Journal: International Journal of Molecular Medicine

Article Title: Hypoxia-induced mitophagy regulates proliferation, migration and odontoblastic differentiation of human dental pulp cells through FUN14 domain-containing 1

doi: 10.3892/ijmm.2022.5128

Figure Lengend Snippet: Primer sequences.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-HIF-1α (1:1,000 dilution; cat. no. AF1009; Affinity Biosciences), rabbit anti-sequestosome 1 (SQSTM1)/p62 (1:1,000 dilution; cat. no. AF5384; Affinity Biosciences), rabbit anti-LC3B (1:1,000 dilution; cat. no. 3868S; Cell Signaling Technology, Inc.), mouse anti-dynamin-related protein 1 (DRP1; 1:1,000 dilution; cat. no. ab56788; Abcam), rabbit anti-translocase of inner mitochondrial membrane 23 (TIMM23; 1:1,000 dilution; cat. no. ab116329; Abcam), mouse anti-TOMM20 (1:1,000 dilution; cat. no. ab56783; Abcam), rabbit anti-Beclin-1 (1:1,000 dilution; cat. no. 3495S; Cell Signaling Technology, Inc.), rabbit anti-autophagy related 5 (ATG5; 1:1,000 dilution; cat. no. 12994S; Cell Signaling Technology, Inc.), rabbit anti-FUNDC1 (1:500 dilution; cat. no. ab224722; Abcam), rabbit anti-phosphorylated (p-) FUNDC1 (Ser 17; 1:500 dilution; cat. no. AF0001; Affinity Biosciences), mouse anti-β-actin (1:5,000 dilution; cat. nos.

Techniques: Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Hypoxia-induced mitophagy regulates proliferation, migration and odontoblastic differentiation of human dental pulp cells through FUN14 domain-containing 1

doi: 10.3892/ijmm.2022.5128

Figure Lengend Snippet: Expressions of inflammatory cytokines, HIF-1α and FUNDC1 in healthy and pulpitis tissues. (A) mRNA expression of IL-1β, IL-6, IL-8 and TNF-α in human healthy and pulpitis tissues. mRNA expression of (B) HIF-1α and (C) FUNDC1 in human healthy and pulpitis tissues. (D) Representative immunostaining images of HIF-1α and FUNDC1 in human healthy or inflamed dental pulp tissues. Scale bars are 100 and 25 µ m, respectively. Results are presented as the means ± SD from ≥ three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. healthy. HIF-1α, hypoxia-inducible factor-1α; FUNDC1, FUN14 domain-containing 1.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-HIF-1α (1:1,000 dilution; cat. no. AF1009; Affinity Biosciences), rabbit anti-sequestosome 1 (SQSTM1)/p62 (1:1,000 dilution; cat. no. AF5384; Affinity Biosciences), rabbit anti-LC3B (1:1,000 dilution; cat. no. 3868S; Cell Signaling Technology, Inc.), mouse anti-dynamin-related protein 1 (DRP1; 1:1,000 dilution; cat. no. ab56788; Abcam), rabbit anti-translocase of inner mitochondrial membrane 23 (TIMM23; 1:1,000 dilution; cat. no. ab116329; Abcam), mouse anti-TOMM20 (1:1,000 dilution; cat. no. ab56783; Abcam), rabbit anti-Beclin-1 (1:1,000 dilution; cat. no. 3495S; Cell Signaling Technology, Inc.), rabbit anti-autophagy related 5 (ATG5; 1:1,000 dilution; cat. no. 12994S; Cell Signaling Technology, Inc.), rabbit anti-FUNDC1 (1:500 dilution; cat. no. ab224722; Abcam), rabbit anti-phosphorylated (p-) FUNDC1 (Ser 17; 1:500 dilution; cat. no. AF0001; Affinity Biosciences), mouse anti-β-actin (1:5,000 dilution; cat. nos.

