TRIM27 cooperates with STK38L to inhibit ULK1‐mediated autophagy and promote tumorigenesis

A

Ubiquitination analysis of HA‐ULK1 in HeLa derivatives generated in Fig 1(H) co‐expressing His‐Ub with or without TRIM27 knockout, the cells were treated with starvation for the indicated time.

B

Western blot analysis of ULK1 levels in HeLa cells with or without TRIM27 knockout. The cells were treated with starvation for the indicated time. The lysates were analyzed using anti‐ULK1, anti‐TRIM27, or anti‐Actin.

C

HA‐ULK1, HA‐ULK1 K568/K571R were stably expressed in ULK1 knockout HeLa cells, the cells were treated with starvation, the lysates were analyzed by Western blot using anti‐HA, or anti‐Actin.

D

List of potential Top 10 proteins that bind to TRIM27 under starvation conditions identified by mass spectrometry.

E

Co‐immunoprecipitation of Flag‐TRIM27 with HA‐ULK1 in HEK293T cells with or without STK38L knockdown, the cells were treated with starvation. Flag‐TRIM27 was immunoprecipitated using anti‐Flag, the bound HA‐ULK1 was detected using anti‐HA.

F

Co‐immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa cells.

G

Co‐immunoprecipitation of Flag‐STK38L with ULK1, or TRIM27 in HEK293T cells with or without starvation treatment. Flag‐STK38L was immunoprecipitated using anti‐Flag, the immunoprecipitates and lysates were analyzed with anti‐ULK1, anti‐TRIM27.

H

Co‐immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa cells with or without STK38L knockout. The cells were treated with starvation before lysis.

I

Western blot analysis of ULK1 levels in starved HeLa cells expressing either Myc‐STK38L, or Flag‐TRIM27, or both. The lysates were analyzed using anti‐ULK1, anti‐Flag, anti‐Myc, or anti‐actin.

J

Ubiquitination analysis of HA‐ULK1 in HEK293T cells co‐expressing Flag‐TRIM27 and His‐Ub, or together with Myc‐STK38L.

K

Western blot analysis of ULK1 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time.

L

Western blot analysis of p62 or LC3 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time. Bafilomycin A1 (BAF A1) was used to inhibit the fusion between autophagosomes and lysosomes.

Figure 4. STK38L negatively regulates starvation‐induced autophagy via promoting binding between TRIM27 and ULK1.