Journal: Communications biology

Article Title: The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

doi: 10.1038/s42003-023-05236-9

Figure Lengend Snippet: Fig. 4 Increase of WIPI1 protein enhances autophagic flux. a Immunoblot analysis of LC3B lipidation in U2OS cells after transfection with control plasmids (9E10) or plasmids encoding 9E10-tagged WIPI1. U2OS cells were transfected with the indicated amounts of plasmids for 48 h before protein extraction and immunoblotting against LC3B, 9E10-tagged WIPI1 or GAPDH, n = 3. Additional immunoblots provided in Supplementary Fig. 5a. b U2OS Cas9 control or U2OS WIPI1 KO cells were transfected with control plasmids (9E10) or plasmids encoding 9E10-tagged WIPI1 for 48 h, followed by treatment with bafilomycin A1 (BafA1) in fed conditions for 3 h. Immunoblotting was conducted against LC3B, 9E10 and tubulin (n = 6, mean ± SD, Two-way ANOVA with Tukey’s multiple comparisons test). WIPI1 deficiency control Taqman qPCR is presented in Supplementary Fig. 6b, and representative Western blots in Supplementary Fig. 6c. c U2OS cells stably expressing GFP-WIPI2 were transfected with control plasmids or plasmids encoding mCherry-tagged WIPI1 for 48 h. The numbers of GFP-WIPI2 puncta cells were assessed by fluorescence microscopy in transfected cells. Welch’s t test, mean ± SD, up to 1272 analysed cells from n = 4 in duplicates. d U2OS Cas9 control or U2-OS WIPI1-KO cells were seeded into 96-well glass bottom plates and transfected with siABL1/2, siDDR1 or nontargeting siRNA (siControl) for 48 h, followed by treatment with either DMEM/FBS (fed) or EBSS (starved) for 3 h. After fixation, cells were stained with DAPI and anti-WIPI2/AF488. By automated confocal LSM, 20 images per well were acquired and between 621 to 2563 single cells (from n = 3) subjected to automated CellProfiler-based image analysis (threshold-based puncta segmentation). For statistical analysis, a two-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD). e G361 cells were transfected with plasmids encoding GFP or GFP-WIPI1 and were fed or starved for 3 h in the presence or absence of bafilomycin A1, followed by anti-WIPI2/AF546 immunofluorescence staining. Confocal LSM stacks were acquired, and the numbers of WIPI2 puncta-positive cells per acquired image (individual data points represent the result derived from each image) were counted (left panel: two-way ANOVA with Tukey’s post-hoc test, mean ± SD, up to 215 single cells from n = 3 for each condition). Indicative of the presence of overexpressed GFP-WIPI1 are elongated, perinuclear autophagic membranes found to colocalize with WIPI2 (right panel: Scale bar: 5 μm, extended image presentation in Supplementary Fig. 5b). Supplementary material is available (Supplementary Fig. 6d; Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.

Article Snippet: The following siRNAs were purchased from OriGene Technologies: c-Abl (ABL1) Human siRNA Oligo Duplex (SR3000017/ SR319232), ABL2 Human siRNA Oligo Duplex (SR3000019/ SR319233), MCK10 (DDR1) Human siRNA Oligo Duplex (SR300547/SR319533), c-Myc (MYC) Human siRNA Oligo Duplex (SR321047), MAX Human siRNA Oligo Duplex (SR302818), and Trilencer-27 Universal scrambled negative control siRNA duplex (SR30003/SR30004).

Techniques: Western Blot, Transfection, Control, Protein Extraction, Stable Transfection, Expressing, Microscopy, Staining, Derivative Assay