Techniques: Expressing, Immunostaining

Journal: International Journal of Molecular Medicine

Article Title: Hypoxia-induced mitophagy regulates proliferation, migration and odontoblastic differentiation of human dental pulp cells through FUN14 domain-containing 1

doi: 10.3892/ijmm.2022.5128

Figure Lengend Snippet: Hypoxia promotes the phosphorylation of FUNDC1 but the knockdown of FUNDC1 influences mitochondria morphology. (A) Representative western blotting images. (B) Protein expression of FUNDC1 and (C) phosphorylation of FUNDC1 (Ser 17) in HDPCs cultured in hypoxia were quantified. (D) Protein expression of FUNDC1 in HDPCs cultured in normoxia after transfection with siNC or three different siRNAs, (E) which was quantified. The results are presented as the means ± SD from ≥ three independent experiments. (F) Transmission electron microscopy images of normal mitochondria in the normoxic group, single-membrane autolysosomes containing partially degraded mitochondria in the hypoxia + NC group (red arrow) and blurred mitochondrial cristae (yellow arrow) in the hypoxia + siFUNDC1 group. Scale bars, 500 nm. *** P<0.001 vs. normoxia. Nor, normoxia; N, nucleus; M, mitochondrion; Go, Golgi; ER, endoplasmic reticulum; NC, negative control; si, small-interfering; p, phosphorylated; FUNDC1, FUN14 domain-containing.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-HIF-1α (1:1,000 dilution; cat. no. AF1009; Affinity Biosciences), rabbit anti-sequestosome 1 (SQSTM1)/p62 (1:1,000 dilution; cat. no. AF5384; Affinity Biosciences), rabbit anti-LC3B (1:1,000 dilution; cat. no. 3868S; Cell Signaling Technology, Inc.), mouse anti-dynamin-related protein 1 (DRP1; 1:1,000 dilution; cat. no. ab56788; Abcam), rabbit anti-translocase of inner mitochondrial membrane 23 (TIMM23; 1:1,000 dilution; cat. no. ab116329; Abcam), mouse anti-TOMM20 (1:1,000 dilution; cat. no. ab56783; Abcam), rabbit anti-Beclin-1 (1:1,000 dilution; cat. no. 3495S; Cell Signaling Technology, Inc.), rabbit anti-autophagy related 5 (ATG5; 1:1,000 dilution; cat. no. 12994S; Cell Signaling Technology, Inc.), rabbit anti-FUNDC1 (1:500 dilution; cat. no. ab224722; Abcam), rabbit anti-phosphorylated (p-) FUNDC1 (Ser 17; 1:500 dilution; cat. no. AF0001; Affinity Biosciences), mouse anti-β-actin (1:5,000 dilution; cat. nos.

Techniques: Phospho-proteomics, Knockdown, Western Blot, Expressing, Cell Culture, Transfection, Transmission Assay, Electron Microscopy, Membrane, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Hypoxia-induced mitophagy regulates proliferation, migration and odontoblastic differentiation of human dental pulp cells through FUN14 domain-containing 1

