sj-docx-2-tar-10.1177_17534666231214134 – Supplemental material for Nebulization versus metered-dose inhaler and spacer in bronchodilator responsiveness testing: a retrospective study

Supplemental material, sj-docx-2-tar-10.1177_17534666231214134 for Nebulization versus metered-dose inhaler and spacer in bronchodilator responsiveness testing: a retrospective study by Rongli Lu, Ying Li, Chengping Hu, Pinhua Pan, Qiaohong Zhao and Ruoxi He in Therapeutic Advances in Respiratory Disease</p

sj-docx-1-tar-10.1177_17534666231214134 – Supplemental material for Nebulization versus metered-dose inhaler and spacer in bronchodilator responsiveness testing: a retrospective study

Supplemental material, sj-docx-1-tar-10.1177_17534666231214134 for Nebulization versus metered-dose inhaler and spacer in bronchodilator responsiveness testing: a retrospective study by Rongli Lu, Ying Li, Chengping Hu, Pinhua Pan, Qiaohong Zhao and Ruoxi He in Therapeutic Advances in Respiratory Disease</p

Rnd3 regulates cell proliferation through NICD signaling in lung adenocarcinoma cells, A549.

<p>(<b>A</b>) Rnd3 mRNA detected by qRT-PCR is down-regulated in A459 compared to HBEC. (<b>B</b>) <b>&</b> (<b>C</b>) A western blot to detect phosphorylated MYPT1, NICD in cells. (<b>D</b>) <b>&</b> (<b>E</b>) The application of compound E blocks the proliferation of A459 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (<b>F</b>) <b>&</b> (<b>G</b>) Transient overexpression of Rnd3 in A549 cells down-regulated NICD and Hes1 in a dosage dependent manner. Data represent means ± S.D. *<i>p</i><0.05 compared to control (group 0); #<i>p</i><0.05 compared to group 1; $<i>p</i><0.05 compared to group 3. Data represent means ± S.D.</p

Rnd3 is down-regulated in non-small lung cancer cell lines, H520 and H358.

<p>(<b>A</b>) Rnd3 mRNA detected by qRT-PCR is down-regulated in H358 and H520 compared to HBEC. (<b>B</b>) Rnd3 protein expression levels in cells by western blot. (<b>C</b>) Densitometry quantification of western band intensity in B. (<b>D</b>), (<b>F</b>), (<b>H</b>) <b>&</b> (<b>I</b>) A western blot to detect phosphorylated MYPT1, phosphorylated MLC2, ROCK1 and NICD in cells. (<b>E</b>), (<b>G</b>) <b>&</b> (<b>J</b>) Densitometry quantification of western band intensity showed up-regulation of Rho Kinase activity and NICD expression in H358 and H520 cells compared to HBEC. Western blots were quantified from three independent experimental repeats. BrdU-positive cells were quantified from 8 images taken from four slides. Data represent means ± S.D.</p

The contents of IFN-, IL-4 and IL-17 from cocultured 16 HBE and lymphocytes were assayed by ELISA (n = 6).

<p>A: Influence of NS1 on the secretion of IFN-, IL-4 and IL-17 from cocultured 16 HBE in the presence of OVA and lymphocytes (**P<0.01 versus GFP group; ## P<0.01 versus GFP-NS1 group) B: Influence of NS1 on the secretion of IFN-, IL-4 and IL-17 from cocultured 16 HBE in the presence of OVA and lymphocytes. (*P<0.05, **P<0.01 versus RFP group; # p<0.05, ## P<0.01 versus RFP-NS2 group).</p

Rnd3 inhibits proliferation by promoting NICD degradation in H520 and H358 cells.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) Hes1 was up-regulated in H520 and H358 cells compared to HBEC cells. (<b>C–F</b>) Hes1 expression decreased when Rnd3 was stably overexpressed in H358 and H520 cells. (<b>G</b>) <b>&</b> (<b>H</b>) Transient overexpression of Rnd3 in H358 cells down-regulated NICD and Hes1 in a dosage dependent manner. (<b>I</b>) <b>&</b> (<b>J</b>) Inhibition of proteasome activity by MG132 (final concentration of 15 µM) abolished the Rnd3 dosage dependent NICD down-regulation in H358 cells. Western blots were quantified from three independent experimental repeats. Data represent means ± S.D. *<i>p</i><0.05 compared to control (group 0); <b>#</b><i>p</i><0.05 compared to group 1; <b>$</b><i>p</i><0.05 compared to group 3. Data represent means ± S.D.</p

Titers of pLenO-GFP-NS1 and pLenO-RFP-NS2 were assayed by flow cytometry.

<p>A, B: Infection of HEK293T cells with different dilutions of pLenO-GFP-NS1 under light microcopy and fluorescence microscopy respectively (×100); C, D: Infection of HEK293T cells with different dilutions of pLenO-RFP-NS2 under light microcopy and fluorescence microscopy respectively. From above to below represent the concentration of 1.0 ul, 0.1 ul, 0.01 ul and representative images of flow cytometry (0.01 ul).</p

The changes of Th subsets were assayed by flow cytometry and real-time PCR.

<p>A, B: Influence of NS1 on changes of Th subsets was assayed by flow cytometry (**P<0.01 versus 16 HBE group; ##P<0.01 versus GFP group; P<0.01 GFP-NS1 group). C. Influence of NS2 on changes of Th subsets was assayed by flow cytometry (**P<0.01 versus 16 HBE group; ##P<0.01 versus RFP group; P<0.01 RFP-NS2 group). D: Influence of NS1 on changes of Th subsets was assayed by real-time PCR (**P<0.01 versus GFP group; ##P<0.01 GFP-NS1 group). E. influence of NS2 on changes of Th subsets was assayed by real-time PCR (**P<0.01 versus RFP group; ##P<0.01 RFP-NS2 group). Data represent Means ± SE of 6 experiments.</p

Efficiencies of lentiviral infections to 16 HBE were assayed by fluorescence microscopy and indirect immunofluorescent technology.

<p>A: Infection of 16 HBE by pLenO-GFP-NS1 (MOI = 10); B: Infection of 16 HBE by pLenO-RFP-NS2 (MOI = 10). From above to below represent images under light microcopy, fluorescence microscopy and indirect immunofluorescent assay (×200).</p

The expression of HLA-DR, CD80 and CD86 on 16 HBE was assayed by flow cytometry.

<p>A, B: The influence of NS1 on the expression of HLA-DR, CD80 and CD86 on 16 HBE with or without OVA treatment (**P<0.01 versus 16 HBE group; ##P<0.01 versus GFP group; $$P<0.01 versus GFP-NS1 group). C: The influence of NS2 on the expression of HLA-DR, CD80 and CD86 on 16 HBE with or without OVA (**P<0.01 versus 16 HBE group). Data represent Means ± SE of 6 experiments.</p