Journal: Communications biology

Article Title: The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

doi: 10.1038/s42003-023-05236-9

Figure Lengend Snippet: Fig. 7 ABL1 deficiency in C. elegans increases ATG-18 gene expression and autophagic flux. a Hermaphrodites of the N2 wild type, abl-1(ok171) mutant, atg-18 (gk378) mutant or abl-1(ok171);atg-18 (gk378) double mutant strains were singly transferred to NGM plates, and eggs per hermaphrodite were counted over the whole reproductive period, n ≥4 (left panels). The number of L4 nematodes was calculated as a percentage of the total number of eggs laid (right panels). One-way ANOVA with Holm-Sidak post-hoc test, mean ± SD, n ≥4. b For the L1 starvation assay, eggs were isolated, and L1 larvae hatched in nutrient-free medium, in which they were kept for up to 34 days. Every 2-3 days, larvae were removed from starvation and spotted onto NGM/ OP50 plates. After 2 days of unrestricted feeding, nematodes that reached the L2 larval stage or later were scored, and the percentage of developing larvae was calculated. The corresponding results for WT, abl-1(ok171) mutant, atg-18 (gk378) mutant or the abl-1(ok171);atg-18 (gk378) double mutant strain are shown. c N2 wild-type, and wild-type (GFP::LGG1) or abl-1(ok171) mutant (GFP::LGG1; abl-1(ok171) L1 larvae expressing the adIS2122 transgene GFP::LGG1 were starved for 16 h. Protein extracts from whole larvae were analysed by immunoblotting against GFP or tubulin (left panels). Relative protein levels of cleaved GFP over tubulin were quantified (right panel, Welch’s t test, mean ± SD, n = 3). d Likewise, wild-type (GFP::LGG1) or abl-1(ok171) mutant (GFP::LGG1; abl-1(ok171)) L1 larvae expressing the adIS2122 transgene GFP::LGG1 were imaged (left panels) and GFP-LGG1 puncta number (middle panels) as well as the mean puncta size (right panels) per nematode determined using CellProfiler. For statistical analysis, an unpaired t-test with Welch’s correction was performed (GFP::LGG1, 61 nematodes; GFP::LGG1; abl-1(ok171, 55 nematodes; mean ± SD). Scale bar = 50 µm. e Total RNA from synchronised and sterilised wild-type or abl-1(ok171) nematodes was extracted on day 1, day 6 and day 11 of adulthood. Relative ATG-18 mRNA levels were analysed (Welch’s t test, mean ± SD, n = 4). Left panel: ATG-18 expression in wild type nematodes. Middle panels: ATG-18 expression in abl-1(ok171) nematodes. Right panel: Additional comparative display of results (left panels, middle panels) of day 1 and day 11 only. Supplementary material is available (Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.

Article Snippet: The following siRNAs were purchased from OriGene Technologies: c-Abl (ABL1) Human siRNA Oligo Duplex (SR3000017/ SR319232), ABL2 Human siRNA Oligo Duplex (SR3000019/ SR319233), MCK10 (DDR1) Human siRNA Oligo Duplex (SR300547/SR319533), c-Myc (MYC) Human siRNA Oligo Duplex (SR321047), MAX Human siRNA Oligo Duplex (SR302818), and Trilencer-27 Universal scrambled negative control siRNA duplex (SR30003/SR30004).

Techniques: Gene Expression, Mutagenesis, Isolation, Expressing, Western Blot

Journal: Communications biology

Article Title: The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

doi: 10.1038/s42003-023-05236-9

Figure Lengend Snippet: Fig. 8 ABL1 deficiency in C. elegans increases lifespan in an ATG18-dependent manner. a For lifespan assessments, eggs were isolated by hypochlorite treatment and grown on NGM/OP50 plates until they reached the L4 larval stage. L4 nematodes were then transferred to NGM/OP50/FUdR plates to sterilise the nematodes, and surviving nematodes were counted every 2–3 days. The resulting lifespan curves are shown (statistical OASIS analysis, Supplementary Table 1). b Likewise, lifespan assessments were conducted while depleting the c-MYC homologue MML-1 in N2 wild type (mml-1 (RNAi)) or the ULK homologue UNC-51 in the abl-1(ok171) strain (abl-1(ok171); unc-51 (RNAi)) by RNA interference. Lifespan curves are shown and statistical OASIS analysis is displayed in Supplementary Table 2). c A predicted model for the regulation of WIPI1 gene expression by the ABL/MYC axis and its impact on autophagy and lifespan in C. elegans. Created with BioRender.com.

Article Snippet: The following siRNAs were purchased from OriGene Technologies: c-Abl (ABL1) Human siRNA Oligo Duplex (SR3000017/ SR319232), ABL2 Human siRNA Oligo Duplex (SR3000019/ SR319233), MCK10 (DDR1) Human siRNA Oligo Duplex (SR300547/SR319533), c-Myc (MYC) Human siRNA Oligo Duplex (SR321047), MAX Human siRNA Oligo Duplex (SR302818), and Trilencer-27 Universal scrambled negative control siRNA duplex (SR30003/SR30004).

