DOI: 10.6084/m9.figshare.27151589.v1
title: Additional file 1 of Long noncoding RNA SNHG16 regulates TLR4-mediated autophagy and NETosis formation in alveolar hemorrhage associated with systemic lupus erythematosus

description: Additional file 1: Fig. S1. SNHG16 expression in LV-SNHG16‑ and sh-SNHG16-transfected cells. a Left, map of pLV-SFFV-SNHG16-PGK-puro, a total of 9.3 kb in length. Right, SNHG16 levels in LV-SNHG16‑transfected 293T cells. b Left, map of pLKO.1-sh-SNHG16-puro, a total of 7.4 kb in length. Right, SNHG16 levels in sh-SNHG16‑transfected MLE-12 cells. Values are mean ± SD. Results in Fig. S1a were representative of 3 independent experiments, and in Fig. S1b were representative of 2 independent experiments with similar findings. Fig. S2. Expression of SNHG16 in purified neutrophils and sorted T cells, monocytes and B cells from PB of healthy individuals. a Flow cytometric graphs of sorted CD3-positive T cells, CD14-positive monocytes and CD19-positive B cells from healthy individual No. 1, No. 2 and No. 3. b PB cell numbers/µL (left) and SNHG16 levels/105 cells (right) in neutrophils, T cells, monocytes and B cells from healthy individual No. 1, No. 2 and No. 3. Values are mean ± SD. Fig. S3. Expression of miR-146a in PBMCs and PBNs from SLE patients. a MiR-146a levels in PBMCs from SLE patients and HC. b A negative correlation between miR-146a levels in PBMCs and SLEDAI-2K activity scores. c MiR-146a levels in PBMCs from HC, Nil. LN, SLE-AH and other AH patients. A negative correlation between miR-146a and d TLR4, e TRAF6 and f NEAT1 levels in PBMCs from SLE patients. g MiR-146a levels in PBNs from SLE patients and HC. h A negative correlation between miR-146a levels in PBNs and SLEDAI-2K activity scores. i MiR-146a levels in PBNs from HC, Nil. LN and AH patients. Values are mean ± SD. Horizontal lines are mean values. Patient numbers, n = 62 for PBMCs from SLE, 15 for PBNs from SLE, 7 for PBMCs from Nil, LN and SLE-AH, 6 for PBMCs from other AH, 5 for PBNs from Nil, LN, 4 for PBN from AH. *p &lt; 0.05. **p &lt; 0.01, ***p &lt; 0.001. Fig. S4. Expression of NEAT1 in SLE patients and pristane-injected mice. NEAT1 levels in a PBMCs and e PBNs from SLE patients and HC. b A positive correlation and f no correlation between NEAT1 levels in PBMCs and PBNs from SLE patients, respectively, and SLEDAI-2K activity scores. d No correlation between NEAT1 and TLR4 levels in PBMCs from SLE patients. NEAT1 levels in c PBMCs and g PBNs from HC, Nil, LN and AH patients. h NEAT1 levels in thioglycolate-induced mouse neutrophils from pristane-induced AH mice. NEAT1 levels in i lung and j spleen tissues from pristane-induced AH mice. Values are mean ± SD. Horizontal lines are mean values. Patient numbers, n = 62 for PBMCs from SLE, 15 for PBNs from SLE, 7 for PBMCs from Nil, LN, SLE-AH, 6 for PBMCs from other AH, 5 for PBNS from Nil, LN, 4 for PBNs from AH. 4 mice per group in h. 5 mice per group in i, j. All results in Fig. S4h, S4i and S4j were representative of 2 independent experiments with similar findings. * p &lt; 0.05. Fig. S5. p53 levels and apoptotic cell ratios in PBMCs from SLE patients. a Left, p53 levels in SLE patients and HC. Right, p53 levels in HC, Nil, LN and AH. b Left, apoptotic cell ratios in SLE patients and HC. Right, apoptotic cell ratios in HC, Nil, LN and AH. Values are mean ± SD. Horizontal lines are mean values. Patient numbers, n = 30 for SLE, n = 5 for Nil, n = 5 for LN, n = 3 for AH. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Fig. S6. Increased autophagy formation by immunoblot assay in PBMCs from SLE patients. a Representative immunoblot assay for Beclin-1, LC and mTOR in PBMCs from SLE patients and HC. b Signal intensity quantitation analysis for Beclin-1, LC and mTOR in PBMCs from SLE patients and HC. Values are mean ± SD. Patient numbers, n = 3 for representative immunoblot assay, n = 10 for signal intensity quantitation analysis. * p &lt; 0.05. Fig. S7. Apoptosis formation in Dox-stimulated MLE-12 cells regulated by SNHG16. a SNHG16 expression in MLE‑12 cells stimulated with various concentrations of IL‑6. b Left, representative photographs of TUNEL IF staining (green) in MLE‑12 cells stimulated with 1 μM Dox. Cell nuclei counterstained with DAPI (blue). Scale bar = 10 µm, magnification ×1000. Middle, quantification of TUNEL‑positive cell percentages in mock and stimulation with 1 μM Dox. Right, apoptotic cell ratios in MLE-12 cells stimulated with various concentration of Dox. c HMGB1 levels in culture supernatants of MLE-12 cells stimulated with various concentration of Dox. d From left to right, expression of SNHG16, TRAF6, p53, Bax and miR-146a levels by qRT-PCR analyses in MLE-12 cells stimulated with various concentration of Dox. e Representative immunoblot assay of p53/Bax (top) and TRAF6 (low) expression in MLE-12 cells stimulated with various concentration of Dox. f MiR-17 and miR-146a levels in SNHG16-overexpressed and -silenced MLE-12 cells. g Left, SNHG16 expression in SNHG16-overexpressed MLE-12 transfectants. Right, from left to right, levels of p53, Bax and SNHG16 and apoptotic cell ratios in 1 μM Dox-stimulated SNHG16-overexpressed MLE-12 transfectants. h Left, SNHG16 expression in SNHG16-silenced MLE-12 transfectants. Right, from left to right, levels of p53, Bax and SNHG16 and apoptotic cell ratios in 1 μM Dox-stimulated SNHG16-silenced MLE-12 transfectants. Values are mean ± SD. Results in Fig. S7a to S7f were representative of 3 independent experiments, and in Fig. S7g and S7h were representative of 2 independent experiments with similar findings. