Journal: Autophagy Reports

Article Title: TRPML3 and TRPML1/3 differentially regulate unconventional MIF secretion through TMED10 and secretory autophagy

doi: 10.1080/27694127.2026.2740425

Figure Lengend Snippet: Relationship between MIF and TMED10. (A) Secretion of MIF was measured in HEK293T cells either untreated or treated with brefeldin A. (B) Secretion of MIF was measured in HEK293T cells untreated or treated with the indicated doses of probenecid. (C) GST or the indicated GST-TMED10 domains coupled to glutathione sepharose beads were incubated with whole cell lysates from HEK293T cells expressing Flag-MIF. Elutes were subjected to SDS-PAGE and immunoblotted with an anti-Flag antibody. (D) GST-MIF coupled to glutathione sepharose beads was incubated with whole cell lysate from HEK293T cells expressing GFP-TMED10 or GFP-TMED10(ΔCT). Elutes were subjected to SDS-PAGE and immunoblotted with anti-GFP antibody. (E) Secretion of MIF was measured in HEK293T cells expressing mock or GFP-TMED10. (F) Secretion of MIF was measured in HEK293T cells treated with control siRNA or siRNA targeting TMED10 for 72 h. (G) A crosslink assay was performed in HEK293T cells expressing GFP-TMED10 with mock or Flag-MIF to examine the oligomeric form of TMED10. (H) Secretion of Flag-MIF was measured in HEK293T cells expressing mock, RAB1A, 1B, and 2A.

Article Snippet: For knockdown experiments, siRNA targeting human TMED10 (BIONEER, 10,972–1), human ATG5 (BIONEER, 9474–1), human SYT5 (BIONEER, 6861–1), and a negative control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) were used.

Techniques: Incubation, Expressing, SDS Page, Control

Journal: Autophagy Reports

Article Title: TRPML3 and TRPML1/3 differentially regulate unconventional MIF secretion through TMED10 and secretory autophagy

doi: 10.1080/27694127.2026.2740425

Figure Lengend Snippet: Effect of autophagy on MIF secretion. (A) HeLa cells expressing GFP-LC3 and mCherry-MIF were treated with cold digitonin (50 µg/ml) and imaged by fluorescence microscopy. (B) Secretion of MIF was measured in HEK293T cells either untreated or treated with 10 μM SAR405. (C) Secretion of MIF was measured in HEK293T cells treated with control siRNA or siRNA targeting ATG5 for 72 h. (D) Secretion of MIF was measured in HEK293T cells either untreated or treated with 100 nM bafilomycin A1. (E) Secretion of MIF was measured in HEK293T cells treated with control siRNA or siRNA targeting SYT5 for 48 h. Knockdown efficiency was confirmed by immunoblotting for SYT5. (F) Secretion of Flag-MIF was measured in HEK293T cells expressing mock or GFP-SEC22B. (G) HeLa cells expressing GFP-SEC22B and mCherry-MIF were treated with cold digitonin (50 µg/ml) and imaged by fluorescence microscopy. (H, upper) HeLa cells co-expressing GFP-MIF and Flag-VAMP4 were treated with cold digitonin (50 µg/ml), immunostained with an anti-Flag antibody, and imaged by fluorescence microscopy. (H, lower) HeLa cells expressing GFP-VAMP8 and mCherry-MIF were treated with cold digitonin (50 µg/ml) and imaged by fluorescence microscopy.

Article Snippet: For knockdown experiments, siRNA targeting human TMED10 (BIONEER, 10,972–1), human ATG5 (BIONEER, 9474–1), human SYT5 (BIONEER, 6861–1), and a negative control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) were used.

Techniques: Expressing, Fluorescence, Microscopy, Control, Knockdown, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B drives GC cell invasion and migration in a PI3K/AKT-dependent manner. (A) Gene set enrichment analysis indicated that genes upregulated in the UPK1B-high group were enriched in the PI3K/AKT pathway. (B) Protein levels of UPK1B in GC cell lines. (C) Knockdown of UPK1B reduced PI3K/AKT activation in MKN45 cells. Silencing UPK1B suppressed the (D) migration/invasion capacity and (E) wound closure rate of MKN45 cells. (F) Overexpression of UPK1B enhanced PI3K/AKT pathway activation in AGS cells, which was attenuated by the PI3K inhibitor LY294002. Inhibition of PI3K/AKT signaling reversed UPK1B-induced (G) migration/invasion capacity and (H) wound closure rate of AGS cells. UPK1B, uroplakin 1B; GC, gastric cancer; p-, phosphorylated; sh, short hairpin RNA; NC, negative control; OE, overexpression.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Migration, Knockdown, Activation Assay, Over Expression, Inhibition, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: CDX2 acts as a transcriptional repressor of UPK1B and its high expression is associated with favorable prognosis of patients with GC. (A) Venn diagram showing overlapping predicted transcriptional regulators of UPK1B from ChEA and ChEA3 databases. (B) Knockdown of CDX2 in AGS cells resulted in increased UPK1B (C) mRNA and (D) protein expression. (E) Overexpression of CDX2 in MKN45 cells reduced UPK1B protein levels. Data from (F) The Cancer Genome Atlas Stomach Adenocarcinoma cohort and (G) the Kaplan-Meier plotter database indicated that high CDX2 expression was associated with improved prognosis of patients with GC. UPK1B, uroplakin 1B; GC, gastric cancer; si, small interfering RNA; NC, negative control; OE, overexpression; HR, hazard ratio; CDX2, caudal-related homeobox transcription factor 2; ChEA, ChIP-X Enrichment Analysis.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Expressing, Knockdown, Over Expression, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B activates PI3K/AKT signaling by antagonizing the inhibitory regulator PIK3IP1 in gastric cancer cells. (A) Venn diagram showing that PIK3IP1 was identified as a putative UPK1B-interacting partner based on BioGRID and HIPPIE protein-protein interaction databases. (B) UPK1B and PIK3IP1 co-localized in the cytoplasm and plasma membrane of MKN45 cells. (C) Interaction between UPK1B and PIK3IP1 in MKN45 cells. (D) Knockdown of PIK3IP1 in MKN45 cells. (E) Silencing PIK3IP1 in UPK1B-knockdown MKN45 cells restored PI3K/AKT pathway activation. Knockdown of PIK3IP1 reversed the decrease in (F) migration/invasion and (G) wound-healing capacity in UPK1B-silenced MKN45 cells. UPK1B, uroplakin 1B; p-, phosphorylated; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; PIK3IP1, PI3K inhibitor interacting protein 1; HIPPIE, Human Integrated Protein-Protein Interaction Reference; IP, immunoprecipitation.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Clinical Proteomics, Membrane, Knockdown, Activation Assay, Migration, Small Interfering RNA, shRNA, Negative Control, Immunoprecipitation