Experimental animals

Nus1flox/flox (C57BL/6JGpt-Nus1em1Cflox/Gpt, Strain NO. T010909) were purchased from GemPharmatech Company (Nanjing, China). Specific pathogen-free (SPF) C57BL/6J pregnant mice were purchased from SLAC ANIMAL (Shanghai, China). All animals were raised under a 12-hour light/dark cycle with free access to food and water. All animal experiments performed in this study were in accordance with the institutional guidelines for the use and care of animals, and all procedures were approved by the Institute Animal Welfare Committee of Soochow University.

Cell culture and drug treatment

MES23.5 dopaminergic cells, murine neuroblastoma Neuro-2a (N2a) cells and human embryonic kidney 293 (HEK293) cells were grown in Dulbecco’s modified Dulbecco’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, New York, NY, USA), streptomycin (100 µg/mL) and penicillin (100 µg/mL) (Gibco). Human neuroblastoma SH-SY5Y cells were cultured in DMEM/F12 medium supplemented with 10% FBS, penicillin (100 µg/mL) and streptomycin (100 µg/mL). Primary cultured astrocytes and microglia were isolated from 3-day-old C57BL/6J mice, and primary cortical neurons were isolated from the cortex of Nus1flox/flox mouse embryos on embryonic day 17. The methods and procedures for obtaining and culturing primary cultured cells were described previously [16, 17].

Reagents

FGF1 (HZ-1327) was purchased from Proteintech (Rosemont, USA). Heparin was a kind gift from Dr. Zhenqing Zhang at Soochow University. MG132 (S1748) was purchased from Beyotime Biotechnology (Shanghai, China). NH4Cl (326372) and 1-methyl-4-phenylpyridinium ion (MPP+, D048) were purchased from Sigma (St. Louis, MO, USA). WM-3835 (S9805) was purchased from Selleck Technology (Houston, TX, USA). Nicotinamide (HY-B0150) and trichostatin A (HY-15144) were purchased from MedChem Express. Except for FGF1 and heparin, which were dissolved in PBS, all the reagents were dissolved in dimethyl sulfoxide (DMSO). A Cre recombinase-expressing lentiviral vector (LV-Cre) and a control lentiviral vector (LV-Ctrl) were purchased from Brain VTA (Wuhan, China).

Plasmid transfection

pcDNA3.1-3xFLAG-NgBR, pcDNA3.1-3xFLAG-RFX1, pcDNA3.1-3xFLAG-RFX2, pcDNA3.1-3xFLAG-RFX3 and RFX1-pcDNA3.1-EGFP-N were purchased from YouBio (Changsha, China). For plasmid transfection, cells were transfected with plasmids via Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

RNA interference

Small interfering RNAs (siRNAs) against the human NUS1, RFX1, KAT7 and STUB1 genes, as well as the mouse Nus1 gene, were synthesized with the sequences shown in Table 1 in the supplemental information. The cells were transfected with siRNAs via Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instructions.

Cell viability assay

Cell viability was measured with a Cell Counting Kit-8 (CCK8) (APExBIO Technology LLC, Houston, USA) according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were treated with two human-derived siRNAs to silence NUS1 expression for 48 h or silence NUS1 expression for 36 h, followed by treatment with FGF1 (100 ng/mL)/heparin (10 µg/mL) or PBS for 12 h. Then, the cells were incubated with 10% CCK8 reagent for 2 h at 37 °C. Cell viability was determined by measuring the absorbance at 450 nm with an enzyme marker.

Cell cytotoxicity assay

The cytotoxicity of lactate dehydrogenase (LDH) release was detected via a CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI, USA). Briefly, SH-SY5Y cells were transfected with two human-derived siRNAs to silence NUS1 expression for 48 h or to silence NUS1 expression for 36 h, followed by FGF1 (100 ng/mL)/heparin (10 µg/mL) treatment or PBS for 12 h. Then, 50 µL of cell supernatant was collected, mixed with 50 µL of CellTiter-Glo and shaken for 20 min at room temperature. Cytotoxicity was determined by measuring the absorbance at 490 nm with an enzyme meter.

ELISA

SH-SY5Y cells were inoculated into 24-well plates and treated with siRNAs against NUS1 for 24 h. Then, the cells were cultured with serum-free medium for 24 h, and 100 µL of the FGF1 content in the culture medium or in cell lysate was measured with an ELISA kit (EK0339, BOSTER, Wuhan, China).