Techniques: Isolation, Gene Expression

Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: The adapter protein Abi1 promotes cell migration. ( A ) Protein expression of human airway smooth muscle (HASM) cells expressing control (Ctrl) shRNA or Abi1 shRNA, or rescue cells was evaluated by immunoblot analysis. Data are mean values of experiments from 5 batches of cell culture. Error bars indicate SD. ( B ) Migration of HASM cells was tracked using time-lapse microscopy. Images were taken every 10 min for 16 h. Migration plots (35–46 cells from each group) were generated using the NIH ImageJ software. Red tracks indicate downward migration whereas black tracks indicate upward migration. ( C )–( E ) Abi1 knockdown (KD) reduced accumulated distance, Euclidean distance and speed of cell migration. Rescue of Abi1 recovered these parameters. ( F ), ( G ) Migration of HASM cells was examined by using the wound healing assay. Scale bar: 100 µm. n = 10 experiments. Error bars indicate SD. * p < 0.05. One ANOVA was used for statistical analysis.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Expressing, Control, shRNA, Western Blot, Cell Culture, Time-lapse Microscopy, Generated, Software, Knockdown, Wound Healing Assay

Journal: PLoS ONE

Article Title: C-Abl Inhibitor Imatinib Enhances Insulin Production by β Cells: C-Abl Negatively Regulates Insulin Production via Interfering with the Expression of NKx2.2 and GLUT-2

doi: 10.1371/journal.pone.0097694

Figure Lengend Snippet: A . NIT-1 cells were cultured in the low glucose media (glucose 4.5 mM) with glucose 8 mM, 16 mM, 8 mM plus Imatinib 3 µM or 16 mM plus Imatinib 3 µM for 6 hrs. The insulin concentration was measured using ELISA. The similar results were obtained in 3 independent experiments. Two-way ANOVA with Bonferroni post-test was performed. B . NIT-1 cells were cultured in the low glucose media (glucose 4.5 mM) with glucose 16 mM or 16 mM plus Imatinib 3 µM for 6 hrs, insulin gene expression was examined by real-time RT-PCR. The levels of insulin gene expression were normalized relative to β actin. The results were reproduced in 3 independent experiments. Student t test was performed. C . In the cultures of B above, C-peptide concentration in each incuation was examined using ELISA. Student t test was performed. D . NIT-1 cells were transfected with c-Abl siRNA or control siRNA for 24 hrs, then were stimulated with 16 mM glucose for 6 hrs. Insulin gene expression was examined by real-time RT-PCR, the data were calculated relative to the group with NIT-1 cells transfected with control siRNA without glucose stimulation. Three independent experiments were performed with similar results. Student t test was performed. *: p<0.05, **:p<0.01,***:p<0.001.

Article Snippet: The c-Abl or control siRNA was incubated with NIT-1 cells following the protocol from the siRNA transfection kit (Santa Cruz Biotech, Inc., Santa Cruz, CA).

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Quantitative RT-PCR, Transfection, Control

Journal: PLoS ONE

Article Title: C-Abl Inhibitor Imatinib Enhances Insulin Production by β Cells: C-Abl Negatively Regulates Insulin Production via Interfering with the Expression of NKx2.2 and GLUT-2

doi: 10.1371/journal.pone.0097694

Figure Lengend Snippet: NIT-1 cells were cultured in low glucose DMEM medium (glucose 4.5 mM) for a few days. A . The NIT-1 cells were harvested and then cultured in low glucose DMEM medium with different conditions: Imatinib 0 µM, 0.3 µM, 1 µM or 3 µM for 6 hrs. The supernatants from all cultures were harvested and measured for insulin production by ELISA. The results presented were from a representative of three independent experiments. Two-way ANOVA with Bonferroni post-test was performed. B . The NIT-1 cells were cultured in low glucose DMEM medium with Imatinib 0 µM, 0.3 µM or 1 µM for 6 hrs. The insulin gene expression was examined by real-time RT-PCR. The levels of insulin gene expression were normalized relative to β actin. The experiments were repeated 3 times with reproducible results. Two-way ANOVA with Bonferroni post-test was performed. C . NIT-1 cells were transfected with c-Abl siRNA or control siRNA for 24 hrs. Then the transfected cells were cultured in low glucose DMEM medium for 6 hrs. An additional group was also included by culturing c-Abl siRNA transfected NIT-1 cells with 1 µM Imatinib. The supernatants were harvested from the above cultures and the insulin concentration was examined by ELISA. One-way ANOVA with post hoc test was performed. Similar results were obtained from at least 3 independent experiments. *: p<0.05, **:p<0.01,***:p<0.001.

Article Snippet: The c-Abl or control siRNA was incubated with NIT-1 cells following the protocol from the siRNA transfection kit (Santa Cruz Biotech, Inc., Santa Cruz, CA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Gene Expression, Quantitative RT-PCR, Transfection, Control, Concentration Assay