Journal: Nature Communications

Article Title: An autophagy enhancer ameliorates diabetes of human IAPP -transgenic mice through clearance of amyloidogenic oligomer

doi: 10.1038/s41467-020-20454-z

Figure Lengend Snippet: a INS-1 cells were transfected with mRFP-GFP-LC3 . The numbers of yellow (autophagosomes) and red puncta (autophagolysosomes) were counted ( t = 9.8, df = 38 for autophagosome; t = 17.2, df = 38 for autophagolysosome) (right). Representative pictures are presented (left). Inset images were magnified. b INS-1 cells were treated with MSL-7 in the presence or absence of bafilomycin A1 (BAF). Immunoblotting using the indicated antibodies (ACTB, β-actin) were conducted. Numbers indicate fold changes normalized to ACTB bands. c – d Nuclear translocation of TFEB ( c ) and TFE3 ( d ) by MSL-7 was counted ( t = 29.9, df = 12 in c ; t = 38.7, df = 12 in d ) (right of c , d ). Representative pictures are shown (left of c , d ). e INS-1 cells were treated with MSL-7, and immunoblotting using the indicated antibodies were conducted. Numbers indicate fold changes normalized to total TFEB bands. f After lysis of INS-1 cells treated with MSL-7 for 4 h, immunoprecipitation (IP) was conducted using anti-TFEB (left) or anti-TFE3 antibody (right). Supernatant was subjected to immunoblot analysis (IB) using the indicated antibodies. Numbers indicate fold changes normalized to total TFEB or TFE3 bands. g Nuclear and cytosolic fractions of lysates from INS-1 cells treated with MSL-7 were subjected to immunoblot analysis using the indicated antibodies. Numbers indicate fold changes normalized to ACTB bands (cytosol) or Lamin bands (nuclear). h After lysis of Tfeb-GFP- transfected (left) or Tfe3-GFP- transfected cells (right) treated with MSL-7, IP using anti-GFP antibody was conducted. i After treatment of Tfeb-GFP- transfected or Tfe3-GFP -transfected cells with MSL-7, confocal microscopy was conducted. Numbers indicate fold changes normalized to GFP bands. j Real-time RT-PCR was performed using mRNA from INS-1 cells treated with MSL-7 for 6 h and specific primers. All data in this figure are the means ± SEM from more than 3 independent experiments performed in triplicate. (scale bar, 5 μm) * P < 0.05; ** P < 0.01; *** P < 0.001 by two-tailed Student’s t -test ( a , c , d , j ). Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used: SQSTM1 (Progen Biotechnik, 1:5000), LC3 (Novus Biologicals, 1:1000), TFEB (Bethyl Laboratories, 1:1000), TFE3 (Sigma Aldrich, 1:1000), phospho-S142-TFEB (Merk Millipore, 1:1000), phospho-(Ser) 14-3-3 binding motif (Cell Signaling Technology, 1:1000), 14-3-3 protein (Cell Signaling Technology, 1:1000), β-actin (ACTB) (Santa Cruz Biotechnology, 1:4000), HA (Cell Signaling Technology, 1:1000) or Lamin A (Santa Cruz Biotechnology, 1:1000).

Techniques: Transfection, Western Blot, Translocation Assay, Lysis, Immunoprecipitation, Confocal Microscopy, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: An autophagy enhancer ameliorates diabetes of human IAPP -transgenic mice through clearance of amyloidogenic oligomer