doi: 10.3892/ijmm.2022.5128

Figure Lengend Snippet: FUNDC1 knockdown prevents hypoxia-induced mitophagy. (A) Representative western blotting images. The protein expression of (B) HIF-1α, (C) p62, (D) LC3II, (E) DRP1, (F) TIMM23, (G) TOMM20, (H) Beclin-1 and (I) ATG5 in HDPCs after hypoxic culture and transfection with NC or siRNA was all quantified. The results are presented as the means ± SD from ≥ three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001. FUNDC1, FUN14 domain-containing; p-, phosphorylated; Nor, normoxia; Hypo, hypoxia; NC, negative siRNA; si, small interfering; HIF-1α, hypoxia-inducible factor-1α; DRP1, dynamin-related protein 1; TIMM23, translocase of inner mitochondrial membrane 23; TOMM20, translocase of outer mitochondrial membrane 20; ATG5, autophagy related 5.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-HIF-1α (1:1,000 dilution; cat. no. AF1009; Affinity Biosciences), rabbit anti-sequestosome 1 (SQSTM1)/p62 (1:1,000 dilution; cat. no. AF5384; Affinity Biosciences), rabbit anti-LC3B (1:1,000 dilution; cat. no. 3868S; Cell Signaling Technology, Inc.), mouse anti-dynamin-related protein 1 (DRP1; 1:1,000 dilution; cat. no. ab56788; Abcam), rabbit anti-translocase of inner mitochondrial membrane 23 (TIMM23; 1:1,000 dilution; cat. no. ab116329; Abcam), mouse anti-TOMM20 (1:1,000 dilution; cat. no. ab56783; Abcam), rabbit anti-Beclin-1 (1:1,000 dilution; cat. no. 3495S; Cell Signaling Technology, Inc.), rabbit anti-autophagy related 5 (ATG5; 1:1,000 dilution; cat. no. 12994S; Cell Signaling Technology, Inc.), rabbit anti-FUNDC1 (1:500 dilution; cat. no. ab224722; Abcam), rabbit anti-phosphorylated (p-) FUNDC1 (Ser 17; 1:500 dilution; cat. no. AF0001; Affinity Biosciences), mouse anti-β-actin (1:5,000 dilution; cat. nos.

Techniques: Knockdown, Western Blot, Expressing, Transfection, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Hypoxia-induced mitophagy regulates proliferation, migration and odontoblastic differentiation of human dental pulp cells through FUN14 domain-containing 1

doi: 10.3892/ijmm.2022.5128

Figure Lengend Snippet: Knocking down FUNDC1 expression weakens the promotion of hypoxia-induced proliferation, migration and odontoblastic differentiation. (A) Representative images of the wound healing assay (scale bar, 1 mm) and (B) percentages of wound closure from three independent experiments are quantified. (C) The number of migratory cells stained with crystal violet solution from three independent experiments was quantified. (D) Representative images of migratory cells are shown. Scale bar, 100 µ m. (E) Viability of HDPCs was detected by Cell Counting Kit-8 analysis. (F) ALP staining and (G) activity in the normoxic, hypoxia + NC and hypoxia + siFUNDC1 groups on days 3. Scale bar, 200 µ m. (H) Protein expression levels of RUNX2, Col I, OSX and OPN in the normoxic or hypoxic group on day 3 were quantified, (I) the corresponding representative western blotting images of which were shown. Results are presented as the means ± SD from ≥ three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001. FUNDC1, FUN14 domain-containing 1; Nor, normoxia; Hypo, hypoxia; NC, negative control; si, small interfering; ALP, alkaline phosphatase; RUNX2, runt-related transcription factor 2; Col I, collagen type I; OSX, osterix; OPN, osteopontin.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-HIF-1α (1:1,000 dilution; cat. no. AF1009; Affinity Biosciences), rabbit anti-sequestosome 1 (SQSTM1)/p62 (1:1,000 dilution; cat. no. AF5384; Affinity Biosciences), rabbit anti-LC3B (1:1,000 dilution; cat. no. 3868S; Cell Signaling Technology, Inc.), mouse anti-dynamin-related protein 1 (DRP1; 1:1,000 dilution; cat. no. ab56788; Abcam), rabbit anti-translocase of inner mitochondrial membrane 23 (TIMM23; 1:1,000 dilution; cat. no. ab116329; Abcam), mouse anti-TOMM20 (1:1,000 dilution; cat. no. ab56783; Abcam), rabbit anti-Beclin-1 (1:1,000 dilution; cat. no. 3495S; Cell Signaling Technology, Inc.), rabbit anti-autophagy related 5 (ATG5; 1:1,000 dilution; cat. no. 12994S; Cell Signaling Technology, Inc.), rabbit anti-FUNDC1 (1:500 dilution; cat. no. ab224722; Abcam), rabbit anti-phosphorylated (p-) FUNDC1 (Ser 17; 1:500 dilution; cat. no. AF0001; Affinity Biosciences), mouse anti-β-actin (1:5,000 dilution; cat. nos.

