Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Endoglin is highly expressed on CAFs in human pancreatic tumors. ( A ) Representative images of human pancreatic cancer (representative from n = 25 PDAC patients) and normal pancreas stained for α-SMA, endoglin, cytokeratin, and vimentin. Endothelial cells (black arrow) and endoglin expressing CAFs (white arrow). ( B ) Immunofluorescent double staining for α-SMA and endoglin in human PDAC tumors. ( C ) Endoglin mRNA expression by human cells; ECRF endothelial cells, MIA PaCa-2, PANC-1 and BxPC-3 PDAC cells and 8 patient derived primary pancreatic CAFs. ( D ) Endoglin protein expression on human pancreatic fibroblasts. Basal and BMP9-induced downstream signaling (pSMAD1) was inhibited with TRC105 (full-length blot shown in Supplementary figure 4A – C ).

Article Snippet: The mouse PDAC cell line KPC-3 (Kras G12D/+ LSL-Trp53 R172H/+ Pdx-1-Cre), (kindly supplied by the department of Immunology, LUMC) with a targeted insertion of codon-optimized Luc-2 (pGL4.10) [luc2] (Promega Leiden, the Netherlands), mouse MC38 cells (kindly supplied by the department of Immunology, LUMC) and primary fibroblasts were all cultured in DMEM/F12 glutamax medium (Invitrogen, Landsmeer, the Netherlands), with 10% fetal bovine serum (FBS) (Gibco, Bleiswijk, the Netherlands), 0.01 M HEPES, 0.1 μg/mL Gentamycin, 40U/mL Penicillin and 40 μg/mL Streptomycin (all Invitrogen Landsmeer, the Netherlands) at 37°C and 5% CO2.

