3D conditions, cells are disassociated and plated on a thin layer of Matrigel. Within 5-7 days, cells grow into a monolayer with distinct stem cell (5-10% of cells), TA cell (5-10% of cells) and differentiated cell (80-90% of cells) niches. Stem cell niches, called nodes, are dense, regularly spaced niches made up of OLFM4/Sox9/LGR5/MYC positive stem cells. (B) Representative image of stem cell niche (node) characterized by OLFM4+ cells with quantification. OLFM4 measured through immunofluorescent staining, population makes up around 10% of monolayers, although almost all cells in node are OLFM4+. Cells were portioned into node or non-node and percent of OLFM4+ cells out of total node or non-node cells was used for quantification. (C) Representative image of transit-Amplifying (TA) cells characterized by Ki-67 immunofluorescent staining. TA cells surround the OLFM4+ stem cells and make up around 10% of the monolayer. (D) Differentiated cells shown by CK20 immunostaining makes up the surrounding monolayer, around 80% of total cells. (E) Total EGFR representative image and quantification of almost all cells in monolayer expressing EGFR uniformly. (F) P-EGFR-Y1068 representative image and quantification preferentially activated in nodes. (G) Representative image and quantification of P-AKT-S473 exclusively activated within nodes and low in differentiated compartment. (H) P-ERK1/2 representative image and quantification. Activation excluded from nodes and on in a small percentage of differentiated cells at any given time (10-20%). (I) Co-stain representative image of P-AKT and P-ERK mutual exclusivity. (J) Quantification of AKT+, ERK+, Double Positive and Double Negative cells within monolayer. Red asterisks marking around 5% total cells that are double positive, uninsulated cells that coactivate AKT and ERK. Three to four biological replicates were performed. Data shown is from analysis of 4 technical replicates with at least 150 total cells quantified per replicate. Data are represented as mean ± SEM. All scale bars are 100uM, significance calculated with Welch’s t-test, ** P ≤ 0.01, *** P ≤ 0.001. See also Figure S1 and S2.

Article Snippet: Antibodies and dyes Rabbit monoclonal OLFM4 (CST, 14369S), Rat monoclonal anti-Ki67 (Invitrogen, 14-5698-82), Mouse monoclonal P-ERK1/2 (Invitrogen, 14-9109-82), Rabbit Monoclonal P-AKT-Ser473 (CST, 4058), Rabbit monoclonal P-RAF1-S259 (Invitrogen, 44-502), Rabbit monoclonal P-EGFR-Y1068 (abcam, ab40815), Mouse monoclonal EGFR (Invitrogen, MA5-13070), Click-iT EdU Cell Proliferation Kit for Imaging, DAPI (Thermofisher, D21490), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32731), Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A21247), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A32728), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 546 (Invitrogen, A11030).

Techniques: Staining, Immunostaining, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT

doi: 10.64898/2026.04.02.715982

Figure Lengend Snippet: (A) Experiment schematic of how pharmacological perturbations are tested. Fully grown 3D organoids are disassociated into single cells and plated into a 384-well plate covered in a thin layer of Matrigel. Organoids are grown for at least 5 days, or until cells make a full monolayer with proper node patterning. Wells are pharmacologically treated for 1 hour to look at short-term signaling activation changes (P-ERK and P-AKT) or 72 hours to assess long-term cell fate consequences to allow proper time for cell differentiation. (B) Immunofluorescence staining of P-ERK1/2 (yellow) and P-AKT (blue) in control and 100nM PMA treated cells. (C) Quantification of percentage of cells in node and outside of nodes with active ERK and AKT. (D) Quantification of ERK/AKT double positive cells and double negative cells. Red asterisks indicate less than 5% of cells activate both pathways, signaling insulation which is maintained in the presence of PMA. (E) Quantification of ERK-KTR biosensor in organoids treated with PMA and control. Organoids were treated with 100nM PMA 1 hour after starting the movie. Graph shows average ERK activity by cytoplasmic/nuclear ration of KTR intensity with ribbon showing 95% CI. (F) Heatmaps showing ERK activity of all cells tracked, with each cell being a horizontal line on the graph. Yellow shows high ERK activity and blue being low activity. (G) Kinase dynamics score (KDS) violin plot calculated as the standard deviation of ERK activity within each cell over the first 5 hours of the movie. (H) Immunofluorescence staining of cell fate markers OLFM4 (yellow, stem cells) and Ki67 (blue, TA cells) in control and 100nM PMA treated cells. (I) Quantification of percent of total cells that are stem or TA cells showing PMA causes a two-fold loss of stem cells. (J) Model showing PMA causes a pulse of ERK activity, leading to loss of AKT, loss of stem cells, and global differentiation of the monolayer. Three to four biological replicates were performed. Data shown is from analysis of 4 technical replicates with at least 150 total cells quantified per replicate. Data are represented as mean ± SEM. All scale bars are 100uM, significance calculated with four-way ANOVA, ** P ≤ 0.01, **** P ≤ 0.0001. See also Figure S2

Article Snippet: Antibodies and dyes Rabbit monoclonal OLFM4 (CST, 14369S), Rat monoclonal anti-Ki67 (Invitrogen, 14-5698-82), Mouse monoclonal P-ERK1/2 (Invitrogen, 14-9109-82), Rabbit Monoclonal P-AKT-Ser473 (CST, 4058), Rabbit monoclonal P-RAF1-S259 (Invitrogen, 44-502), Rabbit monoclonal P-EGFR-Y1068 (abcam, ab40815), Mouse monoclonal EGFR (Invitrogen, MA5-13070), Click-iT EdU Cell Proliferation Kit for Imaging, DAPI (Thermofisher, D21490), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32731), Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A21247), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A32728), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 546 (Invitrogen, A11030).

