Sodium dodecyl sulfate (sds)

Bovine serum albumin (BSA, Solarbio, China) was used as a model protein. One milliliter of protein solution (1 mg/mL BSA/phosphate-buffered saline (PBS)) was pipetted onto the titanium surfaces. After 3 h of incubation at 37°C, the samples were moved into a 24-well plate and washed with PBS （Solarbio, China). Nonadherent proteins were removed, and the rest of the proteins were dissolved in 600 μL of 1% sodium dodecyl sulfate (SDS, Solarbio, China). The protein content in the SDS solution was determined using a bicinchoninic acid (BCA) protein assay kit (Applygen Technologies, Inc., China). The test results were normalized by a BCA standard protein curve.

For the haemolysis studies, rabbit erythrocytes (Fenghui Biology, Hunan, China) were obtained from healthy blood donors. Blood samples mixed with 0.25% potassium oxalate were immediately separated by centrifugation at 1500 rpm for 10 min. Subsequently, the cells were washed three times with phosphate buffered saline (PBS, Yuanpei Biology, Shanghai, China) and diluted to a 5% suspension with PBS. 250 μL of erythrocyte stock dispersion was mixed with 250 μL of PBS containing different concentrations of AMPs and incubated at 37 °C for 1 h. Intact erythrocytes were removed by centrifugation at 10,000 rpm for 5 min. The absorbance of the resulting supernatant was measured at 540 nm. PBS was used as a negative control (0% lysis), and 0.1% SDS (Solarbio Life Sciences) was used as a positive control (100% lysis). The haemolysis rate was determined using Eq. (1). $$HaemolysisRate(\%)=\frac{D_{\mathit{test}}-D_{\mathit{nc}}}{D_{\mathit{pc}}-D_{\mathit{nc}}}\times 100\%$$ where D_test, D_nc, and D_pc are the 540 nm absorbance of the tested sample, negative control, and positive control, respectively.

Clinical isolates were obtained from a tertiary teaching hospital (Wenzhou, China). Reference strains (E. coli ATCC25922, K. pneumoniae ATCC700603, and A. baumannii ATCC19606) were used in all mechanism assays. Antibiotics including rifampicin, linezolid, erythromycin, tetracycline, vancomycin, and polymyxin B (PMB) (Kangtai, Zhejiang, China) were used in this study. Pentamidine, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and their solvent dimethyl sulfoxide (DMSO) were purchased from MedChemExpress (Junction, NJ, USA) and Sigma-Aldrich, USA (St. Louis, MO, USA). The broth microdilution (BMD) method was used to determine the MICs of all agents against all bacteria (Table S2). Sub-MICs of PMB (0.0625 mg/L) or SDS (0.01% v/v) (Solarbio, Beijing, China) were used as membrane permeators and CCCP (5 mg/L) as an efflux inhibitor [21 (link)]. Sub-MICs of CCCP (5 mg/L) and PMB (0.0625 mg/L) were added to serially two-fold-increased concentrations of linezolid, rifampicin, or vancomycin to determine their effects on the MICs of these antibiotics for multiple GNBs.

The outer membrane permeability was measured using a hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN, Aladdin, Shanghai, China) uptake method, as previously described (24 (link)), with some modifications. K. pneumoniae FK7921 was grown to the mid-logarithmic phase with an OD of 0.4 to 0.6 at 600 nm in LB. Equal volumes of bacterial solution were allocated to the designated tubes for the experimental groups with the desired concentrations of pentamidine monotherapy, linezolid monotherapy, or combination therapy as well as for the positive-control groups with colistin (Kangtai, Zhejiang, China), SDS (Solarbio, China), and Triton X-100 (Solarbio, China) and the negative-control group, which contained only the bacteria. The tubes were incubated at 37°C with shaking at 180 rpm for 2 h. The bacterial solutions were then pelleted and washed three times in PBS buffer (pH 7.2) with 5 mM glucose and were subsequently resuspended with 1 mL of 10 μM NPN working solution. 200 μL of the resulting cell suspension was added to each well of a black 96-well plate. After incubation at room temperature for 30 min in darkness, a multimode plate reader (VICTOR Nivo, PerkinElmer, USA) was used to measure the fluorescence at an excitation wavelength and an emission wavelength of 350 and 420 nm, respectively.

Chemical reagents, including Tris, NaCl, and SDS, for molecular biology and buffer preparation were purchased from Solarbio (Beijing, China). Antibodies (Table 4) for Western blot analysis were from Cell Signaling Technology, Abcam, or Santa Cruz Biotechnology. RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM, BI, USA), basal medium eagle (BME) and fetal bovine serum (FBS) were all purchased from BI (Bioind, Israel).Table 4

List of antibodies.

Antibodies	Company	catalog
PBK	Santa Cruz Biotechnology	sc-390399
p-PBK/TOPK(Thr9)	Cell signaling technology	#4941 s
mTOR	Cell signaling technology	#2972 s
p-mTOR(Ser2448)	Cell signaling technology	#5536 s
AKT1	Cell signaling technology	#2938 s
p-AKT(Ser473)	Cell signaling technology	#4051 s
p-p70S6K(Thr421/Ser424)	Cell signaling technology	#9204 s
YB1	Abcam	ab76149
p-YB1(Thr89)	Absin Bioscience Inc	YBX1-T89W191012
p-YB1(Ser209)	Absin Bioscience Inc	YBX1-S209W191012
GAPDH	Proteintech Group	60004-1-Ig
β-tubulin	HUABIO	M1305-2
EF-1α1	Santa Cruz Biotechnology	sc-21758
GFP	Santa Cruz Biotechnology	sc-9996
ACTB	Beijing Zhong Shan Goldenbridge Biotechnology Company Ltd	TA-09