Nicotine (NIC) treatment increases the number of ISCs in the Intestine

(A and B) Image of Ki67-positive cells (Red: Ki67, Blue: DAPI) and their quantification at the crypt base of proximal jejunum (A) or colon (B) of NIC-treated and untreated mice (A:3–4 mice per group, B: 3 mice per group). (C) Olfm4 staining image (Red: Olfm4, Blue: DAPI) and the quantification of Olfm4-positive cells at the crypt base of proximal jejunum with or without NIC treatment (3–4 mice per group). (D) GFP staining image (Green: GFP, Blue: DAPI) and the quantification of LgR5-GFP-positive cells at the crypt base of the colon in NIC or control-treated Lgr5-EGFP-IRES-CreERT2 mice (3 mice per group). (E) Lysozyme staining image (Red: Lysozyme, Blue: DAPI) and the quantification of Lysozyme-positive Paneth cells with or without NIC treatment (3–4 mice per group). Original magnifications: 200× (A-E). Scale bar: 100 µm (A-E). Values represent the mean ± standard error of the mean (SEM). Significant differences are denoted by p values (Student’s t-test). See also Figure S1.

NIC enhances the formation of intestinal organoids from ISCs

(A) Crypts from the proximal small intestine were cultured with 10 μM, 1 μM, and 100 nM NIC or cotinine to allow ISCs to form organoid colonies; the control set contained no NIC or cotinine. Representative images of the organoids and the quantification of organoids number at day 5 (3 wells/ group). (B) Colonic crypts were cultured with or without 1 μM NIC to allow CSCs to form organoid colonies. Representative images of the organoids and the quantification of organoids number at day 5 are shown (3 wells/ group). (C) ISCs and Paneth cells were isolated from the small intestine of Lgr5-EGFPJIRES-CreERT2 mice treated with or without NIC; 2 × 103 cells each were co cultured in the medium containing 10 μM CHIR99021. Representative images of the organoids and the frequency of organoids at day 5 (3 wells/ group). (D) ISCs isolated from the small intestine of Lgr5-EGFP-IRES-CreERT2 mice were cultured in the absence of Paneth cells using the medium containing 10 μM CHIR99021, with or without 1 μM NIC. Representative images of the organoids and the frequency of organoids number at day 5 (3 wells/ group). C: control, N: NIC. Original magnification: 40×. Scale bar: 100 µm. Values represent the mean ± SEM. Significant differences are denoted by p values (Student’s t-test). See also Figure S2.

The effect of NIC is mediated via the α7 subunits of α7-nicotinic acetylcholine receptor (nAChR)

(A) Isolated ISCs were cultured in a medium with or without 1 μM NIC and 10 μM Mecamylamine (3 wells/group). (B) Isolated ISCs were cultured in a medium with or without 1 μM NIC and 3 μM Adiphenine hydrochloride (3 wells/group). (C) In ISCs isolated from control and NIC mice, nAchR mRNA levels were analyzed using quantitative real-time PCR (n = 5 per group). (D) ISC or Paneth cell lysates prepared from control and NIC mice were immunoblotted with antibodies against α7-nAchR and β-actin. (E) Isolated ISCs were cultured in a medium supplemented with or without 10 μM PNU 282987 (3 wells/group). (F) Isolated ISCs were cultured in a medium with or without 1 μM NIC and 1 μM Bungarotoxin (3 wells/group). Values represent the mean ± SEM. Significant differences are denoted by p values (Student’s t-test).

NIC induces Hippo-YAP/TAZ and notch signaling in ISCs

(A) Isolated ISCs cultured using a medium supplemented with or without 1 μM NIC combined with either (A) 1 μM H89 (PKA inhibitor) or (B) 10 nM Go6983 (PKC inhibitor) (3 wells/group).. (C) Crypt lysates isolated from control and NIC-treated mice were immunoblotted using antibodies against YAP, TAZ, and β-actin. (D) In ISCs (n = 4-5 per group) or Paneth cells (n = 4 per group) isolated from control or NIC mice, mRNA levels of genes associated with Hippo-YAP/TAZ and Notch signaling were determined through quantitative real-time PCR. (E) Crypt lysates obtained from control and NIC-treated mice were immunoblotted using antibodies against Notch1, Jagged1, Jagged2, Hes5, and β-actin. (F) Crypt lysates obtained from control and NIC-treated mice were immunoblotted using antibodies against Sox9, TCF4, c-Myc, Cyclin B, Cyclin E, and β-actin. Values represent the mean ± SEM. Significant differences are denoted by p values (Student’s t-test). See also Figure S3.