Techniques: Activation Assay, Cell Differentiation, Immunofluorescence, Staining, Control, Insulation, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT

doi: 10.64898/2026.04.02.715982

Figure Lengend Snippet: (A) Control and 1uM MK2206 treated groups representative images of P-AKT staining. (B) Quantification of P-AKT in nodes and non-nodes showing significant reduction of AKT signaling throughout entire monolayer. (C) Representative image of P-ERK1/2 staining in control and MK2206 treated monolayers. (D) Quantification showing significant increase in ERK1/2 signaling specifically within stem cell niches treated with MK2206. (E) Quantification of ERK/AKT double positive cells and double negative cells. Red asterisks indicate less than 5% of cells activate both pathways, signaling insulation which is maintained with acute AKT inhibition. (F) Quantification of ERK-KTR biosensor in organoids treated with MK2206 and control. Organoids were treated with 1uM MK2206 30 minutes after starting the movie. Graph shows average ERK activity by cytoplasmic/nuclear ration of KTR intensity with ribbon showing 95% CI. (G) Heatmaps showing ERK activity of all cells tracked, with each cell being a horizontal line on the graph. Yellow shows high ERK activity and blue being low activity. (H) Kinase dynamics score (KDS) violin plot calculated as the standard deviation of ERK activity within each cell over the first 5 hours of the movie. (I) Representative image of stem (OLFM4, yellow) and TA (Ki67, blue) cell markers in control and MK2206 treated groups. (J) Quantification of the percentage of total cells with stem or TA makers, showing a loss of stem cells and induction of TA cell fate in MK2206 treated group. (K) Model showing MK2206 causes a pulse of ERK activity, leading to loss of AKT, loss of stem cells, and global differentiation of the monolayer. Data shown is from analysis of 3-4 technical replicates with at least 150 total cells quantified per replicate. Data are represented as mean ± SEM. All scale bars are 100uM, significance calculated with Welch’s t-test (H) or four-way ANOVA (B,D), * P ≤ 0.05,** P ≤ 0.01, **** P ≤ 0.0001. See also Figure S5, S6, and S7.

Article Snippet: Antibodies and dyes Rabbit monoclonal OLFM4 (CST, 14369S), Rat monoclonal anti-Ki67 (Invitrogen, 14-5698-82), Mouse monoclonal P-ERK1/2 (Invitrogen, 14-9109-82), Rabbit Monoclonal P-AKT-Ser473 (CST, 4058), Rabbit monoclonal P-RAF1-S259 (Invitrogen, 44-502), Rabbit monoclonal P-EGFR-Y1068 (abcam, ab40815), Mouse monoclonal EGFR (Invitrogen, MA5-13070), Click-iT EdU Cell Proliferation Kit for Imaging, DAPI (Thermofisher, D21490), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32731), Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A21247), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A32728), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 546 (Invitrogen, A11030).

Techniques: Control, Staining, Insulation, Inhibition, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT

doi: 10.64898/2026.04.02.715982

Figure Lengend Snippet: (A) Representative images of Control, RAF1-S259A phosphomutant, and both conditions treated with 100nM of PMA for 1 hour then fixed and stained after 72 hours. All wells were stained with OLFM4 (stem cell marker, yellow) and Ki-67 (TA cell marker, blue). (B) Quantification of all four conditions showing PMA can rescue the neoplastic cell fate of RAF1-S259A mutant cells. (C) Quantification of ERK-KTR biosensor in control and RAF mutant organoids with and without PMA showing PMA can induce a pulse of ERK activity regardless of baseline activity levels. Graph shows average ERK activity by cytoplasmic/nuclear ration of KTR intensity with ribbon showing 95% CI. ( D) Heatmaps showing ERK activity of all cells tracked, with each cell being a horizontal line on the graph. Yellow shows high ERK activity and blue being low activity. (E) Kinase dynamics score (KDS) violin plot calculated as the standard deviation of ERK activity within each cell over the first 5 hours of the movie showing a pulse of PMA is able to induce dynamics back into the RAF1-S259A mutant cells. (F) Model showing ERK dynamics regulating cell fate through AKT-dependent RAF1-S259 checkpoint. Breakdown of this checkpoint induced a neoplastic cell fate characterized by high kinase signaling load and low kinase signaling dynamics. Inducing increased kinase signaling dynamics causes global differentiation regardless of kinase signaling load, showing that kinase dynamics are epistatic to signaling load. Data shown is from analysis of 4 technical replicates with at least 150 total cells quantified per replicate. All scale bars are 100uM. Data are represented as mean ± SEM. All scale bars are 100uM, significance calculated with four-way ANOVA, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ** P ≤ 0.0001. See also Figure S9 and S10. See also Figure S11.

