Figure 5.
SHMT2 inhibition induces autophagic degradation of TCF3. (A) Representative western blot analysis showing LC3 levels in BL60 cells expressing either SHMT2-specific shRNA or nonspecific control shRNA that were treated with chloroquine at a concentration of 100 µM for 4 hours as indicated. GAPDH served as loading control. (n = 4; P = .01 in Student t test). (B) GFP/RFP ratio of BL60 cells transduced with tfLC3 reporter upon treatment with SHIN1 at a concentration of 2.5 µM, AZD2014 at a concentration of 200 nM, and Torin 1 (TOR1) at a concentration of 500 nM for indicated durations. A reduced ratio represents an increased level of autophagy. Bafilomycin A1 (Baf) treatment at 50 nM was used to inhibit autophagy (n = 3, mean ± SEM; P < .0001 in Tukey’s multiple comparison test for TOR1, AZD2014, and SHIN1 compared with DMSO after 6 hours and 24 hours; P = ns for rescue with Baf after 6 hours and 24 hours). (C) Representative confocal images of LC3 immunofluorescence staining. BL60 cells were treated for 24 hours with 2.5 µM SHIN1 or DMSO as well as for the last 6 hours with 50 nM of Bafilomycin or DMSO and stained for LC3 and DAPI. LC3 was stained with Alexa Fluor 647 (red) and nuclei were counterstained with DAPI (blue). Representative images display the overlay max intensity of the 41 z-stacks of the 647 channel and the average intensity of the z-stacks of the DAPI signal. (D) Quantification of LC3 punctae from microscopic images of BL60 cells treated as described in Figure 5C. Data were normalized to control and reported as percentage (n = 2, with n ≥ 31 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots. P < .0001 according to a Kruskal-Wallis test. (E) Representative western blot analysis showing LC3 levels in BL60 cells upon SHIN1 treatment at 2.5 µM for 48 hours in regular medium and upon supplementation with formate and glycine. Chloroquine treatment was applied at a concentration of 100 µM for 4 hours. GAPDH served as loading control (n = 3; P = .03 in Student t test for ΔLC3-II in SHIN1 vs DMSO in regular medium). (F-G) Representative western blots in BL60 cells showing ULK1 Ser757 phosphorylation, ULK1, TCF3, and SHP-1 after treatment of BL60 cells with 2.5 µM SHIN1 for 48 hours in regular medium and upon supplementation with glycine and formate (3.3 mM and 2 mM, respectively). pULK1 and ULK1 were probed on different membranes, but samples were derived from the same experiment and blots were processed in parallel. GAPDH served as loading control (n = 3; P = .02 in Student t test for pULK1 levels in SHIN1 vs DMSO in regular medium; P = .003 in Student t test for TCF3 levels in regular medium vs glycine/formate supplementation; and P = .008 in Student t test for SHP-1 levels in regular medium vs glycine/formate supplementation). (H) Representative western blots showing TCF3 levels in ATG5 KO compared with control-sgRNA in BL60 cell line upon induction of knockout with 250 ng/mL of doxycycline and 48 hours of SHIN1 treatment at a concentration of 2.5 µM vs DMSO control. GAPDH served as loading control (n = 3; P = .037 in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgCtrl; P = ns in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgATG5). (I) PLA score is shown for PLA of TCF3 and LC3 in SHIN1-treated BL60 cells at a concentration of 2.5 µM for 18 hours compared with DMSO control, in regular medium as well as upon supplementation of glycine/formate at a concentration of 3.3 mM and 2 mM, respectively (n = 4; n ≥ 105 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots (P < .001 in Kruskal-Wallis test). (J) Representative confocal images from PLA for TCF3 and LC3 in BL60 cell line, as described in Figure 5I. Merged images represent the composite images of the PLA of TCF3 and LC3 (red) and the DAPI signal (blue).

SHMT2 inhibition induces autophagic degradation of TCF3. (A) Representative western blot analysis showing LC3 levels in BL60 cells expressing either SHMT2-specific shRNA or nonspecific control shRNA that were treated with chloroquine at a concentration of 100 µM for 4 hours as indicated. GAPDH served as loading control. (n = 4; P = .01 in Student t test). (B) GFP/RFP ratio of BL60 cells transduced with tfLC3 reporter upon treatment with SHIN1 at a concentration of 2.5 µM, AZD2014 at a concentration of 200 nM, and Torin 1 (TOR1) at a concentration of 500 nM for indicated durations. A reduced ratio represents an increased level of autophagy. Bafilomycin A1 (Baf) treatment at 50 nM was used to inhibit autophagy (n = 3, mean ± SEM; P < .0001 in Tukey’s multiple comparison test for TOR1, AZD2014, and SHIN1 compared with DMSO after 6 hours and 24 hours; P = ns for rescue with Baf after 6 hours and 24 hours). (C) Representative confocal images of LC3 immunofluorescence staining. BL60 cells were treated for 24 hours with 2.5 µM SHIN1 or DMSO as well as for the last 6 hours with 50 nM of Bafilomycin or DMSO and stained for LC3 and DAPI. LC3 was stained with Alexa Fluor 647 (red) and nuclei were counterstained with DAPI (blue). Representative images display the overlay max intensity of the 41 z-stacks of the 647 channel and the average intensity of the z-stacks of the DAPI signal. (D) Quantification of LC3 punctae from microscopic images of BL60 cells treated as described in Figure 5C. Data were normalized to control and reported as percentage (n = 2, with n ≥ 31 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots. P < .0001 according to a Kruskal-Wallis test. (E) Representative western blot analysis showing LC3 levels in BL60 cells upon SHIN1 treatment at 2.5 µM for 48 hours in regular medium and upon supplementation with formate and glycine. Chloroquine treatment was applied at a concentration of 100 µM for 4 hours. GAPDH served as loading control (n = 3; P = .03 in Student t test for ΔLC3-II in SHIN1 vs DMSO in regular medium). (F-G) Representative western blots in BL60 cells showing ULK1 Ser757 phosphorylation, ULK1, TCF3, and SHP-1 after treatment of BL60 cells with 2.5 µM SHIN1 for 48 hours in regular medium and upon supplementation with glycine and formate (3.3 mM and 2 mM, respectively). pULK1 and ULK1 were probed on different membranes, but samples were derived from the same experiment and blots were processed in parallel. GAPDH served as loading control (n = 3; P = .02 in Student t test for pULK1 levels in SHIN1 vs DMSO in regular medium; P = .003 in Student t test for TCF3 levels in regular medium vs glycine/formate supplementation; and P = .008 in Student t test for SHP-1 levels in regular medium vs glycine/formate supplementation). (H) Representative western blots showing TCF3 levels in ATG5 KO compared with control-sgRNA in BL60 cell line upon induction of knockout with 250 ng/mL of doxycycline and 48 hours of SHIN1 treatment at a concentration of 2.5 µM vs DMSO control. GAPDH served as loading control (n = 3; P = .037 in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgCtrl; P = ns in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgATG5). (I) PLA score is shown for PLA of TCF3 and LC3 in SHIN1-treated BL60 cells at a concentration of 2.5 µM for 18 hours compared with DMSO control, in regular medium as well as upon supplementation of glycine/formate at a concentration of 3.3 mM and 2 mM, respectively (n = 4; n ≥ 105 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots (P < .001 in Kruskal-Wallis test). (J) Representative confocal images from PLA for TCF3 and LC3 in BL60 cell line, as described in Figure 5I. Merged images represent the composite images of the PLA of TCF3 and LC3 (red) and the DAPI signal (blue).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal