Fig 2: Protein expression of chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in periodontal ligament (PDL) cells at rest and under different loading intensities. Protein expression of BAG3, STUB1, HSPA8, HSPB8, SYNPO2, and Filamin A in PDL cells analyzed via western blot. Cultures were exposed to different amounts of mechanical strain (s) of 2.5, 5, and 10%. The left lanes represent the results from the untreated cells serving as control (C), and the right lanes show the results for the different amounts of strain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 37 kDa) was used as protein loading control and is presented for each band below the molecule of interest. kDa kilodalton. Figures feature representative results from one donorProteinexpression von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Markergenen in PDL(Parodontalligament)-Zellen in Ruhe und bei unterschiedlichen Belastungsgraden. Proteinexpression von BAG3, STUB1, HSPA8, HSPB8, SYNPO2 und Filamin A in PDL-Zellen, analysiert mittels Western-Blot. Die Kulturen wurden unterschiedlichen mechanischen Belastungen (s) ausgesetzt: 2,5, 5 und 10%. Die linken Bahnen zeigen die Ergebnisse der unbehandelten Zellen, die als Kontrolle (C) dienten, die rechten Bahnen die Ergebnisse bei den unterschiedlichen Belastungsgraden. Glyceraldehyd-3-Phosphat-Dehydrogenase (GAPDH, 37 kDa) wurde als Proteinladekontrolle verwendet und ist für jede Bande unterhalb des interessierenden Moleküls angegeben. kDa Kilodalton. Die Abbildungen zeigen repräsentative Ergebnisse von einem Spender

Fig 3: Staining intensity calculation for chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in the periodontal ligament (PDL) of teeth with and without orthodontic tooth movement (OTM). Immunohistochemical quantification of the staining intensities of mechanically loaded (Moved) and control (Control) specimens for BAG3, STUB1, HSPA8, HSPB8, SYNPO2, and Filamin A. Three regions of interest (ROI) were measured to obtain the mean values for diaminobenzodine (DAB) staining intensities within an image. Values represent the mean ± standard error of the mean (SEM) of n = 6 specimens per group. All data were statistically analyzed using one way analysis of variance (ANOVA) and Bonferroni correction. P < 0.05 was considered statistically significantBerechnung der Färbeintensität von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Moleküle in PDL(Parodontalligament)-Zellen von Zähnen mit und ohne kieferorthopädische Zahnewegung (OTM). Immunhistochemische Quantifizierung der Färbeintensitäten von mechanisch belasteten Proben (Moved) und Kontrollproben (Control) für BAG3, STUB1, HSPA8, HSPB8, SYNPO2 und Filamin A. Drei ROI („regions of interest“) innerhalb eines Bildes wurden zur Ermittlung der Mittelwerte für DAB(Diaminobenzodin)-Färbeintensitäten vermessen. Die Werte stellen den Mittelwert ±Standardfehler des Mittelwerts (SEM) von n = 6 Proben pro Gruppe dar. Alle Daten wurden mittels Einwegvarianzanalyse (ANOVA) und Bonferroni-Korrektur statistisch ausgewertet. Als statistisch signifikant wurde p < 0,05 festgelegt

Fig 4: a–u In vivo verification of chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in the periodontal ligament (PDL) of teeth with or without orthodontic tooth movement (OTM). In vivo protein expression of BAG3 (a–c), STUB1 (d–g), HSPA8 (h, i), HSPB8 (j–m), SYNPO2 (n–q), and Filamin A (r–u) in PDL cells visualized by immunohistochemistry on teeth without OTM extracted due to crowding, and on teeth with OTM that were incorporated in a fixed rapid palatal expansion appliance, followed by extraction. Specifications indicated via arrows and arrowheads are explained in the “Results” section. R Root of the tooth, B Bone. Expression intensities were visualized via brown staining with diaminobenzodine (DAB). Magnification × 20a–u In-vivo-Nachweis von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Molekülen im Parodontalligament (PDL) von Zähnen mit bzw. ohne kieferorthopädische Zahnbewegung (OTM). In-vivo-Proteinexpression von BAG3 (a–c), STUB1 (d–g), HSPA8 (h, i), HSPB8 (j–m), SYNPO2 (n–q) und Filamin A (r–u) in PDL-Zellen, immunhistochemisch visualisiert an Zähnen ohne OTM, die bei Engstand extrahiert wurden, und an Zähnen mit OTM, die im Rahmen einer schnellen Gaumennahterweiterung in eine festsitzende Apparatur integriert und anschließend extrahiert wurden. Die durch Pfeile und Pfeilspitzen gekennzeichneten Spezifikationen werden im Abschnitt „Ergebnisse“ erläutert. R Zahnwurzel, B Knochen. Expressionsstärken wurden durch Braunfärbung mit Diaminobenzodin (DAB) visualisiert. Vergr. 20:1

Fig 5: Inhibition of EGFR expression and downstream signaling by 35d through hsp70-mediated lysosomal degradation in EGFR-mutant LUAD tumors with TKI resistance. A and B, EGFR and HSP70 expression in GR6, GR8, and GR10 cells treated with 35d for 24 h were determined by Western blot analysis (A). B, quantitation of EGFR and HSP70 expression in GR6, GR8, and GR10 cells from (A). Results are expressed relative to total protein. Comparisons between each treatment group with its untreated control group were analyzed using Student’s t test followed by the Bonferroni correction for three comparisons. ∗p < 0.05, ∗∗p < 0.01. C, EGFR expression in H1650, H1975, and HEK293 cells treated with 35d for 24 h were determined by Western blot analysis. D, GSEA of RNA-seq data as described in Figure 3A using KOBAYASHI EGFR signaling DN and UP pathway gene sets. E, Western blot showing EGFR expression in GR6 and GR10 cells treated with 35d (2 μM) and hsp70 inhibitor ver-155008 (ver) and pifithrin (pif) at the indicated concentrations (μM) for 24 h. F, fold changes in EGFR, BAG3, HSPBP1, SQSTM1, and STUB1 mRNA levels in GR6 cells treated with 35d in RNA-seq data as described in Figure 3A. G, Western blot showing EGFR expression in GR6 and GR10 cells treated with 35d (2 μM) and lysosome inhibitors hydroxychloroquine (HCQ), bafilomycinA (bafA) and proteasome inhibitors MG132, ixazomib (ixa) at the indicated concentrations for 24 h. H, protein expression of EGFR, hsp70, bag3, and p62 analyzed at the indicated time points in GR6 and GR10 cells treated with 35d (2 μM). Relative protein levels are indicated below the blot. Total protein was used as the loading control. Molecular weight markers are noted next to all immunoblots. Quantitation data are from three independent experiments and presented as mean ± SD (B). I, model depicting EGFR protein degradation in response to 35d treatment. EGFR, epidermal growth factor receptor; GR, gef-resistant; GSEA, gene set enrichment analysis; HSP, heat shock protein; LUAD, lung adenocarcinoma; TKI, tyrosine kinase inhibitor.