doi: 10.1038/s41467-020-20454-z

Figure Lengend Snippet: a Prepro-mIAPP-HA- transfected or Prepro -hIAPP-HA -transfected INS-1 cells were treated with MSL-7 in the presence or absence of bafilomycin A1 (BAF). Lysate was subjected to immunoblotting using anti-HA or anti-β-actin antibody (ACTB). Numbers indicate fold changes of hIAPP dimer normalized to ACTB bands. (Con, control-transfected). b Prepro-mIAPP-HA- transfected or Prepro -hIAPP-HA -transfected cells were treated with MSL-7 in the presence or absence of 3-MA. Oligonucleosome content was determined ( F = 19.5, df treatment = 11, df residual = 36). c–d MSL-7-induced nuclear translocation of TFEB ( c ) and TFE3 ( d ) in monkey islet cells was counted ( t = 33.2, df = 14 in c ; t = 45.7, df = 14 in d ) (right of c , d ). Representative pictures are presented (left of c , d ). e mRFP-GFP-LC3 -transfected monkey islet cells were treated with MSL-7. The numbers of autophagosome ( t = 9.5, df = 44) and autophagolysosome ( t = 10.9, df = 44) were counted (right). Representative pictures are presented (left). Inset images were magnified to show red (autophagolysosomes) and yellow (autophagosomes) puncta. f Monkey islet cells treated with MSL-7 in the presence or absence of 3-MA were subjected to immunostaining using A11 antibody. The number of A11 + puncta was counted ( F = 86.3, df treatment = 2, df residual = 42) (right). Representative pictures are presented (left). g Monkey islet cells treated with MSL-7 in the presence of BAF, cells were immunostained using A11 and anti-LC3 antibodies. The number of LC3 puncta colocalized with A11 + oligomer was counted ( t = 13.9, df = 38) (right). Representative pictures are shown (left). Inset images were magnified to shows LC3 puncta colocalized with A11 + oligomer. h Apoptosis was determined in MSL-7-treated monkey islet cells in the presence or absence of 3-MA ( F = 10.1, df treatment = 3, df residual = 20). All data in this figure are the means ± SEM from more than 3 independent experiments performed in triplicate. (scale bar, 5 μm) * P < 0.05; ** P < 0.01; *** P < 0.001 by one-way ANOVA with Tukey’s test ( b , f , h ) and two-tailed Student’s t -test ( c , d , e , g ). Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used: SQSTM1 (Progen Biotechnik, 1:5000), LC3 (Novus Biologicals, 1:1000), TFEB (Bethyl Laboratories, 1:1000), TFE3 (Sigma Aldrich, 1:1000), phospho-S142-TFEB (Merk Millipore, 1:1000), phospho-(Ser) 14-3-3 binding motif (Cell Signaling Technology, 1:1000), 14-3-3 protein (Cell Signaling Technology, 1:1000), β-actin (ACTB) (Santa Cruz Biotechnology, 1:4000), HA (Cell Signaling Technology, 1:1000) or Lamin A (Santa Cruz Biotechnology, 1:1000).

Techniques: Transfection, Western Blot, Translocation Assay, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: An autophagy enhancer ameliorates diabetes of human IAPP -transgenic mice through clearance of amyloidogenic oligomer

doi: 10.1038/s41467-020-20454-z

Figure Lengend Snippet: a Tfeb -KO or Tfe3 -KO INS-1 cells were treated with MSL-7 for 4 h, and immunostained using anti-TFEB (left) or anti-TFE3 antibody (right). After DAPI staining, cells were subjected to confocal microscopy. b Tfeb -KO or Tfe3 -KO INS-1 cells transfected with mRFP-GFP-LC3 tandem construct were treated with MSL-7 for 16 h, and the numbers of autophagosome and autophagolysosome were counted ( F = 178.7, df treatment = 5, df residual = 114) (right). Representative pictures are presented (left). Inset images were magnified to show red (autophagolysosomes), green, and yellow (autophagosomes) puncta. c Tfeb -KO or Tfe3 -KO INS-1 cells transfected with prepro-mIAPP-HA or prepro -hIAPP-HA were treated with MSL-7 for 16 h. Immunoblot analysis was conducted ( F = 32.9, df treatment = 5, df residual = 12) (left). Densitometric value of HA bands normalized to the respective vehicle-treated cells (−) are presented (right). d Tfeb -KO (left) or Tfe3 -KO (right) INS-1 cells transfected with prepro-mIAPP-HA or prepro -hIAPP-HA were treated with MSL-7 for 16 h without 3-MA, and apoptosis was measured ( F = 5.8, df treatment = 11, df residual = 36 in left panel; F = 11.3, df treatment = 11, df residual = 36 in right panel). e Tfeb -KO or Tfe3 -KO INS-1 cells transfected with prepro-mIAPP-HA or prepro -hIAPP-HA were treated with MSL-7 for 16 h in the presence of 3-MA, and apoptosis was measured ( F = 24.1, df treatment = 11, df residual = 36). All data in this figure are the means ± SEM from more than 3 independent experiments performed in triplicate. (scale bar, 5 μm) * P < 0.05; ** P < 0.01; *** P < 0.001 by one-way ANOVA with Tukey’s test. (n.s. not significant) Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used: SQSTM1 (Progen Biotechnik, 1:5000), LC3 (Novus Biologicals, 1:1000), TFEB (Bethyl Laboratories, 1:1000), TFE3 (Sigma Aldrich, 1:1000), phospho-S142-TFEB (Merk Millipore, 1:1000), phospho-(Ser) 14-3-3 binding motif (Cell Signaling Technology, 1:1000), 14-3-3 protein (Cell Signaling Technology, 1:1000), β-actin (ACTB) (Santa Cruz Biotechnology, 1:4000), HA (Cell Signaling Technology, 1:1000) or Lamin A (Santa Cruz Biotechnology, 1:1000).

