Fig 2: Schematic representations of the roles of Botch and Notch1 in SBI following ICH and the underlying mechanismsBriefly, Botch could significantly reduce ICH-induced SBI by effectively antagonizing Notch1, maintaining Notch1 in the immature full-length.

Fig 3: DUSP6 negatively regulates the activation of MAPKs and positively regulates the level of Notch1 NTM.a Western blot analysis showing changes in the levels of phosphorylated MAPKs (ERK1/2, P38, JNK) and the expression of target genes upon treatment with 100 ng/mL EGF in Caco2 cells overexpressing DUSP6 (DUSP6 OE) and DLD1 cells with DUSP6 knockout (DUSP6 KO) compared to the respective ppy-vector and sg-vector control cells. b Western blots showing the changes in the levels of NTM and components of γ-secretase after treatment with 100 ng/mL EGF. (Data shown in (a) and (b) is representative of three independent experiments). c Assessment of γ-secretase activity in Caco2 and DLD1 cells after treatment with 100 ng/mL EGF for 3 h. (n = 5 wells, representative of three independent experiments). d–f Effects of ERK inhibition on DLD1 DUSP6 KO cells. d Western blot analysis showing changes in the levels of NTM and ERK1/2 phosphorylation in DLD1 KO cells with or without ERK inhibition. The blots were analysed by densitometric analysis before the level of NTM relative to ACTIN is calculated and presented in the bar chart. The data shown is representative of two independent experiments. e Assessment of γ-secretase activity and (f) cell proliferation in vector and DUSP6 KO DLD1 cells with or without ERK inhibition (n = 4 biologically independent samples). * denotes P < 0.05 (two-tailed, nonparametric, Mann–Whitney test). Error bars = mean ± standard deviations. “OE” and “KO” denote overexpression and knockout, respectively. NTM cleaved Notch1 transmembrane/intracellular domain. Source data are provided as a Source Data file.

Fig 4: Notch1 levels coincided with the autophagy status inversely reflected by P62 during renal development in immunofluorescence staining. (A) The morphological evaluation of Notch1 (Alexa 488, green) and P62 (Alexa 647, red) on P1, P5, P7, P14, P28 and P49 in glomeruli during renal development. The arrows show P62 and Notch1 colocalization. Scale bar: lane 1, 10 μm; lane 5, 30 μm. (B–C) Fluorescent intensity of Notch1 and P62 in glomeruli, respectively. Data were presented as the means ± S.D. of ≥3 independent experiments. *P < 0.05.

Fig 5: BMSCs transplantation inhibited SAH-induced upregulation of the Notch1 pathway-related molecules, Notch1, NICD, RBP-Jκ, and Hes-1 at 24 and 72 h post-SAH. Representative image of Western blotting (a) and quantification of Notch1 (b), NICD (c), RBP-Jκ (d), and Hes-1 (e) protein levels after BMSCs treatment at 24 and 72 h post-SAH; n = 6 in each group; data are expressed as the mean ± SEM. *P < 0.05 versus Sham, #P < 0.05, ##P < 0.01 versus SAH+ PBS group. The qRT-PCR analysis of Notch1 (f), RBP-Jκ (g), and Hes-1 (h) mRNA levels at 24 and 72 h post-SAH; n = 3 in each group; data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus Sham, #P < 0.05, ##P < 0.01, ###P < 0.001 versus SAH + PBS group. Typical images of double-immunofluorescence staining for NICD and Iba1 (i) showing that BMSCs treatment suppressed NICD expression in Iba1-positive microglia at 24 and 72 h post-SAH. Scale bar = 50 μm