Quantitative real-time PCR

The procedures for RNA extraction and reverse transcription have been described elsewhere [18]. Using a 7500 real‒time PCR system (Applied Biosystems), qRT‒PCR analysis was performed to measure target RNA abundance quantitatively via Power SYBR Green PCR Master Mix (Vazyme Biotech, Nanjing, China). The sequences of the human primers used for real-time PCR are shown in Table 2 in the supplemental information. The relative mRNA levels of these genes to those of β-actin were calculated via the 2−ΔΔCT method.

Transcriptome sequencing

First, total RNA was extracted from three technical repeats via TRIzol reagent (Invitrogen). Quality control and library construction were performed by Huada Gene Technology Company, and then transcriptome sequencing was performed via the BGISEQ platform. The raw data were filtered to remove reads of low quality, reads with adaptor sequences and reads with high levels of N bases. The clean reads were aligned to the reference genome via HISAT software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed, differentially expressed genes (DEGs) were identified, and expression analysis was conducted on the Dr. Tom analysis system (https://biosys.bgi.com).

Immunoblot analysis and antibodies

After the cells were treated with drugs, siRNAs or lentiviruses, they were lysed in cell lysis buffer (0.5% deoxycholate, 1% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and a protease inhibitor mixture (Roche)). The samples were separated by 8% or 12% SDS‒PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated overnight at 4 °C with the primary antibodies shown in Table 3 in the supplemental information. The following secondary antibodies were used: horseradish peroxidase-conjugated sheep anti-mouse and anti-rabbit antibodies (Amersham Pharmacia Biotech, Piscataway, NJ, Sweden). The proteins were visualized with an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher, Waltham, MA, USA) via a chemiluminescence imaging system (Bioshine ChemiQ 4800, Shanghai, China).

PI staining assay

After SH-SY5Y cells were treated with siRNAs against NUS1 and FGF1/heparin, the cells were incubated with Hoechst 33,342 (Sigma‒Aldrich, St. Louis, MO, USA) and PI (Sigma‒Aldrich, St. Louis, MO, USA) for 5 min. The cells were then observed with an inverted IX71 microscope system (Olympus, Tokyo, Japan).

Immunofluorescence staining

HEK293 cells and primary neurons were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with 0.1% Triton X-100 for 10 min. After three washes with PBS, the cells were blocked with 1% FBS in PBS for 1 h. Next, the cells were incubated with anti-KAT7 (sc-9996, Santa Cruz) or anti-cleaved-caspase 3 (9661 S, Cell Signaling Technology) and anti-MAP2 (MAB3418, Millipore) antibodies together. Subsequently, the cells were stained with 40,6-diamidino-2-phenylindole (DAPI) (Sigma‒Aldrich) for 5 min and then washed three times with PBS. Finally, the cells were observed with a confocal microscope system (Nikon, Tokyo, Japan).

Immunoprecipitation assay

The cells were lysed in cell lysis buffer at 4 °C, sonicated and centrifuged at high speed to collect the supernatant. The supernatant was incubated with protein G agarose (Thermo Fisher Scientific) as well as anti-Flag (A5882, Sigma‒Aldrich) overnight at 4 °C. Protein complexes coupled to protein G agarose were washed three times with lysis buffer and analyzed by immunoblotting.

Flow cytometry assay

After the cells were treated with drugs and siRNAs, the cell precipitate was collected. After the cells were resuspended by adding 195 µL of Annexin V-FITC conjugate, 5 µL of Annexin V-FITC and 10 µL of propidium iodide staining solution were added, mixed well, incubated for 10–20 min at room temperature in the dark, and then detected via a flow cytometer (Cytomics FC 500, Beckman).

Statistical analysis

Quantitative analysis of the immunoblots was performed via Photoshop 7.0 (Adobe, USA). GraphPad Prism 8.0 (GraphPad Software, USA) was used for statistical analysis and plotting. Significant differences were assessed via unpaired t tests or one-way or two-way analysis of variance (ANOVA) for multiple comparisons. The significance criterion was set at P < 0.05. *P < 0.05, **P < 0.01, *** P < 0.001, ns, not statistically significant. These values are shown as the means ± SDs.