Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: TNF-α modulates the WISP1 expression in stroma cells of the human bladder. (A) The RT-qPCR determined the expression of WISP1 in the human bladder cells. (B) Two WISP1 isoforms (WISP1v1 and WISP1v2) were found in the human bladder fibroblast (HBdSF) and bladder smooth muscle (HBdSMC) cells by RT-PCR with the pair of primers as shown. The expressions of WISP1, IL-6, CXCL5, CXCL12, and GDF15 of the HBdSF (C) and HBdSMC (D) cells after 10 ng/ml TNF-α treatment assessed by RT-qPCR. WISP1 (E) and IL-6 (F) secretions after TNF-α (0 – 20 ng/mL) treatments in HBdSF cells. Secretion of WISP1 (G) and IL-6 (F) in HBdSMC_shCOL and HBdSMC_shWISP1 cells treated with/without 10 ng/ml of TNF-α. Data are presented as the mean percentage (± SE; n = 4) in relation to the vehicle-treated HBdSMC_shCOL cells. (I) WISP1 secretion after 20 μM CAPE and/or TNF-α in HBdSF cells determined by ELISA assays. Data are presented as the mean percentage (±SE; n = 4) in relation to the vehicle-treated HBdSF cells. (J) The luciferase activity of the WISP1 reporter vector when HBdSMC cells were treated with TNF-α (10 ng/mL) and/or 20 μM CAPE for 24 h. * p < 0.05; ** p < 0.01; ND: no detectable.

Article Snippet: TaqManTM gene expression master mix and polymerase chain reaction (PCR) FAM dye-labeled TaqMan MGB probes for human WISP1 (Hs04234730_m1 for total isoforms and Hs00180245 for WISP1v1), α-SMA (Hs00426835_g1), IL-6 (Hs00985639_m1), GDF15 (Hs00171132_m1), CXCL5 (Hs01099660_g1), SDF-1/CXCL12 (Hs03676656_m1), NDRG1 (Hs00608387_m1), KAI1 (Hs00356310_m1), Maspin (Hs00985283_m1), E-cadherin (Hs01023894_m1), N-cadherin (Hs00169953_m1), Snail (Hs00195591_m1), Slug (Hs00161904_m1), Vimentin (Hs00185584_m1), and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific Inc. (Vilnius, Lithuania).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Plasmid Preparation

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: WISP1 modulates expressions of α-SMA, IL-6, GDF15, and CXCL5 in bladder stroma cells. The mRNA levels of two WISP1 isoforms (WISP1v1 and WISP1v2) after WISP1 knock-downed in the HBdSF (A) and HBdSMC (B) cells were determined by RT-PCR. The protein levels with quantification analysis of WISP1, GDF15, and α-SMA of HBdSMC (C) and HBdSF (D) cells after mock-knockdown or WISP1-knockdown were assessed by immunoblot assays. The mRNA levels of the WISP1, α-SMA, IL-6, GDF15, and CXCL5 after knockdown of WISP1 in the HBdSMC (E) and HBdSF (F) cells, as indicated, were determined by RT-qPCR. Data are presented as target genes/β-actin of mock-knockdown cells relative to WISP1-knockdown cells. ** p < 0.01.

Article Snippet: TaqManTM gene expression master mix and polymerase chain reaction (PCR) FAM dye-labeled TaqMan MGB probes for human WISP1 (Hs04234730_m1 for total isoforms and Hs00180245 for WISP1v1), α-SMA (Hs00426835_g1), IL-6 (Hs00985639_m1), GDF15 (Hs00171132_m1), CXCL5 (Hs01099660_g1), SDF-1/CXCL12 (Hs03676656_m1), NDRG1 (Hs00608387_m1), KAI1 (Hs00356310_m1), Maspin (Hs00985283_m1), E-cadherin (Hs01023894_m1), N-cadherin (Hs00169953_m1), Snail (Hs00195591_m1), Slug (Hs00161904_m1), Vimentin (Hs00185584_m1), and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific Inc. (Vilnius, Lithuania).

