Journal: Cell reports

Article Title: Longitudinal phosphoproteomics reveals the PI3K-PAK1 axis as a potential target for recurrent colorectal liver metastases.

doi: 10.1016/j.celrep.2024.115061

Figure Lengend Snippet: Figure 4. Correlation analyses of phosphoproteomic data from CRC cell lines and public drug sensitivity data revealed PI3K inhibitors showing sensitivity to PAK1 activity (A) Volcano plot depicting the correlations between 117 drug responses (areas under the curve [AUCs]) and the PAK1 kinase activity scores (NESs) of 30 CRC cell lines. The x axis represents the correlation coefficient (Rho), and the y axis represents the distribution of p values. Inhibitors with shared mechanisms of action are labeled by color. (B) Correlations between the PAK1 NES and CTD2 AUCs for each PI3K inhibitor (IC-87114, AZD6482, TGX-221, XL765, and CAL-101). (C) Experimental design of the pharmacoproteomic analysis of 4 selected CRC cell lines treated with AZD6482. (D) Volcano plot depicting the enrichment of phosphoproteome kinase signatures. The x axis represents the change in the NESs between LoVo (left side) and other cells (HT-29, AK-CO-1, and SNU-C1) (right side). The dot size represents the relative number of scored phosphorylation sites in a signature. The dotted line indicates p = 0.05. (E) Bar plot showing the changes in the NES of PAK1 in response to AZD6482 treatment compared with the vehicle control treatment across the 4 cell lines. Two- tailed Welch’s t test was used to compare LoVo cells with HT-29, AK-CO-1, and SNU-C1 cells; *p < 0.05.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Colorectal cancer liver metastasis tumors and matched normal tumor-adjacent liver tissues Japanese Foundation for Cancer Research (Tokyo, Japan) see Table S1 Chemicals, peptides, and recombinant proteins 1 M triethyl ammonium bicarbonate Sigma Cat. #T7408 50% hydroxylamine Fujifilm WAKO Cat. #088-07221 Acetonitrile Kanto Pure Chemical Cat. #01033-76 Ammonium bicarbonate Fujifilm WAKO Cat. #017-02875 AZD6482 Selleck Cat. #S1462 BYL719 Selleck Cat. #S2814 CAL-101 (synonyms: idelalisib, GS-1101) Selleck Cat. #S2226 cOmpleteTM protease inhibitor cocktail Roche Cat. #11697498001 Copanlisib (synonym: BAY 80–6946) Selleck Cat. #S2802 Ethanol Fujifilm WAKO Cat. #057-00451 Formic acid Fujifilm WAKO Cat. #067-04531 HEPES Dojindo Cat. #340-01371 Iodoacetamide Nacalai Tesque Cat. #19302-54 IPI-145 (Synonym: INK1197) Selleck Cat. #S7028 Iron (III) chloride Sigma Cat. #451649 L-cysteine Nacalai Tesque Cat. #1030912 Lys-C Fujifilm WAKO Cat. #121-02541 Lysyl endopeptidase (LysC) Wako Cat. #129-02541 Matrigel Corning Cat. #354277 Oasis HLB cartridges Waters Cat. #WAT094225 Phosphatase Inhibitor Cocktail 2 Sigma Cat. #P5726 Phosphatase inhibitors Roche Cat. #4906845001 Phosphotyrosine (P-Tyr-1000) MultiMab antibodies Cell Signaling Technology Cat. #8954; RRID: AB_2687925 PhosSTOPTM phosphatase inhibitor cocktail Roche Cat. #4906837001 Protease inhibitors, EDTA-free Roche Cat. #4693132001 Sodium deoxycholate Nacalai Tesque Cat. #20117-12 Sodium dodecyl sulfate Fujifilm WAKO Cat. #192-08672 Sodium lauroyl-sarcosinate Nacalai Tesque Cat. #20117-12 Tandem mass tags–10-plex Thermo Fisher Scientific Cat. #90110 TMT 10-plex Isobaric Label Reagent Set plus TMT11-131C Label Reagent Thermo Scientific Cat. #A34808 TMT 11-plex reagents Thermo Scientific Cat. #A34808 TMTpro 16-plex Label Reagent Set Thermo Scientific Cat. #A44520 Trifluoroacetic acid Sigma Cat. #302031 Trifluoroacetic acid Fujifilm WAKO Cat. #204-02743 Tris(2-carboxyethyl)phosphine (TCEP) Fujifilm WAKO Cat. #209-19861 Trypsin Fujifilm WAKO Cat. #202-20081 Urea Fujifilm WAKO Cat. #219-00175 Critical commercial assays CellTiter-Glo luminescent cell viability kit Promega Cat. #G9241 Detergent-compatible (DC) protein assay kit Bio-Rad Cat. #5000111 (Continued on next page) Cell Reports 43, 115061, December 24, 2024 15