Techniques: Staining, Expressing, Double Staining, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: PDA cells reprogram macrophages in TME through DNA methylation. a Tumor and macrophage co-culture experimental schema. b Nqo-1 and Aldh1a3 methylation was examined by methylation-specific PCR (MSP) in mouse BMDMs after co-culturing with KPC PDA cells. * P < 0.05 (paired t test). c The schema of the candidate gene selection process. d Expression of the key genes in glucose metabolism and OXPHOS pathway in mouse BMDMs after co-culturing with KPC cells. The mRNA expression of these genes was measured by RT-PCR and β-actin was used for normalization. e , f Nqo - 1 and Aldh1a3 methylation after pretreating BMDMs with DAC. * P < 0.05 ( d , e , f , Mann–Whitney U test) . g Methylation of Nqo - 1 and Aldh1a3 in TAMs, CD4 + and CD8 + T cells from primary PDA and BMDMs (consider as M0 macrophages) of the same KPC mice, and BMDMs after co-culturing with KPC cells. * P < 0.05 (ANOVA). Data are means ± SEM from technical duplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: DNA Methylation Assay, Co-Culture Assay, Methylation, Selection, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: DNA methylation of the metabolism genes in macrophages is induced by direct interaction with PDA cells through GARP/TGF-βRII-integrin αV/β8. a , b Nqo-1 and Aldh1a3 methylation in mouse BMDMs in a transwell system separated from KPC cells by an 8-μm or 1-μm pore membrane that, respectively, allows or not allows tumor cells to migrate through and direct contract with macrophages and in BMDMs cultured with TCM. Nqo-1 and Aldh1a3 methylation quantified as described in Supplementary Methods. * P < 0.05 (Mann–Whitney U test). c Lucifer Yellow labeled-KPC cells were co-cultured with unlabeled BMDMs. Thick arrows indicate macrophages that contain Lucifer Yellow spread from KPC cells (thin arrow) around them. Scale bar: 20 μm. d GARP expression on M0, M1-like, and M2-like macrophages measured by immunofluorescent staining with FITC-conjugated anti-GARP antibody. Arrow indicates macrophages that have the highest fluorescence within each image. Scale bar: 20 μm. * P < 0.05 (ANOVA). Histogram (right panel) shows quantification of fluorescence intensity. e Multiplex immunohistochemistry (IHC) was performed on a single slide of human PDA tissues for GARP (in green), CD68 (in red) and CD163 (in purple). A representative among 20 human PDAs tested is shown. Arrows (both panels) indicate GARP-expressing CD68 + CD163 + (M2-like) macrophages ; and arrowheads (left panel) indicate GARP-expressing CD68 + CD163 - (M1-like) macrophages . Notched arrowheads (right panel) indicate CD68 + CD163 + (M2-like) macrophages with little GARP expression. Scale bar: 50 μm. f Multiplex IHC staining of GARP (in green) on F4/80 + (in red) macrophages in PDAs from KPC mice. Scale bar: 50 μm. g TGF-βRII and GARP on cell surface of M0, M1-like, and M2-like macrophages co-stained and analyzed by flow cytometry. h Quantification of the percentages of TGF-βRII on cell surface of M0, M1-like, and M2-like macrophages by flow cytometry. * P < 0.05 (ANOVA). i Integrin subunits ɑV and β8 cell-surface expression was measured by flow cytometry. j IHC staining of PDA and normal pancreas tissues from KPC mice with anti-integrin ɑV and β8 antibodies. Scale bar: 100 μm. Data were from technical triplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: DNA Methylation Assay, Methylation, Membrane, Cell Culture, MANN-WHITNEY, Labeling, Expressing, Staining, Fluorescence, Multiplex Assay, Immunohistochemistry, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: GARP mediates Nqo-1 methylation and M2-like phenotypical changes in M1-like macrophages after co-culturing with PDA cells. a Nqo-1 methylation in mouse WT M1-like macrophages compared to GARP KO M1-like macrophages. * P < 0.05 (paired t test). b RT-PCR of M2 marker genes in WT vs. GARP KO M1-like macrophages after co-cultured with KPC cells. Fold changes of these marker genes in co-cultured vs. monocultured M1-like macrophages were shown. Fold change >1: upregulation; fold change <1: downregulation. All results were first normalized by respective β-actin and then respective monocultured BMDMs. * P < 0.05 (Mann–Whitney U test). c Expression of the M2 cytokine IL-10 in WT vs. GARP KO M1-like macrophages after co-culturing with KPC cells, measured by flow cytometry analysis of percentages of IL-10-positive cells with intracellular staining of IL-10. * P < 0.05 (Mann–Whitney U test). d Fold changes of MSP results of the Nqo-1 gene in co - cultured vs. monocultured M1 - like macrophages treated with RGD or TGF-βRII blocking antibody. * P < 0.05 (ANOVA). e Fold changes of RT-PCR results of M2 marker genes in co-cultur e d vs. monocultured M1-like macrophages treated with RGD or TGF-βRII blocking antibody. Data were first normalized by respective β-actin and then respective monocultured M0 macrophages. * P < 0.05 (ANOVA). f Mitochondrial membrane potentials in mouse M0, M1-like and M2-like macrophages after co-culturing with KPC cells by measuring mean fluorescence intensity of TMRM signals on the PE channel of flow cytometry, comparing mono- vs. co-cultured macrophages. * P < 0.05 (ANOVA). g Glucose uptake activities in M0, M1-like, and M2-like macrophages by measuring mean fluorescence intensity of 2-NBDG signals, comparing mono- vs. co-cultured macrophages. * P < 0.05 (ANOVA). h KPC cells were co-cultured with mouse BMDMs or DAC pretreated BMDMs in upper chamber of a transwell system with 8-μm pore membrane that allows them migrating to the lower chamber. Migrated KPC cells were examined by immunofluorescent staining with FITC-conjugated anti-Pan-CK antibody and counted. Fold changes of migrated KPC cell number in co-cultured vs. monocultured group (normalized as 1) were shown. * P < 0.05 (Mann–Whitney U test). i KPC cells were co-cultured with BMDMs pretreated with DAC, glucose uptake inhibitor WZB-117, or DAC + WZB-117, respectively, in the transwell system. Numbers of migrated KPC cells were counted as described in ( h ) and shown. * P < 0.05 (ANOVA). j Il-10 expression per RT-PCR in untreated, DAC, or WZB-117 pretreated BMDMs before (normalized as 1) and after co-culturing with KPC cells. * P < 0.05 (Mann–Whitney U test). Data are means ± SEM from technical duplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: Methylation, Reverse Transcription Polymerase Chain Reaction, Marker, Cell Culture, MANN-WHITNEY, Expressing, Flow Cytometry, Staining, Blocking Assay, Membrane, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: Downregulation of genes in the metabolic pathway in TAMs from murine PDA. a mRNA expression of metabolism genes as indicated were measured by RT-PCR in TAMs, CD4 + , and CD8 + T cells from primary pancreatic tumors and BMDMs of the same KPC mice. Tumors were identified by ultrasound before sacrifice. β-actin used for normalization. Data are means ± SEM from triplicates and representative of two experiments. * P < 0.05 (ANOVA). b The schematic model of the GARP/integrin-mediated interaction between tumor cells and macrophages in the TME of PDAC and the mechanisms of metabolic, phenotypical, and functional reprogramming of macrophages from M1-like to M2-like macrophages in a DNA methylation-dependent manner