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Fig. S8. TLR4 expression in SNHG16-overexpressed/silenced and miR‑146a‑ overexpressed MLE‑12 cells. a TLR4 mRNA levels in SNHG16-overexpressed (left) and -silenced MLE-12 transfectants (right). b Representative immunoblot assay of TLR4 levels in SNHG16-overexpressed (left) and -silenced MLE-12 transfectants (right). c Left, miR-146a levels in miR‑146a‑overexpressed MLE‑12 transfectants. Right, TLR4 levels in miR‑146a‑overexpressed MLE‑12 transfectants and in such cells overexpressed with SNHG16. Values are mean ± SD. Results in Fig. S8a and S8b were representative of 3 independent experiments, and in Fig. S8c were representative of 2 independent experiments with similar findings. **p &lt; 0.01, ***p &lt; 0.001. Fig. S9. Involvement of SNHG16 in TLR4-mediated NETs formation in mouse neutrophils. a Expression of SNHG16, TLR4, TRAF6 and miR-146a in thioglycolate-induced neutrophils from pristane-injected mice on day 4, day 9 and day 14. b SNHG16 expression in naïve mouse neutrophils stimulated with various concentrations of IL‑6 or 3 μg/mL LPS. c Naïve mouse neutrophils stimulated with various concentrations of HMGB1 or LPS. Left, representative photographs of NETs morphology from naïve mouse neutrophils under 300 ng/mL HMGB1 or 3 μg/mL LPS stimulation. Scale bar = 30 µm, magnification ×400. Middle, quantification of NETs morphology with diffuse/spread NETs percentages, Right, CitH3 concentrations, SNHG16, TRAF6 and miR-146a levels in naïve mouse neutrophils stimulated with various concentrations of HMGB1 or 3 μg/mL LPS. Values are mean ± SD. 4 mice per group in a. All results in Fig. S9 were representative of 2 independent experiments with similar findings. **p &lt; 0.01, ***p &lt; 0.001. Fig. S10. TLR4 expression in SNHG16-overexpressed/silenced HL-60 cells and reversed reduction in NETosis in miR‑146a‑silenced CasRX-SNHG16-transfected HL‑60 cells. a TLR4 mRNA levels in SNHG16-overexpressed (left) and -silenced HL-60 cells (right). b Representative immunoblot assay of TLR4 levels in SNHG16-overexpressed (left) and -silenced HL-60 cells (right). c MiR-17 and miR-146a levels in SNHG16-overexpressed and -silenced HL-60 cells. d Left, miR-146a levels in miR‑146a‑silenced CasRX-SNHG16-transfected HL‑60 cells. Right, quantification of NETs formation percentages (left), CitH3 levels (middle), and PAD4 levels (right) in miR‑146a-silenced CasRx‑SNGH16‑transfected dHL‑60 transfectants stimulated with 500 ng/mL LPS for 4 h. Values are mean ± SD. Results in Fig. S10a to S10c were representative of 3 independent experiments, and in Fig. S10d were representative of 2 independent experiments with similar findings. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Fig. S11. ATG5 expression in LPS-stimulated MLE-12 cells and lung tissues from pristane-induced AH mice. a ATG5 levels in MLE-12 cells under the stimulation of different LPS concentrations for 4 h (left) and 50 µg/mL LPS for different time (right). b ATG5 levels in lung tissues from pristane-induced or saline-injected mice. c ATG5 levels in lung tissues from pristane-induced mice receiving SFFV/SFFV-SNHG16 or sh-luciferase/sh-SNHG16 intra-pulmonary delivery. Values are mean ± SD. 5 mice per group in b. 8 mice per group in c. Results in Fig. S11a were representative of 3 independent experiments, and in Fig. S11b and S11c were representative of 2 independent experiments with similar findings. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Fig. S12. SNHG16 and NEAT1 expression in PBMCs, USCs and biopsied renal tissues from LN patients. a SNHG16 and d NEAT1 levels in PBMCs from HC, Nil and LN patients. b SNHG16 and e NEAT1 levels in USCs from HC, Nil and LN patients. c SNHG16 and f NEAT1 levels in kidney tissues from control and LN. Values are mean ± SD. Horizontal lines are mean values. Patient numbers, for PBMC and USC, n = 15 for Nil, n = 15 for LN, for renal tissue, n = 5 for control, n = 5 for LN. *p &lt; 0.05, ***p &lt; 0.001. Fig. S13. SNHG16 expression in a mouse LN model. a Serial measurement of proteinuria levels in mice at month 0, 1, 3, 5 and 6. Serial measurement of anti‑RNP. b and anti-dsDNA titers. c at month 0, 1, 3, 5 and 6. d Periodic acid-Schiff staining of renal glomeruli at month 6 after saline (left) or pristane injection (right). Arrows indicating normal glomeruli (left) or a glomerulus with GN formation (right). Scale bar = 10 µm, magnification ×400. e Kinetic expression of SNHG16 in the kidneys from saline- and pristane-injected mice at month 0, 1, 3, 5 and 6. Values are mean ± SD. All results in Fig. S13 were representative of 2 independent experiments with similar findings. 5 mice per group in Fig. S11. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001.