Fig 2: SPAG5-AS1 stabilized SPAG5 protein through interacting with USP14 in HG-treated HPCs. A, The silver staining gel manifested that a protein band in the pull-down products of SPAG5-AS1 biotin group was observed in HG-treated HPCs. Western blot assay for the USP14 enrichment in the pulldown of SPAG5-AS1 biotin group and SPAG5-AS1 no-biotin group in HG-treated HPCs. B, RIP analysis confirmed the SPAG5-AS1 level in USP14-binding complexes. C, FISH and IF staining were performed for the localization of SPAG5-AS1 and USP14 protein in HG-treated HPCs. Scale bar: 10 µm. D, Co-IP assay of the SPAG5 and USP14 enrichments in the precipitated products of anti-USP14, under SPAG5-AS1 depletion or sh-NC control. E, Western blot for the SPAG5 and USP14 abundance in the pulldown of SPAG5-AS1 biotin group and no-biotin group under USP14 knockdown or sh-NC control. F, Silence of SPAG5-AS1 reduced the level of p-USP14 (S432) without changing its total protein level in HG-treated HPCs. G, Knockdown of USP14 failed to impact SPAG5-AS1 level in HG-treated HPCs. H, The immunoblot of ubiquitin in the precipitates of SPAG5 and the input level of SPAG5 under the silence of SPAG5-AS1 in HG-treated HPCs treated with or without MG-132. I, Remaining SPAG5 protein level after the treatment of CHX was detected by Western blot and quantitated at 0, 3, 6 and 9 h in HG-treated HPCs transfected with sh-NC, sh-SPAG5-AS1#1 or sh-SPAG5-AS1#1 + pcDNA3.1/USP14. J, The protein level of SPAG5 as well as phosphorylated and total levels of AKT and mTOR in HPCs treated with HG + sh-NC, HG + sh-SPAG5-AS1#1 or HG + sh-SPAG5-AS1#1 + USP14. All experiments were conducted in triplicates. Data are presented as mean ± SD. **P < .01

Fig 3: SPAG5 silence attenuated apoptosis and induced autophagy in high glucose–treated podocytes. A, RT‐qPCR and Western blot data for the SPAG5 level in HPCs under HG treatment compared with the control groups (NG or MA treatment). B, SPAG5 and podocin (podocyte marker) fluorescence intensity under HG treatment versus NG and MA controls was tested by IF staining. Scale bar: 25 μm. C, HPCs were treated with HG, MA or HG, and the HG‐treated HPCs were transfected with sh‐NC or sh‐SPAG5#1/2. RT‐qPCR and Western blots of SPAG5 mRNA and protein in HPCs with indicated treatments and transfections. D, Flow cytometry analysis of apoptotic HPCs with indicated treatments and transfections. E, Apoptosis‐related genes were detected by Western blot in HPCs with indicated treatments and transfections. F‐G, IF staining of LC‐3 and podocin, and Western blotting of the autophagy‐related proteins in HPCs with indicated treatments and transfections. Scale bar: 50 μm. H, The protein levels of SPAG5 and phosphorylated and total AKT and mTOR in HPCs with indicated treatments and transfections. All experiments were conducted in triplicates. Data are presented as mean ± SD. **P < .01

Fig 4: YY1 transcriptionally upregulated SPAG5-AS1/SPAG5 in HG-treated HPCs. A-B, 20 transcription factors (TFs) potentially binding to the promoter of SPAG5-AS1/SPAG5 according to the prediction of human TFDB and PROMO were presented by Venn diagram and analysed via pull-down analysis under NG, MA or HG treatment. C, Western blot analysis of the YY1 level under NG, MA or HG treatment in cytoplasmic or nuclear fraction of HPCs. D, 3 YY1 binding sites on the promoter of SPAG5-AS1/SPAG5 were predicted by JASPAR tool. E, ChIP analysis for the DNA fragments with binding site 1, 2 or 3 in the precipitated products of YY1. F, YY1 protein was detected by Western blot in the pulldown of WT SPAG5-AS1/SPAG5 promoter and SPAG5-AS1/SPAG5 promoter with site 1, 2 or 3 in HG-treated HPCs. G, Luciferase reporter analysis elucidated the influence of the YY1 silence on the luciferase activity of SPAG5-AS1/SPAG5 promoter reporter of each group. H, RT-qPCR result for the levels of YY1 mRNA, SPAG5-AS1 and SPAG5 mRNA responding to knockdown of YY1 in HG-treated HPCs. I, Western blot of YY1, SPAG5, p-AKT (tthr308 and ser2448), t-AKT, p-mTOR and t-mTOR of each group. All experiments were conducted in triplicates. Data are presented as mean ± SD. *P < .05, **P < .01

Fig 5: SPAG5-AS1 promoted apoptosis and attenuated autophagy in HG-treated HPCs through SPAG5/AKT/mTOR pathway. HPCs treated with HG were transfected with sh-NC, sh-SPAG5-AS1#1, sh-SPAG5-AS1#1 + pcDNA3.1/SPAG5 or sh-SPAG5-AS1#1 plus the treatment of MHY1485, an activator of mTOR. A-B, SPAG5 mRNA and protein levels, as well as the phosphorylated and total levels of AKT and mTOR in each group. C-D, Flow cytometry of apoptotic HPCs and Western blotting results of apoptosis-related genes in HPCs in each group. E-F, LC-3 and podocin fluorescence intensity and autophagy-related proteins were analysed by IF staining and Western blotting in all groups. Scale bar: 50 µm. All experiments were conducted in triplicates. Data are presented as mean ± SD. *P < .05, **P < .01