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, DNA Methylation Assay

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A – C ) RT-qPCR analysis to monitor ( A ) efficacy of CRISPR/Cas9-mediated Slfn5 disruption, ( B ) expression of Slfn5 in response to IFN-β treatment (5,000 IU for 6 hours) in Slfn5 WT cells, and ( C ) IFN-β–mediated (5,000 IU for 6 hours) induction of indicated murine ISGs in Slfn5 -WT and Slfn5 -KO cells. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over the corresponding controls, and the graphs represent mean ± SEM of 3 independent experiments. * P < 0.05, *** P < 0.001 by 2-tailed unpaired t test with Welch’s correction ( A and B ) or 1-tailed unpaired t test with Mann-Whitney test ( C ). ( D ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated in 6-well plates and counted on days 1, 2, and 3 after seeding. Data are mean of number of cells ± SEM of 3 independent experiments, each done in duplicate. Two-way repeated-measures ANOVA with Šidák’s multiple-comparison test; ** P < 0.01, **** P < 0.0001. ( E ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated into round-bottom 96-well plates under stem cell–permissive conditions to form 3D spheroids. After 7 days, spheres were imaged using a Cytation 3 cell imaging multi-mode reader to determine cross-sectional area. Data are expressed as percentages of WT parental spheres and represent mean ± SEM of 3 independent experiments, each done in triplicate. Two-tailed unpaired t test with Welch’s correction; * P ≤ 0.05.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Quantitative RT-PCR, CRISPR, Disruption, Expressing, Control, MANN-WHITNEY, Comparison, Imaging, Two Tailed Test

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A ) KPC1199 luciferase-expressing Slfn5 -WT or Slfn5 -KO cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of male and female C57BL/6J mice. Mice were randomized into the following treatment groups: Slfn5 WT PBS ( n = 7), Slfn5 WT IFN-α ( n = 7), Slfn5 KO PBS ( n = 7), and Slfn5 KO IFN-α ( n = 8). Where indicated, mice were injected subcutaneously with 600 ng murine IFN-α per mouse once per week for 2 weeks. Tumor growth was monitored at least once per week by bioluminescence imaging (BLI). Normalized BLI values are shown. On day 17, model-based estimate of the mean difference in BLI signal between WT IFN-α and Slfn5 -KO IFN-α was 3.91 × 10 9 photons/s/cm 2 /sr (95% CI 1.72 × 10 9 to 6.09 × 10 9 ). Two-way ANOVA with Šidák’s multiple-comparison test for day 17. Comparison of BLI signals on day 17 is based on mixed-effects model up to day 17. Data are expressed as mean ± SEM of tumor BLI signals for each genotypic treatment group; **** P < 0.0001. ( B ) Survival curves of mice bearing Slfn5 -WT and Slfn5 -KO pancreatic tumors. Survival was estimated using the method of Kaplan-Meier and groups were compared using the log-rank test; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( C ) Median survival time (days) for indicated genotypic treatment groups was estimated using Simple Survival Analysis (Kaplan-Meier) in GraphPad.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Luciferase, Expressing, Injection, Imaging, Comparison

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: KPC1199 luciferase-expressing CTRL or Slfn5 -KO cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of 6- to 8-week-old male and female mice that were grouped as Rag1 WT + CTRL ( n = 9), Rag1 WT + Slfn5 KO ( n = 11), Rag1 KO + CTRL ( n = 11), and Rag1 KO + Slfn5 KO ( n = 11). In the Rag1 WT + Slfn5 KO group, 5 mice were still alive on day 60. Survival curves of indicated mice are shown. Survival was estimated using the method of Kaplan-Meier and groups were compared using the log-rank test; *** P < 0.001; **** P < 0.0001.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Luciferase, Expressing, Injection