Techniques: Reverse Transcription Polymerase Chain Reaction, Knockdown, Western Blot, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: The relationship between GDF15 and tubulointerstitial fibrosis mediated by TGF β in HK2 cells. (A–D) Screening the differentially expressed genes related to TIF. (E, F) Western blot analysis and quantification of fibronectin, collagen I, and GDF15 in HK2 cells stimulated with 10 ng/mL of TGF‐β1 for 12, 24, and 48 h. (G) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in different groups as indicated. The relative expression levels of the indicated proteins were normalised to that of β‐actin ( n = 3).* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: Western Blot, Quantitative RT-PCR, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 expression in the fibrotic kidney of patients with CKD and UUO mice. (A) Immunohistochemical staining for Collagen I, GDF15 and representative images of Masson's trichrome staining in the kidneys of patients with CKD. (B) Correlation between Collagen I, serum creatinine, eGFR and the expression of renal GDF15 in patients with CKD. (C, E) Western blot analysis and quantification of the GDF15 and GFRAL levels in UUO mice. The relative expression levels of the indicated proteins were normalised to that of β‐actin. (D) Immunohistochemical staining for Collagen I, GDF15 and representative images of Masson's trichrome staining, Picrosirius red staining in the kidneys of UUO mice. (F) Quantitative analysis of immunohistochemistry for GDF15 in the cortex and medulla of UUO mice ( n = 6). (G) Dynamic changes in serum GDF15 concentrations at different time points following UUO intervention in mice and GDF15 levels in the supernatant of TGF‐β1‐stimulated HK2 cells ( n = 3). (H) Co‐staining of GDF15 with AGTR1: A distinct yellow signal in the merged image indicates that GDF15 is primarily localised in AGTR1‐positive tubular epithelial cells. (I) Co‐staining of GDF15 with the proximal tubule marker LTL: Absence of a yellow overlap in the merged image suggests weak GDF15 expression in proximal tubules. (J) Co‐staining of GDF15 with the collecting duct marker AQP2: No overlap signal is observed in the merged image, indicating minimal GDF15 expression in collecting ducts. (K) Co‐staining of GDF15 with WT1, a podocyte marker: The merged image shows no co‐localisation, suggesting that GDF15 is not expressed in glomerular podocytes. (L) Co‐staining of GFRAL with NCC: Strong overlap of red and green signals in the merged image indicates predominant localisation of GFRAL in the distal tubules. (M) Co‐staining of GFRAL with AQP2: No overlapping signal is observed in the merged image, demonstrating that GFRAL is not expressed in collecting ducts. Scale bars for all merged images = 50 μm ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Immunohistochemistry, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: Downregulation of GDF15 enhances TGF β‐stimulated TIF in vitro and UUO‐induced TIF in vivo. (A) Representative images of Masson's trichrome staining, Picrosirius red staining and immunohistochemical staining for Collagen I, Fibronectin, and macrophage infiltration in the kidney homogenates from sham and UUO mice injected with shNC or sh556. (B) Quantitative analysis of immunohistochemistry for Collagen I, Fibronectin and macrophage in different groups as indicated. (C, E) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vitro ( n = 3). The relative expression levels of the indicated proteins were normalised to that of β‐actin. (G) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vitro ( n = 3). (D, F) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vivo. The relative expression levels of the indicated proteins were normalised to that of β‐actin ( n = 6). (H) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vivo ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: In Vitro, In Vivo, Staining, Immunohistochemical staining, Injection, Immunohistochemistry, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: Overexpression of GDF15 ameliorated TGF‐β‐stimulated TIF in vitro and UUO‐induced TIF in vivo. (A) Representative images of Masson's trichrome staining, Picrosirius red staining and immunohistochemical staining for Collagen I, Fibronectin, and macrophage infiltration in the kidney homogenates from sham and UUO mice injected with empty vector or pGV362‐GDF15. (B) Quantitative analysis of immunohistochemistry for Collagen I, Fibronectin and GDF15 in different groups as indicated. (C, E) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vitro ( n = 3). The relative expression levels of the indicated proteins were normalised to that of β‐actin. (G) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vitro ( n = 3). (D, F) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vivo. The relative expression levels of the indicated proteins were normalised to that of β‐actin ( n = 6). (H) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vivo ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: Over Expression, In Vitro, In Vivo, Staining, Immunohistochemical staining, Injection, Plasmid Preparation, Immunohistochemistry, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 ameliorates renal fibrosis through enhancing autophagy. (A) Bioinformatics predicts the mechanism of GDF15: PCA; Differentially clustered volcano