Techniques: Activity Assay, Labeling, Phospho-proteomics, Control, Two Tailed Test

Journal: NPJ Regenerative Medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: a B6- Ahr fl/fl mice and B6- Ahr fl/fl ;Foxn1-cre mice were treated by 5.5 Gy TBI. At days 0 and 28, thymii were collected and cells were dissected by digestion. Total thymus cells were counted. TECs and thymocyte subpopulations were counted according to total cell count and proportions from flow cytometric analysis ( n = 5 in each group). Day 0 indicates the samples were collected right before TBI. b , c Wild-type C57BL/6 mice, pretreated by 5.5 Gy TBI, were intraperitoneally injected with vehicle, CH-223191 or FICZ thrice weekly for 2 weeks. At day 14, thymus cells were counted as described ( n = 5). Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ** * , p < 0.001; n.s., not significant.

Article Snippet: B6- Ahr fl/fl ;Foxn1-cre mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice were ordered from Cyagen (Suzhou, China).

Techniques: Cell Counting, Injection

Journal: NPJ Regenerative Medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: a B6- Ahr fl/fl mice and B6- Ahr fl/fl ;Foxn1-cre mice, pretreated by 5.5 Gy TBI, were intraperitoneally injected with vehicle or rmIL-22 (200 ng) thrice weekly for 2 weeks. At day 14, thymus cells were counted as described ( n = 5). b , c Thymii were collected from irradiated mice. qPCR was performed to detect mRNA levels of IL-22 and IL-22RA1 ( n = 4). The values of control groups were set as 1, and the values of other groups were 2 –ΔΔCT relative to controls. d Thymic stromal cells were isolated from irradiated mice ( n = 3). Western blot was performed with indicated antibodies. Band intensity was analyzed using ImageJ. e Cryosections of thymii were stained by immunofluorescence with anti-IL-22RA1 ( n = 4). Fluorescence intensity was analyzed using ImageJ. Scale bar: 50 μm. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ** * , p < 0.001; n.s., not significant.

Article Snippet: B6- Ahr fl/fl ;Foxn1-cre mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice were ordered from Cyagen (Suzhou, China).

Techniques: Injection, Irradiation, Isolation, Western Blot, Staining, Immunofluorescence, Fluorescence

Journal: NPJ Regenerative Medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: a , b Thymic stromal cells were isolated from wild-type C57BL/6 mice treated by 5.5 Gy TBI. Day 0 indicates the samples were collected right before TBI. a qPCR was performed to detect mRNA levels ( n = 5). The values of Day 0 were set as 1. b Western blot was performed with indicated antibodies. c , d B6- Il22ra1 fl/fl mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice, pretreated by 5.5 Gy TBI, were intraperitoneally injected with vehicle, rmIL-22 (200 ng) or FICZ (0.1 mg/kg) thrice weekly for 2 weeks. At day 14, c thymus cells were counted as described ( n = 5). d H&E staining was performed on slides of thymus. Scale bar: 500 μm. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ** * , p < 0.001; n.s., not significant.

Article Snippet: B6- Ahr fl/fl ;Foxn1-cre mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice were ordered from Cyagen (Suzhou, China).