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A – J ) Immunophenotypic analysis of tumor-bearing pancreases by multicolor flow cytometry. ( A , C , E , G , and I ) CTRL ( n = 10) and Slfn5 -KO ( n = 9) KPC1199 luciferase-expressing cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of C57BL/6J mice and 7 days after cell implantation CTRL and Slfn5 -KO tumor–bearing pancreases were harvested and processed for immunophenotypic analysis by multicolor flow cytometry. ( B , D , F , H , and J ) CTRL ( n = 10) and Slfn5 -KO ( n = 11) KPC1199 luciferase-expressing cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of C57BL/6J mice and 21 days after cell implantation CTRL and Slfn5 -KO tumor–bearing pancreases were harvested and processed for immunophenotypic analysis by multicolor flow cytometry. Scatter dot plots show the percentage of tumor-infiltrating cells. ( A and B ) Immunosuppressive cells, i.e., TAMs (left panel), Tregs (middle panel), and TAMCs (right panel). ( C and D ) Innate myeloid immune cells, i.e., M1 macrophages (left panel) and M2 macrophages (right panel). ( E and F ) Effector cells, i.e., NK cells (left panel), CD8 + T cells (middle panel), and CD4 + T cells (right panel). ( G and H ) Activation markers (IFN-γ, GZMB, CD69) of NK cells, and ( I and J ) of CD8 + cells. Data are expressed as mean ± SEM of percentages of indicated immune infiltrates as detailed in . Two-tailed unpaired t test with Mann-Whitney test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Flow Cytometry, Luciferase, Expressing, Injection, Activation Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A Schematic diagram for the construction of colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 ). B ALKBH5 knockin efficacy in colon crypts by anti-ALKBH5 (anti-ALKBH5 antibody). C Ki67 abundance as determined by IF staining (anti-Ki67 antibody, n = 89 crypts for WT mice, n = 89 crypts for A5-cKI mice. Student’s t -test, two-sided.) D MUC2 abundance as determined by IF staining (anti-MUC2 antibody, n = 127 crypts for WT mice, n = 89 crypts for A5-cKI mice. Student’s t -test, two-sided). E ChgA abundance as determined by IF staining (anti-ChgA antibody, n = 100 crypts for WT mice, n = 65 crypts for A5-cKI mice. Student’s t -test, two-sided). F Apoptosis as determined by TUNEL staining (anti-TUNEL probe, n = 58 crypts for WT mice, n = 58 crypts for A5-cKI mice. Student’s t -test, two-sided). G AOM-DSS-induced CRC study design. The diagram is created in BioRender. Chou, H. (2025) https://BioRender.com/uax7xbx . H Representative images of colon tumors after harvesting. I Colon tumor number (Student’s t -test, two-sided) and load (Student’s t -test, two-sided), ( n = 11 for WT mice and n = 12 for A5-cKI mice). J ALKBH5 knock in efficacy in tumor tissues was validated by IF staining (anti-ALKBH5 antibody). K Western blot of Alkbh5 and LGR5 and CD133 expression in tumor tissues (upper panel). Quantitative analysis of ALKBH5, LGR5 and CD133 ( n = 5, each dot represents an independent mouse. Student’s t -test, two-sided) protein expression, normalized to GAPDH (lower panel). L Representative images of H&E staining and pathological grading into LGD, HGD and adenocarcinoma (Chi-square, two-sided). Presented data is representative of 2 independent batches of transgenic mice. M Ki67 expression as determined by IF staining (anti-Ki67 antibody, n = 9 for WT mice, n = 7 for A5-cKI mice. Student’s t -test, two-sided). N Apoptosis as determined by TUNEL staining (anti-TUNEL probe, n = 8 for WT mice, n = 9 for A5-cKI mice. Student’s t -test, two-sided). O LGR5 (anti-LGR5 mRNA probe) expression as determined by FISH staining ( n = 8 for WT mice, n = 8 for A5-cKI mice. Student’s t -test, two-sided). P CD133 (anti-CD133 antibody) expression as determined by IF staining ( n = 9 for WT mice, n = 6 for A5-cKI mice. Student’s t -test, two-sided). Q Self-renewal capacity of primary CRC organoid (WENR medium) from WT mice and A5-cKI mice under a light microscope (left panel). Surface area of CRC organoids from WT mice and A5-cKI mice (right panel, n = 107 organoids for WT mice, n = 79 organoids for A5-cKI mice. Student’s t -test, two-sided). R KI67 + proportion of primary CRC organoid from WT mice and A5-cKI mice as determined by flow cytometry ( n = 4, each dot represents an independent sample. Student’s t -test, two-sided). S Apoptosis cells of primary CRC organoid from WT mice and A5-cKI mice as determined by flow cytometry ( n = 4, each dot represents an independent sample. Student’s t -test, two-sided). T CD133 + proportion of primary CRC organoid from WT mice and A5-cKI mice as determined by flow cytometry ( n = 4, each dot represents an independent sample. Student’s t -test, two-sided). Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: Knock-In, Staining, TUNEL Assay, Western Blot, Expressing, Transgenic Assay, Light Microscopy, Flow Cytometry, Standard Deviation