Techniques: Expressing, Migration, Wound Healing Assay, Staining, Cell Counting, Activity Assay, Western Blot, Negative Control

Journal: Cell Death Discovery

Article Title: NTRK1 knockdown induces mouse cognitive impairment and hippocampal neuronal damage through mitophagy suppression via inactivating the AMPK/ULK1/FUNDC1 pathway

doi: 10.1038/s41420-023-01685-7

Figure Lengend Snippet: A , B Nissl staining revealed thatNTRK1 knockdown reduced the number of Nissl bodies in the mouse hippocampal CA3 region. n = 3. C – H The results of NeuN, GFAP, and Iba1 IF staining suggested that NTRK1 knockdown reduced the number of NeuN-positive neurons while increasing the number of GFAP and Iba1-positive neurons in mouse hippocampus. n = 3. I – L WB results indicated thatNTRK1 knockdown inactivated the AMPK/ULK1/FUNDC1 pathway and suppressed mitophagy in mouse hippocampus. n = 3. ** P < 0.01.

Article Snippet: The primary antibodies used were rabbit anti-NTRK1 (1:1000, PAB17918, AmyJet Scientific, Wuhan, China), rabbit anti-AMPK (1:1000, A-ABV10093, AmyJet Scientific, Wuhan, China), rabbit anti-p-AMPK (1:1000, YT639, Biolab Technology, Beijing, China), rabbit anti-ULK1 (1:1000, ABP56727, AmyJet Scientific, Wuhan, China), rabbit anti-p-ULK1 (1:1000, YB-19599, Yubo Biotechnology, Shanghai, China), rabbit anti-FUNDC1 (1:1000, PAB23345, AmyJet Scientific, Wuhan, China), rabbit anti-p-FUNDC1 (1:1000, AF0001, Affinity Biosciences, USA), rabbit anti-LC3 (1:1000, A-ABV11461, AmyJet Scientific, Wuhan, China), rabbit anti-P62 (1:1000, VB2368–100, AmyJet Scientific, Wuhan, China), rabbit anti-Parkin (1:1000, A-AJ1587a, AmyJet Scientific, Wuhan, China), rabbit anti-PINK1 (1:1000, PAB4484, AmyJet Scientific, Wuhan, China), rabbit anti-GAPDH(1:1000, A-AJ1089a, AmyJet Scientific, Wuhan, China), and rabbit anti-COXIV (1:1000, SPC-1320D, AmyJet Scientific, Wuhan, China).

Techniques: Staining

Journal: Cell Death Discovery

Article Title: NTRK1 knockdown induces mouse cognitive impairment and hippocampal neuronal damage through mitophagy suppression via inactivating the AMPK/ULK1/FUNDC1 pathway

doi: 10.1038/s41420-023-01685-7

Figure Lengend Snippet: A , B WB revealed that O304 treatment abrogated the suppression effect of NTRK1 knockdown on the activity of the AMPK/ULK1/FUNDC1 pathway in mouse neurons. n = 3. C , D WB also indicated that O304 treatment counteracted the suppression effect of NTRK1 silencing on mitophagy in mouse neurons. n = 3. E , F The results of TOMM20/LC3-II fluorescence staining revealed that the O304 treatment reversed the suppression effect of NTRK1 knockdown on the expression of Parkin in mouse neuronmitochondria. n = 3. ** P < 0.01.

