Journal: bioRxiv

Article Title: Comparison studies between Cesium-137 and X-ray irradiators in epithelial injury using in vitro and in vivo models

doi: 10.64898/2026.04.17.719248

Figure Lengend Snippet: RT-qPCR analysis of ( a ) Cdkn1a expression levels after 6Gy, ( b ) Cdkn1a expression levels after 8Gy, ( c ) Mki67 expression levels after 6Gy, ( d ) Mki67 expression levels after 8Gy, ( e ) Lgr5 expression levels after 6Gy, ( f ) Lgr5 expression levels after 8Gy, ( g ) Olfm4 expression levels after 6Gy, ( h ) Olfm4 expression levels after 8Gy, ( i ) Alpi1 expression levels after 6Gy, ( j ) Alpi1 expression levels after 8Gy, ( k ) Chga expression levels after 6Gy, and ( l ) Chga expression levels after 8Gy were calculated as a fold change at 6, 24, 48 and 72 h with either 137 Cs or X-ray irradiation. Actb was used as a housekeeping gene (control). Data points represent the average of three independent experiments, with the mean ±SD indicated. Significance was determined by the student’s test followed by an analysis of the normal distribution (Tukey’s test), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Commercially available TaqMan primers detecting mouse ChgA (Mm00514341-FAM), Alpi1 (Mm01285814-FAM), Olfm4 (Mm01320260-FAM), Cdkn1a (Mm00432448-FAM), Lgr5 (Mm00438890-FAM), Mki67 (Mm01278617-FAM), Actb (Mm04394036-VIC) and human CDKN1A (Hs00355782-FAM), KLF4 (Hs00358836-FAM), KLF5 (Hs0000000-FAM), BCL2 (Hs01048932-FAM), BAX (Hs00180269-FAM) and HPRT1 (Hs02800695-VIC) transcripts were used.

Techniques: Quantitative RT-PCR, Expressing, Irradiation, Control

Journal: bioRxiv

Article Title: Comparison studies between Cesium-137 and X-ray irradiators in epithelial injury using in vitro and in vivo models

doi: 10.64898/2026.04.17.719248

Figure Lengend Snippet: Representative images of immunohistochemical staining for OLFM4 ( a ) and SOX9 ( b ) in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 6-, 24-, 48-, and 96-h using 137 Cs irradiator, or X-ray irradiator, or 12Gy X-ray ABD irradiation. Scale bar = 100 µm.

Article Snippet: Commercially available TaqMan primers detecting mouse ChgA (Mm00514341-FAM), Alpi1 (Mm01285814-FAM), Olfm4 (Mm01320260-FAM), Cdkn1a (Mm00432448-FAM), Lgr5 (Mm00438890-FAM), Mki67 (Mm01278617-FAM), Actb (Mm04394036-VIC) and human CDKN1A (Hs00355782-FAM), KLF4 (Hs00358836-FAM), KLF5 (Hs0000000-FAM), BCL2 (Hs01048932-FAM), BAX (Hs00180269-FAM) and HPRT1 (Hs02800695-VIC) transcripts were used.

Techniques: Immunohistochemical staining, Staining, Irradiation

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