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A Schematic diagram for the construction of colon stem cell-specific Alkbh5 knockout mice (Alkbh5 flox/flox LGR5-Cre ERT2 ). B AOM-DSS-induced CRC workflow. The diagram is created in BioRender. Chou, H. (2025) https://BioRender.com/uax7xbx . C Representative images of colon tumors after harvesting. D Tumor number (Student’s t -test, two-sided) and load (Student’s t -test, two-sided). ( n = 11 for WT mice and n = 9 for A5-cKO mice). E Western blot validation of Alkbh5 knockout and LGR5, CD133, CD44 and EpCAM in tumor tissues (left panel). Quantitative analysis of ALKBH5, LGR5, CD133, CD44 and EpCAM ( n = 5, each dot represents an independent mouse. Student’s t -test, two-sided) protein expression, normalized to GAPDH (right panel). F Representative images of H&E staining and pathological analysis of LGD, HGD and adenocarcinoma (Chi-square, two-sided). Presented data is representative of 2 independent batches of transgenic mice. G Ki67 abundance as determined by IHC staining (anti-KI67 antibody, n = 32 for WT mice, n = 16 for A5-cKO mice. Student’s t -test, two-sided). H LGR5 (anti-LGR5 mRNA probe) expression as assessed by FISH staining ( n = 8 for WT mice, n = 6 for A5-cKO mice. Student’s t -test, two-sided). I CD133 (anti-CD133 antibody) expression as assessed by IF staining ( n = 8 for WT mice, n = 10 for A5-cKO mice. Student’s t -test, two-sided). J Growth of primary organoids (WENR medium) from Alkbh5 knockout mice observed under microscope (left panel). Surface area of CRC organoids from WT mice and A5-cKO mice (right panel, n = 92 organoids for WT mice, n = 8 organoids for A5-cKO mice. Student’s t -test, two-sided). K CD133 + proportion of primary CRC organoid from WT mice and A5-cKO mice as determined by flow cytometry ( n = 4, each dot represents an independent sample. Student’s t -test, two-sided). L LGR5 EGFP+ proportion of primary CRC organoid from WT mice and A5-cKO mice as determined by flow cytometry ( n = 8 for WT and n = 6 for A5-cKO, each dot represents an independent sample. Student’s t -test, two-sided). Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: Knock-Out, Western Blot, Biomarker Discovery, Expressing, Staining, Transgenic Assay, Immunohistochemistry, Microscopy, Flow Cytometry, Standard Deviation

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A In vitro LDA on POP66 (Chi-Square, two-sided) and CSC28 (Chi-Square, two-sided) with ALKBH5 knockdown. B In vitro LDA on POP66 (Chi-Square, two-sided) and CSC28 (Chi-Square, two-sided) with ALKBH5 overexpression. C LGR5, CD133 and CD44 protein expression in POP66 and CSC28 with ALKBH5 knockdown or overexpression. D In vivo LDA on POP66 with ALKBH5 knockdown (Chi-Square, two-sided). E In vivo LDA on CSC28 after ALKBH5 knockdown (Chi-Square, two-sided). F Effect of ALKBH5 knockdown on the growth of CRC organoids PDO-816 (left panel) ( n = 19 views for shNC organoids, n = 20 views for shA5-1 organoids, n = 20 views for shA5-2 organoids. one-way ANOVA) and PDO-828 ( n = 15 views for shNC organoids, n = 15 views for shA5-1 organoids, n = 15 views for shA5-2 organoids. one-way ANOVA) (EN medium). LGR5, CD133 and CD44 protein expression in PDO816 and PDO828 with ALKBH5 knockdown (right panel). G Effect of ALKBH5 overexpression on the growth of CRC organoids PDO-816 (left panel) ( n = 15 views for EV organoids, n = 15 views for A5-OE organoids. Student’s t -test, two-sided) and PDO-828 ( n = 16 views for EV organoids, n = 16 views for A5-OE organoids. Student’s t -test, two-sided) (EN medium). LGR5, CD133 and CD44 protein expression in PDO816 and PDO828 with ALKBH5 overexpression (right panel). Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: In Vitro, Knockdown, Over Expression, Expressing, In Vivo, Standard Deviation