Techniques: Isolation, Western Blot, Injection, Staining

Journal: NPJ Regenerative Medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: In the cGVHD models, BALB/c mice were used as donors. Wild type C57BL/6 ( a ), B6- Il22ra1 fl/fl and B6- Il22ra1 fl/fl ;Foxn1-cre mice ( b ) were recipients. Wild type C57BL/6 recipients were treated by vehicle or FICZ thrice weekly for 2 weeks. a , b Survival ( n = 15 in each group) and cGVHD score (Day 60) of recipient mice were monitored. c Histological analyses (Day 90): H&E staining, Masson staining and collagen immunofluorescence staining ( n = 4). Ashcroft score was determined by inflammation and fibrosis. Area of collagenous and fluorescence intensity was analyzed using ImageJ. Scale bar: 50 μm. d , e At day 90, thymus cells were counted as described ( n = 7 or 5). f At day 90, blood samples were collected and flow cytometry was used to analyze T-cell subpopulations ( n = 7 or 5). g At day 90, T cells were isolated from spleen cells of recipients. Lymphocytes were isolated from spleen cells of normal C57BL/6 mice. The isolated recipients’ T cells were cultured alone (control) or co-cultured (MLR) with normal lymphocytes (1:1) for 72 h. Cell viability was detected by CCK-8 assay ( n = 7 or 5). Survival is compared using log-rank test. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; n.s., not significant.

Article Snippet: B6- Ahr fl/fl ;Foxn1-cre mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice were ordered from Cyagen (Suzhou, China).

Techniques: Staining, Immunofluorescence, Fluorescence, Flow Cytometry, Isolation, Cell Culture, CCK-8 Assay

Journal: NPJ Regenerative Medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: a , b Thymic stromal cells were isolated from wild-type C57BL/6 mice treated by 5.5 Gy TBI. Day 0 indicates the samples were collected right before TBI. a qPCR was performed to detect mRNA levels ( n = 5). The values of Day 0 were set as 1. b Western blot was performed with indicated antibodies. c , d B6- Il22ra1 fl/fl mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice, pretreated by 5.5 Gy TBI, were intraperitoneally injected with vehicle, rmIL-22 (200 ng) or FICZ (0.1 mg/kg) thrice weekly for 2 weeks. At day 14, c thymus cells were counted as described ( n = 5). d H&E staining was performed on slides of thymus. Scale bar: 500 μm. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ** * , p < 0.001; n.s., not significant.

Article Snippet: B6- Ahr fl/fl ;Foxn1-cre mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice were ordered from Cyagen (Suzhou, China).

Techniques: Isolation, Western Blot, Injection, Staining

Journal: NPJ Regenerative Medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: In the cGVHD models, BALB/c mice were used as donors. Wild type C57BL/6 ( a ), B6- Il22ra1 fl/fl and B6- Il22ra1 fl/fl ;Foxn1-cre mice ( b ) were recipients. Wild type C57BL/6 recipients were treated by vehicle or FICZ thrice weekly for 2 weeks. a , b Survival ( n = 15 in each group) and cGVHD score (Day 60) of recipient mice were monitored. c Histological analyses (Day 90): H&E staining, Masson staining and collagen immunofluorescence staining ( n = 4). Ashcroft score was determined by inflammation and fibrosis. Area of collagenous and fluorescence intensity was analyzed using ImageJ. Scale bar: 50 μm. d , e At day 90, thymus cells were counted as described ( n = 7 or 5). f At day 90, blood samples were collected and flow cytometry was used to analyze T-cell subpopulations ( n = 7 or 5). g At day 90, T cells were isolated from spleen cells of recipients. Lymphocytes were isolated from spleen cells of normal C57BL/6 mice. The isolated recipients’ T cells were cultured alone (control) or co-cultured (MLR) with normal lymphocytes (1:1) for 72 h. Cell viability was detected by CCK-8 assay ( n = 7 or 5). Survival is compared using log-rank test. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; n.s., not significant.

Article Snippet: B6- Ahr fl/fl ;Foxn1-cre mice and B6- Il22ra1 fl/fl ;Foxn1-cre mice were ordered from Cyagen (Suzhou, China).

Techniques: Staining, Immunofluorescence, Fluorescence, Flow Cytometry, Isolation, Cell Culture, CCK-8 Assay