Article Snippet: The primary antibodies used were rabbit anti-NTRK1 (1:1000, PAB17918, AmyJet Scientific, Wuhan, China), rabbit anti-AMPK (1:1000, A-ABV10093, AmyJet Scientific, Wuhan, China), rabbit anti-p-AMPK (1:1000, YT639, Biolab Technology, Beijing, China), rabbit anti-ULK1 (1:1000, ABP56727, AmyJet Scientific, Wuhan, China), rabbit anti-p-ULK1 (1:1000, YB-19599, Yubo Biotechnology, Shanghai, China), rabbit anti-FUNDC1 (1:1000, PAB23345, AmyJet Scientific, Wuhan, China), rabbit anti-p-FUNDC1 (1:1000, AF0001, Affinity Biosciences, USA), rabbit anti-LC3 (1:1000, A-ABV11461, AmyJet Scientific, Wuhan, China), rabbit anti-P62 (1:1000, VB2368–100, AmyJet Scientific, Wuhan, China), rabbit anti-Parkin (1:1000, A-AJ1587a, AmyJet Scientific, Wuhan, China), rabbit anti-PINK1 (1:1000, PAB4484, AmyJet Scientific, Wuhan, China), rabbit anti-GAPDH(1:1000, A-AJ1089a, AmyJet Scientific, Wuhan, China), and rabbit anti-COXIV (1:1000, SPC-1320D, AmyJet Scientific, Wuhan, China).

Techniques: Activity Assay, Fluorescence, Staining, Expressing

Journal: Cell Death Discovery

Article Title: NTRK1 knockdown induces mouse cognitive impairment and hippocampal neuronal damage through mitophagy suppression via inactivating the AMPK/ULK1/FUNDC1 pathway

doi: 10.1038/s41420-023-01685-7

Figure Lengend Snippet: A – C The OFT test revealed that BAY-3827 treatment further prolonged the immobility duration and reduced the rearing and crossing counts of the mice with NTRK1 knockdown. n = 6. D , E The results of TST test and FST test suggested that BAY-3827 treatment further prolonged the immobility duration of the mice with NTRK1 knockdown. n = 6. F , G Virtual radial arm maze test suggested that BAY-3827 treatment intensified the working and reference memory damage in themice with NTRK1 knockdown. n = 6. H – J Morris water maze test demonstrated that BAY-3827 treatment enhanced the damaged of the spatial memory ability in the mice with NTRK1 knockdown. n = 6. K , L WB demonstrated that BAY-3827 treatment further decreased the AMPK/ULK1/FUNDC1 pathway activity in the hippocampus of the mice with NTRK1 knockdown. n = 3. * P < 0.05. ** P < 0.01.

Article Snippet: The primary antibodies used were rabbit anti-NTRK1 (1:1000, PAB17918, AmyJet Scientific, Wuhan, China), rabbit anti-AMPK (1:1000, A-ABV10093, AmyJet Scientific, Wuhan, China), rabbit anti-p-AMPK (1:1000, YT639, Biolab Technology, Beijing, China), rabbit anti-ULK1 (1:1000, ABP56727, AmyJet Scientific, Wuhan, China), rabbit anti-p-ULK1 (1:1000, YB-19599, Yubo Biotechnology, Shanghai, China), rabbit anti-FUNDC1 (1:1000, PAB23345, AmyJet Scientific, Wuhan, China), rabbit anti-p-FUNDC1 (1:1000, AF0001, Affinity Biosciences, USA), rabbit anti-LC3 (1:1000, A-ABV11461, AmyJet Scientific, Wuhan, China), rabbit anti-P62 (1:1000, VB2368–100, AmyJet Scientific, Wuhan, China), rabbit anti-Parkin (1:1000, A-AJ1587a, AmyJet Scientific, Wuhan, China), rabbit anti-PINK1 (1:1000, PAB4484, AmyJet Scientific, Wuhan, China), rabbit anti-GAPDH(1:1000, A-AJ1089a, AmyJet Scientific, Wuhan, China), and rabbit anti-COXIV (1:1000, SPC-1320D, AmyJet Scientific, Wuhan, China).

Techniques: Activity Assay