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A Dot blot assay of CSC28 with ALKBH5 knockdown and overexpression. B Work-flow for systematic identification of ALKBH5 downstream targets. C Top enriched differential pathways in ALKBH5-knockdown versus control CSC28 cells by RNA-seq. D The top enriched motifs based on m 6 A peaks in ALKBH5-knockdown CSC28 cells and control cells. P values for m6A motif were derived using the Fisher’s exact test algorithm in HOMER software (upper panel). Differential m 6 A methylated peaks after ALKBH5 knockdown or overexpression, as determined by MeRIP-seq (lower panel). E Top differential enriched pathways in ALKBH5-knockdown versus control CSC28 cells by Ribo-seq. F Integrated analysis of RNA-seq, MeRIP-seq and Ribo-seq datasets (left panel). The correlation between ALKBH5 and FAM84A mRNA in TCGA (Pearson, two-sided) GEPIA (Pearson, two-sided) datasets and foldchange in RNA-seq (Student’s t -test, two-sided) and Ribo-seq (Student’s t -test, two-sided) (middle panel). The correlation between ALKBH5 and FAM84A mRNA in GSE39582 (Pearson, two-sided) and GSE17536 (Pearson, two-sided) (right panel). G UCSC snapshots of MeRIP-seq reads of FAM84A. Normalized to RNA input. H m 6 A abundance on FAM84A as revealed by MeRIP-qPCR. Schematic design of MeRIP-qPCR assay (left panel). m 6 A modification on FAM84A as determined by MeRIP-qPCR (For POP66, n = 3; For CSC28, n = 6, each dot represents an independent sample. One-way ANOVA) (middle panel). m 6 A modification on FAM84A after overexpression of WT ALKBH5 (A5-OE) or mutant ALKBH5 (A5 H204A ) in CSC ( n = 3, each dot represents an independent sample. One-way ANOVA) (right panel). I Direct Binding between ALKBH5 and FAM84A mRNA, as determined by RIP-qPCR ( n = 3, each dot represents an independent sample. Student’s t -test, two-sided). J FAM84A expression as determined by qPCR ( n = 3, each dot represents an independent sample. One-way ANOVA). K m 6 A levels of FAM84A mRNA (left panel), the binding of FAM84A mRNA to ALKBH5 (middle panel) and FAM84A mRNA expression (right panel) in WT and ALKBH5-cKI mice tumor as revealed by MeRIP-qPCR ( n = 3, each dot represents an independent mouse. Student’s t -test, two-sided), ALKBH5-RIP-qPCR (Presented data is representative of 3 independent biological replicates. Student’s t -test, two-sided) and qPCR ( n = 3, each dot represents an independent mouse. Student’s t -test, two-sided), respectively. The diagram is created in BioRender. Chou, H. (2025) https://BioRender.com/csvnp3n . L Schematic diagram of site-specific RNA targeting using dCas13b-ALKBH5 fusion proteins with gRNA close to the target site. M Establishment of dCas13b-ALKBH5 system in colorectal CSCs as determined by western blot (left panel). FAM84Am 6 A modification levels (right panel) in colorectal CSCs co-transfected with dCas13b-ALKBH5 and the gRNA (For POP66, n = 4; for CSC28, n = 3, each dot represents an independent sample. Student’s t -test, two-sided). Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: Dot Blot, Knockdown, Over Expression, Control, RNA Sequencing, Derivative Assay, Software, Methylation, Modification, Mutagenesis, Binding Assay, Expressing, Western Blot, Transfection, Standard Deviation

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A FAM84A expression as determined by western blot in CSC. B FAM84A protein expression as determined by western blot in tumor tissues from transgenic mice. C Correlation between ALKBH5 and FAM84A protein expression in the TMA patient cohort ( N = 164, Chi-square, two-sided). D FAM84A mRNA stability as determined by qPCR after ALKBH5 knockdown in POP66. RNA decay rate was normalized to expression at 0 hr. ( n = 3, each dot represents an independent sample. Two-way ANOVA) E FAM84A mRNA stability as determined by qPCR after ALKBH5 overexpression in POP66. RNA decay rate was normalized to expression at 0 hr. ( n = 3, each dot represents an independent sample. Two-way ANOVA) F Plasmid design for the luciferase reporter assay with FAM84A WT 5’UTR (upper panel), luciferase activity of FAM84A WT reporter with ALKBH5 knockdown and FAM84A WT reporter with the overexpression of WT ALKBH5 (A5-OE) or mutant ALKBH5 (A5 H204A ) in POP66 ( n = 3, each dot represents an independent sample. One-way ANOVA) (lower panel). G Plasmid design for luciferase reporter assay with mutated 5’UTR (FAM84A Mut ) sequences added to the 5’ end of luciferase gene (upper panel), luciferase activity of FAM84A Mut reporter with ALKBH5 knockdown and FAM84A Mut reporter with the overexpression of WT ALKBH5 (A5-OE) in POP66 ( n = 3, each dot represents an independent sample. One-way ANOVA) (lower panel). H Heatmap for m 6 A reader screening. I Schematic diagram for RIP-qPCR assay (left panel). Binding between IGF2BP1 protein and FAM84A mRNA was determined by RIP-qPCR in POP66 ( n = 3, each dot represents an independent sample. Student’s t -test, two-sided) (right panel). J Effect of IGF2BP1 knockdown on the stability of FAM84A mRNA in POP66 with or without ALKBH5 knockout ( n = 3, each dot represents an independent sample. Two-way ANOVA). K Effect of IGF2BP1 knockdown on FAM84A mRNA ( n = 3, each dot represents an independent sample. One-way ANOVA) (left panel) and protein (right panel) in POP66 with or without ALKBH5 knockout. Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: Expressing, Western Blot, Transgenic Assay, Knockdown, Over Expression, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Binding Assay, Knock-Out, Standard Deviation