Techniques: Staining, Confocal Microscopy, Transfection, Construct, Western Blot

Journal: Pharmaceutical Biology

Article Title: Andrographolide emeliorates maltol aluminium-induced neurotoxicity via regulating p62-mediated Keap1-Nrf2 pathways in PC12 cells

doi: 10.1080/13880209.2021.1883678

Figure Lengend Snippet: Andro upregulated the expression of p62 and LC3 proteins and mRNA in PC12 cells induced by Al(mal) 3 . Cells were incubated with 700 μM Al(mal) 3 and 5 or 10 μM Andro for 24 h. The expression of p62 and LC3 proteins were detected by western blot, GAPDH was used as loading control (A). Quantitative analysis of p62 (B) and the ratio of LC3-II to LC3-I (C) protein expression levels. The levels of p62 and LC3 (D) mRNA were analysed using RT-qPCR * p < 0.05, ** p < 0.01 versus the control, # p < 0.05, ## p < 0.01 versus Al(mal) 3 group was considered statistically significant differences ( n = 3).

Article Snippet: The membranes were then blocked with 5% (w/v) fat-free milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies at 1:1000 dilution ratio: APP(1007-5, huaanbio, China), BACE1(5606, CST, USA), p-Tau (Ser396) (AF3148, Affinity, USA), Tau (AF1249, Beyotime, Shanghai, China), Keap1 (sc-514914, Santa Cruz, USA) Nrf2 (ab62352, Abcam, USA), p62 (AF5384, Affinity, USA), LC3 (bs-8878R, Bioss, China), GAPDH (FD0063, Fudebio, China).

Techniques: Expressing, Incubation, Western Blot, Quantitative RT-PCR

Journal: Pharmaceutical Biology

Article Title: Andrographolide emeliorates maltol aluminium-induced neurotoxicity via regulating p62-mediated Keap1-Nrf2 pathways in PC12 cells

doi: 10.1080/13880209.2021.1883678

Figure Lengend Snippet: Effect of silencing p62 or Nrf2 on the expression of p62, LC3, Nrf2 proteins and mRNA in PC12 cells co-treated with Al(mal) 3 and Andro. After transfection with Nrf2 or p62 siRNA, cells were incubated with 700 μM Al(mal) 3 and 10 μM Andro for 24 h. The expression of p62 (A), LC3 (B) and Nrf2 (E) were detected by western blot, GAPDH was used as loading control. The levels of p62 (B), LC3 (C) and Nrf2 (F) mRNA were analysed using RT-qPCR. # p < 0.05, ## p < 0.01 versus the control, * p < 0.05, ** p < 0.01 versus Al(mal) 3 + Andro group was considered statistically significant differences ( n = 3).

Article Snippet: The membranes were then blocked with 5% (w/v) fat-free milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies at 1:1000 dilution ratio: APP(1007-5, huaanbio, China), BACE1(5606, CST, USA), p-Tau (Ser396) (AF3148, Affinity, USA), Tau (AF1249, Beyotime, Shanghai, China), Keap1 (sc-514914, Santa Cruz, USA) Nrf2 (ab62352, Abcam, USA), p62 (AF5384, Affinity, USA), LC3 (bs-8878R, Bioss, China), GAPDH (FD0063, Fudebio, China).

Techniques: Expressing, Transfection, Incubation, Western Blot, Quantitative RT-PCR

Journal: Endocrinology

Article Title: Deficiency of C1QL1 reduced murine ovarian follicle reserve through intraovarian and endocrine control.

doi: 10.1210/endocr/bqac048

Figure Lengend Snippet: Figure 5. Deficiency of C1QL1 promoted apoptosis and inhibited autophagy in the ovary. (A) Representative images of TUNEL staining of ovarian sections. The green signals indicate representative cells with positive TUNEL staining, which was mainly detected in the large atretic follicles. (B) The intensity of TUNEL-positive signals was shown in charts (n = 4 per group). (C) Representative images of immunofluorescence staining of ovarian sections with LC3B antibody. LC3B-positive signals were found in granulosa cells and oocytes and were weakened in C1QL1-deficient ovaries. (D) The intensity of LC3B-positive signals are shown (n = 4 per group). (E) Apoptosis- and autophagy-related proteins in the ovaries were analyzed by Western blotting. Representative blots are shown in the left panel, and the amount of protein normalized to β-tubulin, which is presented as mean ± SEM, is shown in the right panel. n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significance.

Article Snippet: The sections and granulosa cells were stained with anti-rabbit LC3B antibody and Alexa Fluor 594-conjugated secondary antibody and mounted with DAPI (Boster).

Techniques: TUNEL Assay, Staining, Immunofluorescence, Western Blot