plot; Top 15 of GO enrichment. (B) Western blot analysis of the LC3B, Beclin1 and p62 in TGF‐β‐stimulated TIF with transfection of shNC or sh865 ( n = 3). (C, F) Western blot analysis and quantification of the LC3B, Beclin1 in UUO‐induced TIF with injecting shNC or sh556 ( n = 6). (D) Western blot analysis of the LC3B, Beclin1 and p62 in TGF‐β‐stimulated TIF with transfection of empty vector or pGV362‐GDF15 ( n = 3). (E, G) Western blot analysis and quantification of the LC3B, Beclin1 in UUO‐induced TIF with injecting empty vector or pGV362‐GDF15 ( n = 6). (H) Western blot analysis the Collagen I and Fibronectin in TGF‐β‐stimulated TIF with transfection of empty vector, pGV362‐GDF15 and chloroquine treatment ( n = 3). (I) qRT‐PCR analysis of fibronectin and collagen I levels in different groups as indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 ameliorates renal fibrosis through improving lysosomal neogenesis. (A) GSEA enrichment. (B) Downregulation of kidney GDF15 in the UUO model, electron microscopy detected the number of lysosomes (i, ii), the scale bars in the upper‐row images represent 2 μm, and those in the lower‐row images represent 500 nm and immunohistochemical staining analysis of the LAMP (iii). (C) qRT‐PCR analysis of Lamp1 and Lamp2 in different groups as indicated. (D) Overexpression of kidney GDF15 in the UUO model, electron microscopy detected the number of lysosomes (i, ii), the scale bars in the upper‐row images represent 2 μm, and those in the lower‐row images represent 500 nm and immunohistochemical staining analysis of the LAMP (iii). (E) qRT‐PCR analysis of Lamp1 and Lamp2 in different groups as indicated ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: Electron Microscopy, Immunohistochemical staining, Staining, Quantitative RT-PCR, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 promoted autophagy and lysosome biogenesis by inhibiting the PI3K/Akt/mTOR pathway. (A) Downregulation of kidney GDF15 in the UUO model, Western blot analysis and quantification of TFEB. The relative expression levels of the indicated proteins were normalised to that of β‐actin. (B) qRT‐PCR analysis of TFEB and PASP in different groups as indicated. (C) Overexpression of kidney GDF15 in the UUO model, Western blot analysis and quantification of TFEB. The relative expression levels of the indicated proteins were normalised to that of β‐actin. (D) qRT‐PCR analysis of TFEB and PASP in different groups as indicated. (E) Western blot analysis of p‐mTOR, mTOR, p‐Akt, Akt, p‐PI3K, and PI3K levels in TGF β‐stimulated TIF with transfection of empty vector or pGV362‐GDF15. (F, G) Effects of GDF15 silencing and PI3K inhibition on the PI3K signalling pathway and the expression of fibrosis‐ and autophagy‐related proteins. HK‐2 cells were transfected with shGDF15 (sh865) or control vector (shNC) for 48 h, followed by treatment with the PI3K inhibitor LY294002 (final concentration 20 μM) for 24 h. Western blot and quantification was performed to detect the expression levels of p‐PI3K, PI3K, fibronectin, collagen I, TFEB, and the internal control β‐actin. All data were normalised to β‐actin ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Inhibition, Control, Concentration Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 mediates autophagy activation and anti‐fibrotic effects through GFRAL. (A) Western blot analysis and quantification of GFRAL protein expression in HK2 cells following siRNA‐mediated knockdown. siNC served as the negative control, and siGFRAL#1, #2, and #3 represent different interference sequences. (B) Western blot analysis and quantification of fibronectin, collagen I, and GDF15 protein expression in various treatment groups (Vector, GDF15, GDF15 + siNC, GDF15 + siGFRAL) under TGF‐β stimulation. β‐actin was used as the loading control. (C) Western blot analysis and quantification of LC3B, P62, and Beclin1 protein levels in each group. (D) Western blot analysis and quantification of TFEB protein expression in each group. (E) Transmission electron microscopy was used to observe the structural changes of lysosomes in HK2 cells from each group. The scale bars in the upper‐row images represent 1 μm, and those in the lower‐row images represent 500 nm. (F) qRT‐PCR analysis of Lamp1 and Lamp2 in different groups as indicated. (G) Western blot analysis and quantification of PI3K/AKT/mTOR signalling pathway proteins, including p‐mTOR, mTOR, p‐AKT, AKT, p‐PI3K, and PI3K. β‐actin was used as the loading control ( n = 3).* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking non‐specific binding with 5% BSA for 1 h, we incubated the membranes overnight at 4°C with primary antibodies against the following: collagen IA (1:1000; BA0325; Boster), Beclin1 (1:1000; T55092 ; Abmart), P62 (1:1000; T55546 ; Abmart), LC3B (1:1000; T55992 ; Abmart), total PI3K (1:1000; T40064 ; Abmart), phospho‐PI3K (1:1000; T40065 ; Abmart), total mTOR (1:1000; T55306 ; Abmart), phospho‐mTOR (1:1000; T56571 ; Abmart), TFEB (1:1000; TA7015; Abmart), total Akt (1:1000; 60203–2‐Ig; Proteintech), phospho‐Akt (1:1000; 66444–1‐Ig; Proteintech), fibronectin (1:1000; ab2413; Abcam), GDF15 (1:500; BS3818‐R; Bioss), and anti‐GFRAL (1:1000; ab214929; Abcam).

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Negative Control, Plasmid Preparation, Control, Transmission Assay, Electron Microscopy, Quantitative RT-PCR