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A Representative images of FAM84A staining in TMA cohort (left panel). Low FAM84A protein expression predicts poor survival of CRC patients (Log-rank test) (right panel). B FAM84A knockout rescued the inhibitory effect of ALKBH5 depletion on self-renewal as determined by in vitro LDA in POP66 and CSC28 (Chi-Square, two-sided). C FAM84A knockout rescued the inhibitory effect of ALKBH5 depletion on CD133, LGR5 expression as determined by western blot in POP66. D ALKBH5-induced self-renewal as determined by in vitro LDA in POP66 and CSC28 (Chi-Square, two-sided). E ALKBH5-induced CD133, LGR5 expression was abolished by the ectopic expression of FAM84A in POP66 and CSC28. F FAM84A interacting proteins was isolated by co-immunoprecipitation, followed by silver staining and mass spectrometry analysis. The diagram is created in BioRender. Chou, H. (2025) https://BioRender.com/d48babm . G The binding between FAM84A and β-catenin as determined by IP. H Abundance of total β-catenin and active β-catenin as determined by western blot in CSC28. I Abundance of active β-catenin as determined by western blot analysis of cytoplasm and nuclear protein fractions from CSC28. J Interaction between β-catenin with GSK-3β and Axin-2 as revealed by co-immunoprecipitation assay in CSC28. K β-catenin expression and ubiquitination in CSC28. L Expression of total β-catenin as determined by western blot with or without MG132 treatment in CSC28. M β-catenin protein expression in POP66 and CSC28 with ALKBH5 and/or FAM84A overexpression. N ubiquitination levels in POP66 and CSC28 with ALKBH5 and/or FAM84A overexpression. O β-catenin protein expression in tumor tissues from WT mice and A5-cKO mice ( n = 5, each dot represents an independent mouse. Student’s t -test, two-sided). P Nuclear active β-catenin levels as determined by IF (anti-active β-catenin antibody) staining in ALKBH5-cKI mice ( n = 5 for WT mice and n = 8 for A5-cKI mice. Student’s t -test, two-sided) compared with wildtype mice. Q Nuclear active β-catenin levels as determined by IF (anti-active β-catenin antibody) staining in A5-cKO mice ( n = 9 WT mice and n = 10 for A5-cKO mice. Student’s t -test, two-sided) compared with wildtype mice. Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: Staining, Expressing, Knock-Out, In Vitro, Western Blot, Isolation, Immunoprecipitation, Silver Staining, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Over Expression, Standard Deviation

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A ROC curve based on ALKBH5 expression level in chemo-responder and non-responder groups ( GSE72968 ). B ALKBH5 and FAM84A protein expression with 5-FU or OXA treatment. C IC 50 of Oxaliplatin (OXA) ( n = 6, each dot represents an independent sample) (upper panel) and 5-FU ( n = 6, each dot represents an independent sample for both POP66 and CSC28) (lower panel) as determined by cell viability assay with or without ALKBH5 overexpression in CSC. D Flow cytometry of OXA-induced apoptosis in POP66 and CSC28 ( n = 3, each dot represents an independent sample. One-way ANOVA) with or without ALKBH5 overexpression (upper panel). ALKBH5 overexpression abolished 5-FU-induced apoptosis in POP66 and CSC28 ( n = 3, each dot represents an independent sample. One-way ANOVA) (lower panel). E Expression of apoptosis markers in POP66 with or without ALKBH5-overexpression after 5-FU (upper panel) Oxaliplatin (lower panel) treatment. F Flow cytometry of OXA-induced apoptosis in POP66 and CSC28 ( n = 3, each dot represents an independent sample. One-way ANOVA) with or without ALKBH5 knockdown. G Flow cytometry of 5-FU-induced apoptosis in POP66 and CSC28 ( n = 3, each dot represents an independent sample. One-way ANOVA) with or without ALKBH5 knockdown. H Expression of apoptosis markers in POP66 and CSC28 with or without ALKBH5-knockdown after OXA I 5-FU treatment. J Effect of ALKBH5 knockdown in combination with OXA ( n = 16 views for shNC, n = 14 views for shA5-1, n = 20 views for shA5-2, n = 20 views for shNC+OXA, n = 15 views for shA5-1 + OXA, n = 12 views for shA5-2 + OXA. One-way ANOVA) (upper panel) and 5-FU ( n = 20 views for shNC, n = 20 views for shA5-1, n = 20 views for shA5-2, n = 20 views for shNC+5-FU, n = 20 views for shA5-1 + 5-FU, n = 20 views for shA5-2 + 5-FU. One-way ANOVA) (lower panel) treatment in PDO-816. K Effect of ALKBH5 knockdown in combination of OXA ( n = 3, each dot represents an independent sample. One-way ANOVA) (upper panel) and 5-FU ( n = 4, each dot represents an independent sample. One-way ANOVA) (lower panel) by flow cytometry of apoptosis in PDO-816. Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: Expressing, Viability Assay, Over Expression, Flow Cytometry, Knockdown, Standard Deviation

