Fig 1: LIN28A enhanced HG-induced cell apoptosis, ROS generation and inflammatory cytokine secretion. (A) The expression of LIN28A in DN and normal tissues was analyzed by qRT-PCR and western blotting (n=6). (B) IHC staining of LIN28A. (C) Protein levels of LIN28A in HK-2 cells treated with NG, HM or HG were examined by western blotting (n=4). (D) Western blotting analysis of LIN28A in HK-2 cells (n=4). HK-2 cells were transfected with vector, oe-LIN28A, si-NC or si-LIN28A and subsequently treated with HG for 48 h (E) LIN28A was detected using western blotting (n=4). (F) Cell viability was examined by MTT assays (n=4). (G) TUNEL staining (TUNEL, green. Scale bar=100 µM) were examined. (H) Cellular ROS levels were examined by a fluorometric ROS sensor (red, n=4. Scale bar=100 µM). (I) Secretion of TNF-a and IL-6 into culture supernatants (n=4). *P< 0.05, **P< 0.01 and ***P< 0.001.
Fig 2: MALAT1-mediated aggravation of HG-induced injury was dependent on LIN28A. HK-2 cells were transfected with si-NC, si-MALAT1, si-MALAT1 + vector or si-MALAT1 + oe-LIN28A and subsequently treated with HG for 48 h (A) qRT-PCR analysis of LIN28A (n=4). (B) Protein levels of LIN28A were assessed by western blotting (n=4). (C) Cell viability was examined by MTT assays (n=4). (D) TUNEL staining (TUNEL, green. Scale bar=100 µM) were examined. (E) Cellular ROS levels were examined by a fluorometric ROS sensor (red, n=4. Scale bar=100 µM). (F) Secretion of TNF-a and IL-6 into culture supernatants (n=4). *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 3: MALAT1 interacted with LIN28A in HK-2 cells. (A) qRT-PCR analysis of MALAT1 in HK-2 cells transfected with si-NC, si-LIN28A, si-LIN28A + vector or si-LIN28A + oe-LIN28A (n=4). (B) The enrichment of MALAT1 in anti-LIN28A-immunoprecipitated fractions from HK-2 and HEK-293T cells was analyzed by qRT-PCR (n=3). (C) LIN28A was pulled down by the MALAT1 sense probe (n=3). (D) MALAT1 stability in HK-2 cells in response to actinomycin D (n=4). *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 4: MALAT1 facilitated the interaction of LIN28A and Nox4 to stabilize Nox4 mRNA in HK-2 cells. (A) LIN28A was pulled down by the Nox4 sense probe (n=3). (B) The enrichment of Nox4 in anti-LIN28A-immunoprecipitated fractions from HK-2 cells was analyzed by qRT-PCR (n=3). (C) Levels of LIN28A and Nox4 mRNA in HK-2 cells transfected with si-NC or si-LIN28A (n=4). (D) Nox4 mRNA stability in response to actinomycin D was determined by qRT-PCR (n=3). (E) The abundance of MALAT1, GAPDH and U1 snRNA in cytoplastic and nuclear fractions (n=4). The expression of Nox4 was analyzed by qRT-PCR (F, n=4) and western blotting (G, n=4). (H) RIP assays for analyzing the interaction between LIN28A and Nox4 in HK-2 and HEK-293T cells (n=3). (I) Nox4 mRNA stability in response to actinomycin D was determined by qRT-PCR in HK-2 and HEK-293T cells (n=3). HK-2 cells were transfected with si-NC, si-MALAT1, si-MALAT1 + vector or si-MALAT1 + oe-LIN28A and subsequently treated with HG for 48 h The expression of Nox4 was determined by qRT-PCR (J, n=4) and western blotting (K, n=4). *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 5: Knockdown of MALAT1 alleviated renal tubular injury by suppressing LIN28A and the Nox4/AMPK/TOR signaling axis in rats with DN. SD rats with DN were intravenously injected with shMALAT1 lentiviral particles and sequentially fed a high glucose and fat diet for 5 months. (A) Body weight of rats (n=6 each group). (B) Kidney to body weight ratio (n=6 each group). (C) Fasting blood glucose (n=6 each group). (D) Urinary albumin to creatinine ratio (n=6 each group). (E) The concentration of BUN and creatinine in serum (n=6 each group). (F) H&E staining of kidney sections (Scale bar=50 µM). (G) Masson’s Trichrome staining of kidney sections (Scale bar=50 µM). (H) TUNEL staining for cell apoptosis analysis in kidney sections (Scale bar=50 µM). (I) qRT-PCR analysis of MALAT1 in the kidneys (n=6). (J) Protein levels of LIN28A, Nox4, p-AMPK, AMPK, p-mTOR, mTOR, Bcl-2 and Bax in the kidneys (n=6). (K) The concentrations of TNF-a and IL-6 in serum (n=6). *P < 0.05, **P < 0.01 and ***P < 0.001.
Supplier Page from Abcam for Anti-Lin28 antibody