Inactivation of Hippo-YAP/TAZ and Notch signaling suppresses NIC-induced Colony Formation in mice

(A) Isolated ISCs were cultured using a medium with or without 1 μM Nicotine and 5 nM K-975 (3 wells/group). (B) Isolated ISCs were cultured using a medium with or without 1 μM Nicotine and 1 μM MK-0752 (3 wells/group). (C) Isolated ISCs were cultured using a medium with or without 10 μM PNU282987 and 5 nM K-975 (3 wells/group). (D) Isolated ISCs were cultured using a medium with or without 10 μM PNU282987 and 1 μM MK-0752 (3 wells/group). (E) Isolated ISCs were cultured in a medium with or without 1 nM Ingenol-3-angelate and 5 nM K-975 (3 wells/group). (F) Isolated ISCs were cultured in a medium with or without 1 nM Ingenol-3-angelate and 1 μM MK-0752 (3 wells/group). (G) Schematic model of NIC-associated signaling pathway in ISCs. The model traces a signaling cascade via α7-nAchR, PKC, Hippo-YAP/TAZ and Notch signaling, and Wnt/β-catenin signaling in ISCs. Values represent the mean ± SEM. Significant differences are denoted by p values (Student’s t-test).

Dibenzazepine (DBZ) treatment suppresses the NIC-induced expansion of ISCs in vivo

(A) Schematic representation of the treatment showing daily injection of DBZ (1 mg/kg body weight) for 2 weeks. (B) Immunoblotting analysis of crypt lysate isolated from DBZ-and vehicle-treated mice in control and NIC-treatment groups using Hes5, YAP, TAZ, and β-actin antibodies. (C and D) Immunostained Ki67-positive and (C) (Red, Ki67; Blue, DAPI) Olfm4 positive cells (D) (Red, Olfm4; blue, DAPI) and their quantification in the proximal jejunum of DBZ-or vehicle-treated mice (NIC-treated and untreated) (n = 3 per group). Original magnifications: 200× (C and D). Scale bar: 100 µm (C and D). Values represent the mean ± SEM. Significant differences are denoted by p values (Student’s t-test). See also Figure S4.

DBZ inhibits intestinal tumor growth by NIC

(A) Schematic representation of Apcflox/flox; Lgr5-eGFP-IRES-CreERT2 (Lgr5CreER Apcfl/fl) tumor initiation. Mice were treated with control or NIC more than 8 weeks before a single Tamoxifen injection (30 mg/kg body weight), continued for 4 weeks before tissue collection. (B) Macroscopic quantification of the number and area of polyps in the entire intestine of control or NIC-treated Lgr5CreER Apcfl/fl mice. (C) Representative images (Red: β-catenin, Blue: DAPI) and the quantification of the number of β-catenin positive adenomatous lesions in the entire intestine of control or NIC-treated Lgr5CreER Apcfl/fl mice. (D) Schematic presentation of Lgr5CreER Apcfl/fl tumor initiation. Control or NIC-treated Lgr5CreER Apcfl/fl mice were subjected to a single Tamoxifen injection (30 mg/kg body weight), followed by daily DBZ or vehicle injections continued for 4 weeks before tissue collection. (E) Macroscopic quantification of the number of polyps in the entire intestine of DBZ or vehicle-treated Lgr5CreER Apcfl/fl mice (NIC-treated and untreated). (F) Representative images (Red: β-catenin, Blue: DAPI) and the quantification of the number of β-catenin positive adenomatous lesions in the entire intestine of DBZ or vehicle-treated Lgr5CreERApcfl/fl mice (NIC-treated and untreated). Original magnifications: 200× (C, and F). Scale bar: 50 µm (C, and F). Values represent the mean ± SEM. Significant differences are denoted by p values (Student’s t-test).