Journal: Nature Communications

Article Title: Targeting of the m 6 A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer

doi: 10.1038/s41467-025-67502-0

Figure Lengend Snippet: A Schematic diagram showing the treatment strategy for AOM/DSS-induced CRC in transgenic mice. The diagram is created in BioRender. Chou, H. (2025) https://BioRender.com/uax7xbx . B Colon tumor number (One-way ANOVA) and load (One-way ANOVA) at end point. ( n = 10 for WT, n = 6 for A5-cKO, n = 16 for WT + 5-FU, n = 12 for A5-cKO+5-FU, n = 15 for WT + OXA, n = 13 for A5-cKO+OXA mice). C Determination of Ki67 (anti-KI67 antibody) by IHC stanning of tumor tissues from transgenic mice ( n = 18 views for WT, n = 11 views for A5-cKO, n = 9 views for WT + 5-FU, n = 10 views for A5-cKO+5-FU, n = 16 views for WT + OXA, n = 13 views for A5-cKO+OXA mice. One-way ANOVA). D Determination of active β-catenin expression (anti- active β-catenin antibody) by IHC stanning of tumor tissues from transgenic mice ( n = 18 views for WT, n = 8 views for A5-cKO, n = 13 views for WT + 5-FU, n = 12 views for A5-cKO+5-FU, n = 10 views for WT + OXA, n = 10 views for A5-cKO+OXA mice. One-way ANOVA). E Expression of apoptosis markers in WT mice and A5-cKO mice after 5-FU (upper panel) or OXA (lower panel) treatment. ( n = 3, each dot represents an independent mouse) F ALKBH5 knockdown potentiated OXA efficacy in POP66 xenografts as indicated by image of tumor (left panel), tumor growth curve (Two-way ANOVA) (middle panel) and tumor weight (One-way ANOVA) (right panel) ( n = 8/group). G ALKBH5 knockdown potentiated OXA efficacy in CSC28 xenografts as indicated by image of tumor (left panel), tumor growth curve (Two-way ANOVA) (middle panel) and tumor weight (One-way ANOVA) (right panel) ( n = 8/group). H Determination of Ki67 (anti-KI67 antibody) expression by IHC staining of POP66 xenografts ( n = 8 for shNC, n = 8 for shNC+OXA, n = 8 for shA5, n = 8 for shA5+OXA. One-way ANOVA) I Determination of apoptosis by TUNEL staining of POP66 xenografts ( n = 9 for shNC, n = 10 for shNC+OXA, n = 6 for shA5, n = 7 for shA5+OXA. One-way ANOVA) J Construction of VNP-siRNA. K The treatment protocol in xenograft model. The diagram is created in BioRender. Chou, H. (2025) https://BioRender.com/4sm73ib . L Representative image of POP66 xenografts (left panel), tumor growth curve (Two-way ANOVA) (middle panel) and tumor weight (One-way ANOVA) (right panel) treated with VNP-siALKBH5 and/or OXA ( n = 8/group). M Expression of FAM84A, active β-catenin and stemness markers after VNP-siALKBH5 treatment as determined by western-blot (upper panel) ( n = 5 independent mouse.) Expression of apoptosis markers (lower panel) in POP66 with or without VNP-siALKBH5 after OXA treatment. ( n = 3 independent mouse.) N Representative image of CSC28 xenografts (left panel), tumor growth curve (Two-way ANOVA) (middle panel) and tumor weight (One-way ANOVA) (right panel) treated with VNP-siALKBH5 and/or OXA ( n = 8/group). O Expression of FAM84A, active β-catenin and stemness markers after VNP-siALKBH5 treatment as determined by western-blot (upper panel). ( n = 5 independent mouse.) Expression of apoptosis markers (lower panel) in CSC28 with or without VNP-siALKBH5 after OXA treatment. ( n = 3 independent mouse.). Centers and error bars represent mean and Standard deviation, respectively.

Article Snippet: Conditional Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 ) were generated by Shanghai Model Organisms Company, which were crossed to LGR5-Cre ERT2 mice to achieve colon stem cell-specific Alkbh5 knockin mice (Rosa26 lsl- Alkbh5 LGR5-Cre ERT2 , Alkbh5-cKI).

Techniques: Transgenic Assay, Expressing, Knockdown, Immunohistochemistry, TUNEL Assay, Staining, Western Blot, Standard Deviation