Article Snippet: Antibodies and dyes Rabbit monoclonal OLFM4 (CST, 14369S), Rat monoclonal anti-Ki67 (Invitrogen, 14-5698-82), Mouse monoclonal P-ERK1/2 (Invitrogen, 14-9109-82), Rabbit Monoclonal P-AKT-Ser473 (CST, 4058), Rabbit monoclonal P-RAF1-S259 (Invitrogen, 44-502), Rabbit monoclonal P-EGFR-Y1068 (abcam, ab40815), Mouse monoclonal EGFR (Invitrogen, MA5-13070), Click-iT EdU Cell Proliferation Kit for Imaging, DAPI (Thermofisher, D21490), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32731), Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A21247), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A32728), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 546 (Invitrogen, A11030).

Techniques: Control, Staining, Marker, Mutagenesis, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Epithelial MHC II antigen presentation dynamically informs intestinal homeostasis and injury

doi: 10.64898/2026.03.18.712348

Figure Lengend Snippet: (A) Schematic of the experimental workflow. MHC II deletion on IECs (MHC II ΔIEC ) was induced by tamoxifen administration. Villus, crypt, and LP compartments of the small intestine (SI) were isolated and subjected to MHC II-bound peptide purification followed by LC-MS/MS. Data represent n = 4 mice unless specified otherwise. (B) Loss of immunopeptidome compartmentalization shown by Principal Component Analysis (PCA) of immunopeptides from the villus, crypt, and LP immune compartments in MHC II ΔIEC compared to MHC II fl/fl mice. Axes indicate variance explained; n = 2 mice per genotype. (C) Number of compartment-specific MHC II-bound peptides in MHC II fl/fl and MHC II ΔIEC mice. Data are shown as mean ± SEM, and statistical analysis was performed using multiple unpaired t-tests with Holm-Šídák correction (α = 0.05). * P < 0.05. (D) MHC II ligandome shift represented by a Venn diagram showing the distribution of the discovered peptides across intestinal compartments of MHC II ΔIEC . Counts are pooled across replicates after filtering. (E) Cellular reprogramming identified using Reactome pathway enrichment analysis of unique source proteins from the villus peptidome of MHC II fl/fl compared to MHC II ΔIEC mice by the ReactomePA v1.16.2 R package . Significance was assessed using Benjamini-Hochberg correction, q < 0.05. (F) The overlap between the source proteins of the LP immunopeptidome in MHC II fl/fl and MHC II ΔIEC mice is shown in an area-proportional Venn diagram. Counts are pooled across replicates. (G) Identified peptide cores from ECM-remodeling proteins. Rows are cores followed by source proteins. Identifications represent the number of biological replicates in which a peptide core was detected (per compartment). (H) Immunofluorescence analysis of distal SI sections from MHC II fl/fl and MHC II ΔIEC mice. Representative images (left panels) show Ly6G (green), E-Cadherin (white), Olfm4 (red), and nuclei (blue). Scale bar 20 µm. Quantification of Ly6G + cells per field of view (FOV; 20x), where each point represents individual FOV (Right panel). Statistical analysis was performed using a two-tailed unpaired Student’s t-test ( n = 2 mice per group); data shown as mean ± SEM. * P < 0.05. (I) Neutrophil infiltration in MHC II ΔIEC mice compared to MHC II fl/fl controls assessed by flow cytometry analysis of the distal SI (left panels). Percentage of CD11b + Ly6G + neutrophils out of CD45 + cells displayed as mean ± SEM (right panel). Statistical analysis was done using a two-tailed unpaired t-test with Welch’s correction on n = 4 mice of MHC II fl/fl vs. n = 3 mice of MHC II ΔIEC . * P < 0.05. (J) H&E staining of distal SI sections from MHC II fl/fl and MHC II ΔIEC mice (left panels) and quantification of inflammatory lesions per 10 mm of mucosal layer shown as mean ± SEM (right panel). Scale bar: 50µm. Statistical significance was assessed using two-tailed unpaired t-test . (K) Fibroblast accumulation in MHC II ΔIEC mice was identified by immunofluorescence staining of distal SI sections stained for PDPN (green) and CD45 (purple). Nuclei stained with DAPI (blue). Scale bar: 20µm.

Article Snippet: Sections were deparaffinized with standard techniques, incubated with primary antibodies for Ly6G (1:100, Biolegend 127606), PDPN (1:100, BioLegend 127402), Olfm4 (1:200) (Cell Signaling Technology, 39141S), Mmp9 (1:100, HUA-ET1704-69-HuaBio) and E-cadherin (1:100) (BD, BD610182) overnight at 4°C, followed by secondary antibodies incubation (1:400, Abcam) at room temperature for 30 min and Hoechst 33342 (1:1000) (TargetMol, T5840).

Techniques: Isolation, Purification, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Two Tailed Test, Flow Cytometry, Staining