Additional file 1 of Mxc, a Drosophila homolog of mental retardation-associated gene NPAT, maintains neural stem cell fate
Creators: name: Sang, Rong
name: Wu, Cheng
name: Xie, Shanshan
name: Xu, Xiao
name: Lou, Yuhan
name: Ge, Wanzhong
name: Xi, Yongmei
name: Yang, Xiaohang
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Knockingdown mxc does not induce apoptosis. (A-B’) Third‐instar larval brains of wt (A-A’) and mxc RNAi (B-B’), labeled by anti‐Dpn (red) and anti‐Caspase3(green). Only background Caspase3 signals were detected in the wt (A) and mxc RNAi (B) larvalbrains. Scale bars: 20 μm. Figure S2. No cell size change in mxc RNAi NBs.Statistical data of cell size of the NB in wt and mxc RNAi third-instar larval brains. The data areplotted as mean ± SD. No significant difference using a Student’s ttest, p = 0.3439. Both of the numbers of neuroblasts counted N = 15. FigureS3. Schematic of the components of Histone locus body (HLB). The HLBis formed via a hierarchical recruitment of components. Mxc and FLASH are thecore components of HLB formation. Mute, Slbp, U7 snRNP and other components arerecruited by Mxc and FLASH for canonical histone gene transcription and pre-RNAprocessing 5,16. Figure S4. Absence of mxc impairs HLBformation in NBs. (A-C”) Third‐instarlarval NBs of wt (A-A”), mxcRNAi (B-B”), and mxc RNAi with UAS-Mxc (C-C”) labeled by anti‐Mute (red), MPM2 antibody (green) and Dapi(blue). In wt NBs (A-A”), MPM2signals and Mute signals were colocalized and formed nuclear foci (arrowheads).In mxc RNAi NBs (B-B”), no MPM2 puncta were detected (B’). The Mute patterns (arrowheads) wereless condensed and multiple smaller puncta were observed in the NBs (B”). Overexpression of Mxc in mxcRNAi background (C-C”) rescued the phenotype, MPM2 and Mute were colocalized atthe nuclear foci (arrowheads). Scale bars: 10 μm. (D-E”) Third‐instarlarval NBs of wt (D-D”) and mxc16a-1(E-E”), labeled by anti‐Mute(red), MPM2 antibody (green) and Dapi (blue). Multiple smaller puncta were observed in mxc16a-1NBs and these MPM2 and Mute signals were not colocalized (E-E”). Scale bars: 10μm. (F-G”) MARCM clones of wt (F-F”) and mxc22a-6 (G-G”)in third‐instar larval brains, labeled by MPM2 (red),anti-Mute (blue) and anti-GFP (green). In mxc22a-6NBs, noMPM2 puncta were detected(G’), and Mute patterns were also shown as multiple puncta (G”). Scale bars: 10μm. Figure S5. Knocking down mxc or histone genes leads to DNA DSBs. (A-D’)Confocal images of the third‐instarlarval brains of wt (A-A’), mxcRNAi (B-B’), His3 RNAi (C-C’) and His4 RNAi (D-D’), labeled byanti‐H2Av pS137 (red) and anti‐Dpn (green). In wt larval central brain (A), no obviously phosphorylated H2Avsignals were detected. Strong signals (arrowheads) were detected in the centralbrains of mxc RNAi (B), His3 RNAi (C) and His4 RNAi (D)lines. Scale bars: 20 μm. Figure S6. Autophagy was activated in mxcknockdown or mutant NBs. (A-B) Third‐instarlarval brains of wt (A) and mxc16a-1(B), labeled by anti‐Dpn (red) and anti‐GFP (green). These lines contained UAS-Atg8a-GFPdriven by wor-GAL4. In the wtlarval central brain (A), only the basic signal of Atg8a-GFP was detected. Inthe mxc16a-1 larval central brain (B), obvious Atg8a puncta(arrowheads) were detected in the cytoplasm of the NB. Scale bars: 20 μm. (C) Statistical analysis of autophagy levelsin the NBs of wt and mxc16a-1larval central brains. The data are plotted as mean ± SD. ****p &lt;0.0001 using a Student’s t test, p = 7.441E-11. Numbers of NBscounted N = 57, N = 50, respectively.(D-E) Third‐instar larval brains of wt (D) and mxc RNAi (E), labeledby anti‐Dpn (red) and anti‐GFP(green). These lines contained UAS-Atg8a-GFP which was driven by wor-GAL4.Obvious Atg8a-GFP puncta (arrowheads) were also detected in the cytoplasm of mxcknockdown NB. Scale bars: 20 μm.